Immunogenic compositions comprising porphyromonas gingivalis proteins and/or peptides and methods

Provided herein are methods and immunogenic compositions useful for protecting mammals from infection and pathology of P. gingivalis. Specifically, arginine-specific proteases of Porphyromonas gingivalis and peptides derived therefrom offer protection against infection. Immunogenic compositions comprising a 50 kDa arginine-specific protease, the high molecular weight complex or peptides from one of the foregoing proteins are capable of protecting against P. gingivalis infection and/or gingivitis and/or periodontitis caused thereby in mammals, including humans.

BACKGROUND OF THE INVENTION 
The field of this invention is immunogenic compositions comprising 
bacterial proteases and/or peptides derived therefrom, more particularly 
those of Porphyromonas gingivalis, most particularly the arginine-specific 
proteases and immunogenic compositions containing Arg-gingipains and/or 
peptides derived therefrom, and the lysine-specific proteases termed 
Lys-gingipains herein and immunogenic compositions containing 
Lys-gingipain(s) and/or peptides derived therefrom. Those immunogenic 
compositions are useful in the protection of a mammal, including a human, 
from infection and pathology caused by P. gingivalis. 
Porphyromonas gingivalis (formerly Bacteroides gingivalis) is an obligately 
anaerobic bacterium which is implicated in periodontal disease. P. 
gingivalis produces several distinct proteolytic enzymes; its proteinases 
are recognized as important virulence factors, together with other factors 
such as lipopolysaccharide and a polysaccharide capsule, fimbriae, 
lectin-like adhesins, hyaluronidase, keratinase, superoxide dismutase and 
hemagglutinating and hemolyzing activities. A number of physiologically 
significant proteins, including collagen, fibronectin, immunoglobulins, 
complement factors C3, C4, C5, and B, lysozyme, iron-binding proteins, 
plasma proteinase inhibitors, fibrin and fibrinogen, and factors of the 
plasma coagulation cascade system, are hydrolyzed by P. gingivalis 
proteases. Broad proteolytic activity plays a role in the evasion of host 
defense mechanisms and the destruction of gingival connective tissue in 
progressive periodontitis [Saglie et al. (1988) J. Periodontal. 
59:259-265]. 
Progressive periodontitis is characterized by acute tissue degradation 
promoted by collagen digestion and a vigorous inflammatory response 
characterized by excessive neutrophil infiltration [White and Maynard 
(1981) J. Periodontal Res. 16:259-265]. Gingival crevicular fluid 
accumulates in periodontitis as periodontal tissue erosion progresses at 
the foci of the infection, and numerous plasma proteins are exposed to 
proteinases expressed by the bacteria at the injury site. Neutrophils are 
recruited to the gingiva, in part, by the humoral chemotactic factor C5a. 
The complement components C3 and C5 are activated by complex plasma 
proteases with "trypsin-like" specificities called convertases 
[Muller-Eberhard (1988) Ann. Rev. Biochem. 57:321-347]. The human plasma 
convertases cleave the .alpha.-chains of C3 and C5 at a specific site 
generating biologically active factors known as anaphylatoxins (i.e. C3a 
and C5a). The anaphylatoxins are potent proinflammatory factors exhibiting 
chemotactic and/or spasmogenic activities as well as promoting increased 
vascular permeability. The larger products from C3 and C5 cleavage (i.e. 
C3b and C5b) participate in functions including complement cascade 
activation, opsonization, and lytic complex formation. 
There are conflicting data as to the number and types of proteinases 
produced by P. gingivalis. In the past, proteolytic activities of P. 
gingivalis were classified into two groups; those enzymes which 
specifically degraded collagen and the general "trypsin-like" proteinases 
which appeared to be responsible for other proteolytic activity. Chen et 
al. (1992) J. Biol. Chem. 267, 18896-18901 reported the first rigorous 
purification and biochemical characterization of an arginine-specific P. 
gingivalis protease; the purification of a lysine-specific proteinase of 
P. gingivalis is described by Pike et al. (1994) J. Biol. Chem. 
269:406-411 [see also Potempa et al. (1995) Perspectives in Drug Discovery 
and Design 2:445-458]. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide immunogenic compositions 
comprising at least one peptide corresponding in sequence to the 
N-terminus of at least one arginine-specific proteinase derived from P. 
gingivalis, preferably from Arg-gingipain, termed Arg-gingipain-1 (or 
RGP-1), having an apparent molecular mass of 50 kDa as estimated by sodium 
dodecyl sulfate polyacrylamide gel electrophoresis and an apparent 
molecular mass of 44 kDa as estimated by gel filtration chromatography, 
and enzymological properties as described hereinbelow. In a specifically 
exemplified RGP protein, the protein is characterized by an N-terminal 
amino acid sequence as given in SEQ ID NO:1 
(YTPVEEKQNGRMIVIVAKKYEGDIKDFVDWKNQR) and by a C-terminal amino acid 
sequence as given in SEQ ID NO:2 (ELLR). A second Arg-specific gingipain 
has an N-terminal sequence as given in SEQ ID NO:24 
(YTPVEEKENGRMIVIVAKKY), it differs from the sequence as given in SEQ ID 
NO:10 in that position 7 is Glu rather than Gln. 
Within the scope of the present invention are methods for protecting a 
mammal, including a human, from periodontitis and/or other pathology 
caused at least in part by P. gingivalis, said method comprising the step 
of administering to said mammal an immunogenic composition comprising at 
least one peptide corresponding in sequence to the amino-terminus of at 
least one of RGP-1, RGP-2, HMW RGP, or one or more peptides derived from 
one or more of the foregoing proteins or having amino acid sequence(s) 
taken from the amino acid sequence(s) of one or more of the foregoing 
proteins, wherein said peptide or protein, when used in an immunogenic 
composition in an animal, especially a mammal or human, confers protection 
against infection by and/or periodontitis caused at least in part by P. 
gingivitis. Preferred immunogenic compositions for protecting mammals 
(e.g., man) from P. gingivalis infection do not include a hemagglutinin 
protein or peptide. 
A further object of this invention are immunogenic compositions comprising 
an N-terminal peptide derived from the catalytic subunit of a high 
molecular weight Arg-gingipain (HMW RGP), which comprises a proteolytic 
component essentially as described hereinabove and at least one 
hemagglutinin component. A nucleotide sequence encoding the HMW RGP 
complex polyprotein is given in SEQ ID NO:5, nucleotides 949-6063 and the 
deduced amino acid sequence is given in SEQ ID NO:6. As specifically 
exemplified, the mature HMW RGP has a 50 kDa protease component (same as 
RGP-1) having a complete deduced amino acid sequence as given in SEQ ID 
NO:6 from amino acid 228 through amino acid 719 or in SEQ ID NO:4, amino 
acids 228-719. HMW RGP further comprises at least one hemagglutinin 
component. The encoded RGP-hemagglutinin complex is transcribed as a 
prepolyprotein, with the amino acid sequence of at least one hemagglutinin 
protein as given in SEQ ID NO:6 from amino acid 720-1091, from 1092-1492 
and/or from 1430-1704. 
Compositions and immunogenic preparations including but not limited to 
vaccines, comprising at least one peptide antigen derived from the 
N-terminus of an Arg-gingipain from P. gingivalis and/or a peptide derived 
from an Arg-gingipain, and/or a Lys-gingipain and a suitable carrier 
therefor are provided. Such immunogenic compositions and vaccines are 
useful, for example, in immunizing an animal, including a human, against 
infection by and/or the inflammatory response and tissue damage caused by 
P. gingivalis in periodontal disease. The vaccine preparations comprise an 
immunogenic amount of an Arg-specific proteinase, Lys-gingipain, or an 
immunogenic peptide fragment or subunit of either one or both of said 
Arg-gingipains and Lys-gingipains or other P. gingivalis protease. Such 
vaccines may comprise one or more N-terminal peptides from Arg-gingipains 
and/or one or more Lys-gingipains and/or an Arg-gingipain or Lys-gingipain 
in combination with another protein or other immunogen. By "immunogenic 
amount" is meant an amount capable of eliciting the production of 
antibodies directed against one or more Arg-gingipain and/or Lys-gingipain 
catalytic subunit (or one or more peptides whose amino acid sequence is 
derived from the foregoing proteins) in an individual or animal to which 
the vaccine has been administered. 
Oligopeptides of the present invention include those of about 30 amino 
acids or less, and include those comprising sequences as given in SEQ ID 
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID 
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID 
NO:21, SEQ ID NO:23 and SEQ ID NO:24. These oligopeptides can be 
formulated into vaccine compositions which are effective in protecting an 
animal, including a human, from infection by P. gingivalis and from 
periodontitis caused by P. gingivalis.

DETAILED DESCRIPTION OF THE INVENTION 
Abbreviations used herein for amino acids are standard in the art: X or Xaa 
represents an amino acid residue that has not yet been identified but may 
be any amino acid residue including but not limited to phosphorylated 
tyrosine, threonine or serine, as well as cysteine or a glycosylated amino 
acid residue. The abbreviations for amino acid residues as used herein are 
as follows: A, Ala, alanine; V, Val, valine; L, Leu, leucine; I, Ile, 
isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, 
Met, methionine; G, Gly, glycine; S, Ser, serine; T, Thr, threonine; C, 
Cys, cysteine; Y, Tyr, tyrosine; N, Asn, asparagine; Q, Gln, glutamine; D, 
Asp, aspartic acid; E, Glu, glutamic acid; K, Lys, lysine; R, Arg, 
arginine; and H, His, histidine. Other abbreviations used herein include 
Bz, benzoyl; Cbz, carboxybenzoyl; pNA, p-nitroanilide; MeO, methoxy; Suc, 
succinyl; OR, ornithyl; Pip, pipecolyl; SDS, sodium dodecyl sulfate; TLCK, 
tosyl-L-lysine chloromethyl ketone; TPCK, tosyl-L-phenylalanine 
chloromethyl ketone; S-2238, D-Phe-Pip-Arg-pNA, S-2222, 
Bz-Ile-Glu-(.gamma.-OR)-Gly-pNA; S-2288, D-Ile-Pro-Arg-pNA; S-2251, 
D-Val-Leu-Lys-pNA; Bis-Tris, 
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; FPLC, 
fast protein liquid chromatography; HPLC, high performance liquid 
chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; 
EGTA, [ethylene-bis(oxyethylene-nitrile)tetraacetic acid; EDTA, 
ethylenediamine-tetraacetic acid; Z-L-Lys-pNa, Z-L-Lysine-p-Nitroanilide; 
HMW, high molecular weight. 
Arg-gingipain (RGP) is the term given to a P. gingivalis enzyme with 
specificity for proteolytic and/or amidolytic activity for cleavage of a 
peptide and/or an amide bond, in which L-arginine contributes the carboxyl 
group. The Arg-gingipains described herein have identifying 
characteristics of cysteine dependence, inhibition response, Ca.sup.2+ 
-stabilization and glycine stimulation. Particular forms of Arg-gingipain 
are distinguished by the apparent molecular masses of the mature proteins 
(as measured without boiling before SDS-PAGE). See also Chen et al (1992) 
supra. Arg-gingipains of the present invention have no amidolytic or 
proteolytic activity for peptide and/or amide bonds in which L-lysine 
contributes the --COOH moiety. 
Antibodies specific for RGPs are produced in adult periodontitis patients, 
with the majority being reactive with antigenic determinants in the 
hemagglutinin/adhesin domain of RGP-1, [Curtiss et al. (1996) Infect. 
Immun. 64:2532]. Although patients with a history of destructive disease 
frequently demonstrate an elevated IgG response to P. gingivalis, these 
antibodies are apparently ineffective at limiting continued disease 
progression [Turner et al. (1989) Microbios 60:133; Yoshimura et al. 
(1987) Microbiol. Immunol. 31:935; Gunsolley et al. (1990) J. Periodontol. 
61:412; Naito et al. (1987) Infect. Immun. 55:832]. In several animal 
studies, induction of an immune response to certain components of P. 
gingivalis exacerbates disease [McArthur and Clark (1993) J. Periodontol. 
64:807]. Animal experiments described herein have demonstrated the 
protective effect of P. gingivalis-specific antibodies produced against 
peptides derived from N-terminus of RGP-1 (FIG. 1). 
Arg-gingipain (RGP-1) is the name given herein to a protein characterized 
as having a molecular mass of 50 kDa as measured by SDS-PAGE and 44 kDa as 
measured by gel filtration over Sephadex G-150, having amidolytic and/or 
proteolytic activity for substrates having L-Arg in the P.sub.1 position, 
i.e. on the N-terminal side of the peptide bond to be hydrolyzed, 
dependent on cysteine (or other thiol groups for full activity), having 
sensitivity to cysteine protease group-specific inhibitors including E64, 
iodoacetamide, iodoacetic acid, and N-methylmaleimide, leupeptin, 
antipain, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, TLCK, 
TPCK, p-aminobenzamidine, N-chlorosuccinamide, and chelating agents 
including EDTA and EGTA, but being resistant to inhibition by human 
cystatin C, .alpha.2-macroglobulin, .alpha.1-proteinase inhibitor, 
antithrombin III, .alpha.2-antiplasmin, serine protease group-specific 
inhibitors including diisopropylfluorophosphate, phenylmethyl 
sulfonylfluoride and 3,4-diisochlorocoumarin. The amidolytic and/or 
proteolytic activities are stabilized by Ca.sup.2+ and stimulated by 
glycine-containing peptides and glycine analogs. Arg-gingipain-1 (RGP-1) 
is the 50 kDa protein whose purification and characterization was 
disclosed in Chen et al. (1992) supra and Wingrove et al. (1992) supra. 
Arg-gingipain-2 (RGP-2) is a 50 kDa arginine-specific proteinase whose 
purification is first described hereinbelow. RGP-1 is distinguished from 
RGP-2 in that RGP-1 is not retained during chromatography over DE-52; 
RGP-2 is eluted from Whatman DE-52 with salt. A comparison of the primary 
structures of RGP-1 and RGP-2 is presented in Table 2. 
An exemplified Arg-gingipain termed HMW RGP herein has an apparent 
molecular mass of 95 kDa as determined by SDS-PAGE without boiling of 
samples. When boiled, it dissociates into components of 50 kDa, 43 kDa, 27 
kDa and 17 kDa. Arg-gingipain-1 (RGP-1) is the name given to the 50 kDa, 
enzymatically active component of the high molecular weight complex. 
The complete amino acid sequence of the exemplified mature RGP-1 is given 
in SEQ ID NO:6, from amino acids 228-719. A second exemplary amino acid 
sequence is given in SEQ ID NO:4, amino acids 1 through 510. The complete 
coding sequence for the HMW RGP precursor polyprotein is given in SEQ ID 
NO:5, nucleotides 949-6063. In nature these proteins are produced by 
Porphyromonas gingivalis; they can be purified from cells or from culture 
supernatant using the methods provided herein. These proteins can also be 
produced recombinantly in suitable host cells genetically engineered to 
contain and express the exemplified (or synonymous) coding sequences. 
As used herein with respect to RGP-1 or RGP-2, a substantially pure 
Arg-gingipain preparation means that there is only one protein band 
visible after silver-staining an SDS polyacrylamide gel run with the 
preparation, and the only amidolytic and/or proteolytic activities are 
those with specificity for L-arginine in the P.sub.1 position relative to 
the bond cleaved. A substantially pure high molecular weight Arg-gingipain 
preparation has only one band (95 kDa) on SDS-PAGE (sample not boiled) or 
four bands (50 kDa, 43 kDa, 27 kDa, 17 kDa; sample boiled). Using a higher 
resolution tricine SDS-PAGE system, an additional component of 19 kDa has 
been detected in HMW RGP [Pavloff et al. (1995) supra]. No amidolytic or 
proteolytic activity for substrates with lysine in the P.sub.1 position is 
evident in a substantially pure HMW RGP. Substantially pure Arg-gingipain 
is substantially free of naturally associated components when separated 
from the native contaminants which accompany them in their natural state. 
Thus, Arg-gingipain that is chemically synthesized or recombinantly 
synthesized in a cellular system different from the cell from which it 
naturally originates will be substantially free from its naturally 
associated components. 
Techniques for chemical synthesis of polypeptides are described, for 
example, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2154. A 
chemically synthesized Arg-gingipain protein or peptide derived therefrom 
is considered an "isolated" polypeptide or peptide. 
Recombinantly produced RGP-1 and HMW RGP can be obtained by culturing host 
cells genetically engineered to contain and express the non-naturally 
occurring (recombinant) polynucleotides comprising nucleotide sequences 
encoding an Arg-gingipain as described herein under conditions suitable to 
attain expression of the proteinase-encoding sequence. See, e.g., U.S. 
Pat. No. 5,523,390, incorporated by reference herein. 
Example 1 below and U.S. Pat. No. 5,523,390 describe the purification of a 
50 kDa RGP-1 and HMW RGP from P. gingivalis culture supernatant, i.e., 
from a natural source. Various methods for the isolation of an 
Arg-gingipain from other biological material, such as from nonexemplified 
strains of P. gingivalis or from cells transformed with recombinant 
polynucleotides encoding such proteins, may be accomplished by methods 
known in the art. Various methods of protein purification are known in the 
art, including those described, e.g., in Guide to Protein Purification, 
ed. Deutscher, Vol. 182 of Methods in Enzymology (Academic Press, Inc., 
San Diego, 1990) and Scopes, Protein Purification: Principles and Practice 
(Springer-Verlag, New York, 1982). 
Further analysis of the high molecular weight fractions containing 
Arg-specific amidolytic and proteolytic activity revealed that HMW RGP 
contained proteins of 44 kDa, subsequently identified as a hemagglutinin, 
and 27 kDa and 17 kDa, which are also postulated to have hemagglutinating 
activity. The empirically determined N-terminal amino acid sequence of the 
complexed 44 kDa protein corresponds to amino acids 720-736 of SEQ ID 
NO:6. 
Purified RGP-1 exhibits an apparent molecular mass of about 50 kDa as 
determined by SDS-polyacrylamide gel electrophoresis. The size estimate 
obtained by gel filtration on high resolution agarose (Superose 12, 
Pharmacia, Piscataway, N.J.) is 44 kDa. N-terminal sequence analysis 
through 43 residues gave a unique structure which showed no homology with 
any other proteins, based on a comparison in the protein NBRS data base, 
release 39.0. The sequence obtained is as follows: 
YTPVEEKQNGRMIVIVAKKYEGDIKDFVDWKNQR (SEQ ID NO:1). The C-terminal amino 
acid sequence of the gingipain-1 (major form recognized in zymography 
SDS-PAGE, 0.1% gelatin in gel), was found to be ELLR (SEQ ID NO:2). This 
corresponds to the amino acids encoded at nucleotides 3094-3105 in SEQ ID 
NO:3 and nucleotides 3094-3105 in SEQ ID NO:5, consistent with 
autoproteolytic processing of the precursor polyprotein to produce the 
mature 50 kDa RGP-1 protein. Without wishing to be bound by theory, it is 
proposed that SEQ ID NO:3 comprises the coding sequence for RGP-1, the 
enzymatically active component of the high molecular weight form of 
Arg-gingipain. This is consistent with the observation that there are at 
least two genes with substantial nucleic acid homology to the 
Arg-gingipain-specific probe. 
Because progressive periodontitis is characterized by tissue degradation, 
collagen destruction and a strong inflammatory response, and because P. 
gingivalis exhibits complement-hydrolyzing activity, purified RGP-1 was 
tested for proteinase activity using purified human complement C3 and C5 
as substrates [see Wingrove et al. (1992) J. Biol. Chem. 269:18902-18907]. 
RGP-1 selectively cleaved the C3 .alpha.-chain. C3a biological activity in 
the C3 digestion mixture was not observed, and the C3a-like fragment 
released from the .alpha.-chain was extensively degraded by RGP-1. When 
human C5 is subjected to prolonged digestion by RGP-1, functional C5a 
accumulates in the digestion mixture. RGP-1 injected into guinea pig skin 
enhances vascular permeability at concentrations greater than 10.sup.-8 M 
and causes neutrophil accumulation at the site of injection. This activity 
was dependent on proteolytic activity of the RGP-1 protein. The results 
demonstrate the ability of RGP-1 to elicit an inflammatory response. 
The N-terminal amino acid sequence of the 50 kDa component of the HMW RGP 
is identical to the first 22 amino acids of the 50 kDa RGP-1. 
Characterization of the HMW RGP activity showed the same dependence on 
cysteine (or other thiols) and the same spectrum of response to potential 
inhibitors. Although the HMW RGP and RGP-1 amidolytic activity was 
stimulated by Gly-Gly, the response for RGP-2 was only about half that 
observed for RGP-1 and HMW RGP. 
The cloning and coding sequences for Arg-gingipain are described in U.S. 
Pat. No. 5,523,390. SEQ ID NO:3 herein is the DNA sequence of the 3159 bp 
PstI/BamHI fragment from P. gingivalis strain HG66 (W83). An exemplified 
sequence encoding mature RGP-1 extends from 1630-3105. The first 
nucleotide belongs to the PstI cloning site. The first ATG appears at 
nucleotide 949 and is followed by a long open reading frame (ORF) of 2210 
nucleotides. The first ATG is following by 8 others in frame (at 
nucleotides 1006, 1099, 1192, 1246, 1315, 1321, 1603, and 1609). Which of 
these initiation codons are used in translation of the Arg-gingipain-2 
precursor can be determined by expression of the polyprotein in bacteria 
and subsequent N-terminal sequence analysis of preprotein intermediates. 
The primary structure of the mature Arg-gingipain is derived from the 
empirical N-terminal and C-terminal sequences and molecular mass. Thus, a 
mature RGP has an amino terminus starting at nucleotide residue 1630 in 
SEQ ID NO:3 and at amino acid 228 in SEQ ID NO:4; both mature proteins are 
cleaved after an Arg. The 50 kDa and the 44 kDa bands from Ez-L-Arg-pNa 
activity peaks are identical in sequence to the deduced amino acid 
sequence of gingipain, encoded respectively at nucleotides 1630-1695 and 
at nucleotides 3106-3156. The carboxyl terminus is most likely derived 
from autoproteolytic processing after the Arg residue encoded at 3103-3105 
where the coding sequence of hemagglutinin starts (nucleotide 3106). The 
deduced 492 amino acids of RGP-1 give rise to a protease molecule with a 
calculated molecular weight of 54 kDa, which correlates well with the 
molecular mass of 50 kDa determined by SDS-PAGE analysis. 
The skilled artisan recognizes that other P. gingivalis strains can have 
coding sequences for a protein with the distinguishing characteristics of 
an Arg-gingipain; those coding sequences may be identical to or synonymous 
with the exemplified coding sequence, or there may be some variation(s) in 
the encoded amino acid sequence. An Arg-gingipain coding sequence from a 
P. gingivalis strain other than H66 can be identified by, e.g. 
hybridization to a polynucleotide or an oligonucleotide having the whole 
or a portion of the exemplified coding sequence for mature gingipain, 
under stringency conditions appropriate to detect a sequence of at least 
70% homology. 
SEQ ID NO:5 presents the nucleotide sequence encoding the complete 
prepolyprotein sequence, including both the protease component and the 
hemagglutinin component(s) of HMW RGP. The coding sequence extends from an 
ATG at nucleotide 949 through a TAG stop codon ending at nucleotide 6063 
in SEQ ID NO:5. The deduced amino acid sequence is given in SEQ ID NO:6. 
Cleavage of the precursor protein after the Arg residue at 227 amino acid 
residues into the precursor protein removes the N-terminal precursor 
portion and after the Arg residue at amino acid 719 releases a low 
molecular weight Arg-gingipain catalytic component and at least one 
hemagglutinin component. 
The cloning and sequencing of the lysine-specific gingipain (KGP) is 
described in U.S. Pat. No. 5,475,077, which is incorporated by reference 
herein. The coding sequence of the 60 kDa active component of the 
Lys-gingipain complex extends through nucleotide 2863 in SEQ ID NO:7. The 
amino acid sequence identical to the amino-terminal sequence of the 44, 27 
and 17 kDa Lys-gingipain complex components, at least one of which is 
believed to function as a hemagglutinin, is encoded at nucleotides 
2864-2938 in SEQ ID NO:7. Without wishing to be bound by any particular 
theory, it is believed that an Arg-specific protease processes the 
polyprotein which is (in part) encoded within the nucleotide sequence of 
SEQ ID NO:7. The predicted molecular mass of 55.9 kDa for a 509 amino acid 
protein encoded from nucleotides 1336-2863 is consistent with the 
empirically determined estimate of 60 kDa (SDS-PAGE). 
Both HMW KGP (see U.S. Pat. No. 5,475,077), and HMW RGP can to 
erythrocytes, laminin and fibrinogen even if the catalytic domains are 
inactivated. However, TLCK-inactivated 50 kDa RGP cannot bind although the 
active form can degrade fibrinogen, fibronectin and laminin. Without 
wishing to be bound by theory, it is postulated that three nearly 
identical repeated sequences of HMW KGP and HMW RGP mediate this adhesion. 
Polyclonal antibodies have been made in response to a chemically 
synthesized peptide encompassing the repeated sequence 
(YTYTVYRDGKIKEGLTATTEDDGVATG-NHEYCVEKYTAGSVSPKVC) (SEQ ID NO:9), which is 
close to a consensus sequence for the three repeating domains of HMW RGP 
and HMW KGP. These antibodies do not affect the catalytic activities of 
these proteases. 
An Arg-gingipain coding sequence was also isolated from P. gingivalis W50. 
A 3.5 kb BamHI fragment was sequenced; it exhibited 99% nucleotide 
sequence identity with the 3159 bp fragment of P. gingivalis W83 (HG66) 
DNA containing Arg-gingipain coding sequence. A comparison of the deduced 
amino acid sequences of the encoded Arg-gingipains revealed 99.9% 
identity. 
Regardless of the affinity for Arg-Sepharose and the differences in 
specific activities, the purified form of RGP-2 gave in SDS-PAGE a single 
band with molecular mass of 48.5 kDa, slightly lower than for the 
catalytic domain of HMW RGP (50.0 kDa). It is also slightly lower than for 
RGP-1, where the molecular mass was refined using laser densitometry 
scanning of the gel to 49.0 kDa from the previously reported 50 kDa. 
In contrast to the uniform molecular mass, analysis of the purified forms 
of RGP-2 by means of zymography on gelatin SDS-PAGE revealed reciprocal 
heterogeneity in active band patterns and substantial differences in an 
electrophoretic mobility in comparison to RGP-1. The major activity zone 
of the latter gingipain was located in the 68-70 kDa area of the gel and 
did not have equivalent neither in starting material nor in the activity 
peaks separated by gel filtration chromatography. This indicates that the 
contribution of RGP-1 to the total proteolytic activity of P. gingivalis 
H66 is relatively minor, a conclusion which is in keeping with the low 
activity against Bz-L-Arg-pNA recovered in Vo of the DE-52 (300 activity 
units) as compared to the activity eluted from the column with NaCl (5,819 
activity units). 
Partial primary structure analyses of the 48.5-50 kDa forms of Arg-specific 
gingipain show that the amino-termini of three forms of RGP-2, which have 
been sequenced up to 50 amino acid residues and with one exception, Glu9 
instead Gln9, have identical primary structures (RGP-1 and the catalytic 
domain of HMW RGP). To further characterize possible structural 
differences between the Arg-Sepharose affinity variants of RGP-2 and 
RGP-1, a sample of each enzyme was S-ethylpyridylated and subjected to 
autodigestion or trypsin digestion. Due to the RGPs' strict specificity 
for Arg-X peptide bonds, autodigestion resulted in a discrete peptide band 
pattern with relatively high molecular masses within the range from 3 kDa 
to 27 kDa. The pattern was identical for the affinity variants of RGP-2, 
but it showed some differences in comparison to RGP-1, despite striking 
similarities of the overall peptide maps. 
The structures of RGP-2 variants was further investigated by reverse phase 
HPLC (C18 column) after tryptic digestion of the S-pyridylethylated 
proteins. Exactly the same peptide maps were again obtained, indicating 
that at the primary structure level, the Arg-Sepharose affinity variants 
of RGP-2 are indistinguishable. In contrast, the peptide map of RGP-1 
differs slightly from that of RGP-2. Several HPLC-purified tryptic 
peptides derived from RGP-1 and RGP-2 have been subjected to 
amino-terminal sequence analyses and in both cases, the same sequence 
overlapping with the following fragments of the catalytic domain of HMW 
RGP as inferred from DNA structure: 61-Gln-80-Lys, 92-Ser-112-Arg, 
142-Trp-184-Lys, 194-Asn-230-Lys. In one case, however, the peptide of 
RGP-2 which did not have an equivalent in the reverse phase HPLC peptide 
map of RGP-1 gave unique, though related, sequence, that differed from the 
latter one in 13 out of 29 compared amino acid residues (Table 2). 
Although RGP-1 and RGP-2 are closely related proteins, they differ in 
primary structure and therefore must be the products of different genes. 
SEQ ID NO:3 and SEQ ID NO:5 both represent sequences from P. gingivalis. 
However, it is understood that there will be some variations in the amino 
acid sequences and encoding nucleic acid sequences for Arg-gingipains from 
different P. gingivalis strains. The ordinary skilled artisan can readily 
identify and isolate Arg-gingipain-encoding sequences from other strains 
where there is at least 70% homology to the specifically exemplified 
sequences herein using the sequences provided herein taken with what is 
well known to the art, e.g., polymerase chain reaction and/or nucleic acid 
hybridization techniques. Also within the scope of the present invention 
are Arg-gingipain where the protease (or proteolytic component) has at 
least about 85% amino acid sequence identity with an amino acid sequence 
exemplified herein. 
It is also understood by the skilled artisan that there can be limited 
numbers of amino acid substitutions in a protein without significantly 
affecting function, and that nonexemplified gingipain-1 proteins can have 
some amino acid sequence diversion from the exemplified amino acid 
sequence. Such naturally occurring variants can be identified, e.g., by 
hybridization to the exemplified (mature) RGP-1 or HMW RGP coding sequence 
(or a portion thereof capable of specific hybridization to Arg-gingipain 
sequences) under conditions appropriate to detect at least about 70% 
nucleotide sequence homology, preferably about 80%, more preferably about 
90% and most preferably 95-100% sequence homology. Preferably the encoded 
Arg-gingipain protease or proteolytic component has at least about 85% 
amino acid sequence identity to an exemplified Arg-gingipain amino acid 
sequence. 
It is well known in the biological arts that certain amino acid 
substitutions can be made in protein sequences without affecting the 
function of the protein. Generally, conservative amino acids are tolerated 
without affecting protein function. Similar amino acids can be those that 
are similar in size and/or charge properties, for example, aspartate and 
glutamate and isoleucine and valine are both pairs of similar amino acids. 
Similarity between amino acid pairs has been assessed in the art in a 
number of ways. For example, Dayhoff et al. (1978) in Atlas of Protein 
Sequence and Structure, Volume 5, Supplement 3, Chapter 22, pages 345-352, 
which is incorporated by reference herein, provides frequency tables for 
amino acid substitutions which can be employed as a measure of amino acid 
similarity. Dayhoff et al.'s frequency tables are based on comparisons of 
amino acid sequences for proteins having the same function from a variety 
of evolutionarily different sources. 
In another embodiment of the present invention, polyclonal and/or 
monoclonal antibodies capable of specifically binding to a proteinase or 
fragments thereof are provided. The term antibody is used to refer both to 
a homogenous molecular entity, or a mixture such as a serum product made 
up of a plurality of different molecular entities. Monoclonal or 
polyclonal antibodies specifically reacting with the Arg-gingipains can be 
made by methods known in the art. See, e.g., Harlow and Lane (1988) 
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding 
(1986) Monoclonal Antibodies: Principles and Practice, 2ed., Academic 
Press, New York; and Ausubel et al. (1987) vide infra. Also, recombinant 
immunoglobulins may be produced by methods known in the art, including but 
not limited to, the methods described in U.S. Pat. No. 4,816,567. 
Monoclonal antibodies with affinities of 10.sup.8 M.sup.-1, preferably 
10.sup.9 to 10.sup.10 or more are preferred. 
Antibodies specific for Arg-gingipains are useful, for example, as probes 
for screening DNA expression libraries or for detecting the presence of 
Arg-gingipains in a test sample. Frequently, the polypeptides and 
antibodies will be labeled by joining, either covalently or noncovalently, 
a substance which provides a detectable signal. Suitable labels include 
but are not limited to radionuclides, enzymes, substrates, cofactors, 
inhibitors, fluorescent agents, chemiluminescent agents, magnetic 
particles and the like. U.S. patents describing the use of such labels 
include, but are not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. 
Antibodies specific for Arg-gingipain(s) and capable of inhibiting its 
proteinase activity are useful in treating animals, including man, 
suffering from periodontal disease. Such antibodies can be obtained by the 
methods described above and subsequently screening the 
Arg-gingipain-specific antibodies for their ability to inhibit proteinase 
activity. 
Compositions and immunogenic preparations, including vaccine compositions, 
comprising substantially purified recombinant Arg-gingipain(s) or an 
immunogenic peptide of an Arg-gingipain capable of inducing protective 
immunity in a suitably treated mammal and a suitable carrier therefor are 
provided. Alternatively, hydrophilic regions of the proteolytic component 
or hemagglutinin component(s) of Arg-gingipain can be identified by the 
skilled artisan, and peptide antigens can be synthesized and conjugated to 
a suitable carrier protein (e.g., bovine serum albumin or keyhole limpet 
hemocyanin) if needed for use in vaccines or in raising antibody specific 
for Arg-gingipains. Immunogenic compositions are those which result in 
specific antibody production when injected into a human or an animal. Such 
immunogenic compositions or vaccines are useful, for example, in 
immunizing an animal, including humans, against infection and/or 
inflammatory response and tissue damage caused by P. gingivalis in 
periodontal disease. The vaccine preparations comprise an immunogenic 
amount of one or more Arg-gingipains or an immunogenic fragment(s) or 
subunit(s) thereof. Such vaccines can comprise one or more Arg-gingipains 
or in combination with another protein or other immunogen, or an epitopic 
peptide derived therefrom. A preferred peptide has an amino acid sequence 
identical to the N-terminal sequence of RGP-1. An "immunogenic amount" 
means an amount capable of eliciting the production of antibodies directed 
against Arg-gingipain(s) in an individual to which the vaccine has been 
administered. 
Immunogenic carriers can be used to enhance the immunogenicity of the 
proteinases, proteolytic components, hemagglutinins or peptides derived in 
sequence from any of the foregoing. Such carriers include but are not 
limited to proteins and polysaccharides, liposomes, and bacterial cells 
and membranes. Protein carriers may be joined to the proteinases or 
peptides derived therefrom to form fusion proteins by recombinant or 
synthetic means or by chemical coupling. Useful carriers and means of 
coupling such carriers to polypeptide antigens are known in the art. 
The immunogenic compositions and/or vaccines may be formulated by any of 
the means known in the art. They are typically prepared as injectables, 
either as liquid solutions or suspensions. Solid forms suitable for 
solution in, or suspension in, liquid prior to injection may also be 
prepared. The preparation may also, for example, be emulsified, or the 
protein(s)/peptide(s) encapsulated in liposomes. Where mucosal immunity is 
desired, the immunogenic compositions advantageously contain an adjuvant 
such as the nontoxic cholera toxin B subunit (see, e.g., U.S. Pat. No. 
5,462,734). Cholera toxin B subunit is commerically available, for 
example, from Sigma Chemical Company, St. Louis, Mo. Other suitable 
adjuvants are available and may be substituted therefor. It is preferred 
that an adjuvant for an aerosol immunogenic (or vaccine) formulation is 
able to bind to epithelial cells and stimulate mucosal immunity. 
Among the adjuvants suitable for mucosal administration and for stimulating 
mucosal immunity are organometallopolymers including linear, branched or 
cross-linked silicones which are bonded at the ends or along the length of 
the polymers to the particle or its core. Such polysiloxanes can vary in 
molecular weight from about 400 up to about 1,000,000 daltons; the 
preferred length range is from about 700 to about 60,000 daltons. Suitable 
functionalized silicones include (trialkoxysilyl) alkyl-terminated 
polydialkylsiloxanes and trialkoxysilyl-terminated polydialkylsiloxanes, 
or example, 3-(triethyoxysilyl) propyl-terminated polydimethylsiloxane. 
See U.S. Pat. No. 5,571,531, incorporated by reference herein. Phosphazene 
polyelectrolytes can also be incorporated into immunogenic compositions 
for transmucosal administration (intranasal, vaginal, rectal, respiratory 
system by aerosol administration) (See U.S. Pat. No. 5,562,909). 
Alternatively, mucosal immunity can be triggered by the administration to 
mucosal surfaces, for example, orally, of recombinant avirulent bacterial 
cells which express a protective epitope derived from a P. gingivalis 
protease, for example, RGP-1, HMW RGP or RGP-2, of particular interest is 
the expression of at least about 15 amino acids from the N-terminus of the 
RGP-2 or the N-terminus of a catalytic subunit of HMW RGP or HMW KGP. 
Avirulent Salmonella typhi and avirulent Salmonella typhimurium strains, 
suitable vectors and suitable promoters for driving expression are known 
to the art. The protective epitopes are advantageously expressed as 
fusions with other proteins, such as Salmonella flagellin, tetanus toxin 
fragment C, and E. coli LamB or MalE. 
The active immunogenic ingredients are often mixed with excipients or 
carriers which are pharmaceutically acceptable and compatible with the 
active ingredient. Suitable excipients include but are not limited to 
water, saline, dextrose, glycerol, ethanol, or the like and combinations 
thereof. The concentration of the immunogenic polypeptide in injectable 
formulations is usually in the range of 0.2 to 5 mg/ml. 
In addition, if desired, the vaccines may contain minor amounts of 
auxiliary substances such as wetting or emulsifying agents, pH buffering 
agents, and/or adjuvants which enhance the effectiveness of the vaccine. 
Examples of adjuvants which are effective include, but are not limited to, 
aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); 
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as 
nor-MDP); 
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn 
-glycero-3hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as 
MTP-PE); and RIBI, which contains three components extracted from 
bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall 
skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The 
effectiveness of an adjuvant may be determined by measuring the amount of 
antibodies directed against the immunogen resulting from administration of 
the immunogen in vaccines which are also comprised of the various 
adjuvants. Such additional formulations and modes of administration as are 
known in the art may also be used. 
RGP-1 and/or RGP-2 or HMW RGP and/or epitopic fragments or peptides of 
sequences derived therefrom or from other P. gingivalis proteins having 
primary structure similar (more than 90% identity) to HMW RGP or HMW KGP 
may be formulated into vaccines as neutral or salt forms. Pharmaceutically 
acceptable salts include, but are not limited to, the acid addition salts 
(formed with free amino groups of the peptide) which are formed with 
inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic 
acids, e.g., acetic, oxalic, tartaric, or maleic acid. Salts formed with 
the free carboxyl groups may also be derived from inorganic bases, e.g., 
sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic 
bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, 
histidine, and procaine. 
The immunogenic compositions or vaccines are administered in a manner 
compatible with the dosage formulation, and in such amount as 
prophylactically and/or therapeutically effective. The quantity to be 
administered, generally in the range of about 100 to 1,000 .mu.g of 
protein per dose, more generally in the range of about 5 to 500 .mu.g of 
protein per dose, depends on the subject to be treated, the capacity of 
the individual's immune system to synthesize antibodies, and the degree of 
protection desired. Precise amounts of the immunogen may depend on the 
judgment of the physician or dentist and may be peculiar to each 
individual, but such a determination is within the skill of such a 
practitioner. 
The vaccine or other immunogenic composition can be given in a single dose 
or multiple dose schedule. A multiple dose schedule is one in which a 
primary course of vaccination may include 1 to 10 or more separate doses, 
followed by other doses administered at subsequent time intervals as 
required to maintain and or reinforce the immune response, e.g., at 1 to 4 
months for a second dose, and if needed, a subsequent dose(s) after 
several months. 
When mice were immunized (see Example 8) and subsequently challenged with 
live P. gingivalis in the subcutaneous (SC) chamber model for growth and 
invasion of P. gingivalis, there was significant protection against 
infection where the experimental animals were immunized with heat-killed 
whole cells of P. gingivalis, RGP-2, HMW RGP, and peptides derived from 
the catalytic domain or N-terminus of a 50 kDa Arg-gingipain or an adhesin 
domain of HMW RGP, with infection being measured by recovery of viable P. 
gingivalis from the SC chambers (See Example 8, Table 4). 
All control (unimmunized) mice yielded viable bacteria during the course of 
infection. When mice were immunized with heat-killed P. gingivalis A7436 
whole cells, HMW RGP, RGP-2 or Peptide A (N-terminal sequence of catalytic 
subunit of HMW RGP, SEQ ID NO:10), no viable bacteria were recovered at 
day 7. Partial protection was afforded by Peptide B, the catalytic domain 
peptide (SEQ ID NO:11) and by Peptide C, the hemagglutinin domain of HMW 
RGP (SEQ ID NO:12). 
When protection was assessed by the survival or absence of lesions in the 
SC chamber model, Peptide B gave partial protection while the remaining 
treatments gave full protection (see Table 5 in Example 8). 
Humans (or other mammals) immunized with Arg-gingipains or Lys-gingipains 
and/or peptides having amino acid sequences derived from a low molecular 
weight Arg-gingipain or a HMW RGP, are protected from infection and 
invasion by P. gingivalis as assessed in this animal model. Preferably the 
hemagglutinin domain is not contained in the immunogenic composition. 
Female Balb/c mice were immunized with either RGP-1, RGP-2, or 
MAP-conjugated RGP-derived peptides by direct injection into stainless 
steel chambers implanted subcutaneously (Example 8), and subsequently 
challenged by injection of live P. gingivalis into chambers. Non-immunized 
animals or animals immunized with a scrambled peptide control and 
challenged with P. gingivalis developed ulcerated necrotic lesions on 
their abdomens, exhibited severe cachexia with ruffled hair, hunched 
bodies, and weight loss, with 14/22 and 5/8 deaths (Table 7). In contrast, 
animals immunized with MAP-conjugated Peptide A, corresponding to the 
N-terminus of the catalytic domain of RGPs (FIG. 4), followed by challenge 
with P. gingivalis were completely protected from abscess formation and 
death (Table 7). Similar results were obtained in animals that had been 
immunized with either whole P. gingivalis cells, RGP-1, or RGP-2. However, 
immunization with peptides corresponding to either a sequence encompassing 
the catalytic cysteine residue of RGPs (Peptide B) or an homologous 
sequence within the catalytic domains of RGPs and KGP (Peptide C), 
followed by challenge with P. gingivalis, did not protect animals, nor did 
a peptide corresponding to the binding site within the 
adhesin/hemagglutinin domain of RGP-1 (Peptide D) FIG. 4, Table 1, SEQ ID 
NO:14) which has been shown to be directly involved in the hemagglutinin 
activity of this gingipain [Curtiss et al. (1966) Infect. Immun. 64:2532]. 
Immunization with either peptide A, RGP-1, RGP-2, or P. gingivalis whole 
cells, followed by challenge with live bacteria resulted in a decrease in 
the number of mice from which this organism could be cultured (Table 8). 
In contrast, P. gingivalis was readily cultured from chamber fluid 
obtained from 20/22 non-immunized mice up to the time of death (Table 8) 
and from animals challenged after immunization with Peptides B, C, and D. 
In non-immunized animals P. gingivalis levels increased relative to the 
initial inoculum (10.sup.8 to 10.sup.12 CFU) throughout the course of the 
experiments (Table 3), while in animals immunized with Peptide A, RGP-1, 
RGP-2, or whole cells, P. gingivalis decreased in numbers (from 10.sup.8 
to &lt;10.sup.6). Taken together, these results indicate that immunization 
with a peptide corresponding to the N-terminal catalytic domain of RGPs 
can limit the ability of P. gingivalis to colonize and invade with the 
same efficiency as immunization with active proteinases or whole bacteria. 
Immunization with the N-terminal peptide of Arg-gingipain induced a 
moderate IgG response to RGP-1 and RGP-2 (Table 9). The absence of a 
response to whole cells may be due to the lack of exposure of this epitope 
on cell surfaces so that the N-terminus of the membrane-associated RGP-1 
catalytic domain is not available for antibody binding. The IgG response 
obtained following immunization with Peptide D, representing a portion of 
the adhesin/hemagglutinin domain of RGP-1, was comparable to that induced 
by the N-terminal peptide; however, protection against P. gingivalis 
challenge was not observed when this peptide was used as an immunogen 
(Tables 1 and 2). Immunization with RGP-1 induced a high IgG titer to all 
antigens examined except for RGP-2 (Table 9). The low titer to RGP-2 may 
be due to the absence of the highly immunogenic adhesin/hemagglutinin 
domain in this enzyme [Okamoto et al. (1996) J. Biochem. 120:398; 
Barkocy-Gallagher et al. (1996) J. Bacteriol. 178:2734]. Immunization with 
whole cells induced a good response to RGP-1 and KGP with essentially no 
binding to RGP-2. Postchallenge serum IgG titers were higher for all 
immunization groups when compared to the chamber fluid IgG titer 3 weeks 
postimmunization, reflecting the effect of challenge with P. gingivalis. 
Competitive ELISA assays, using either RGP-1 or KGP as competing soluble 
antigens, indicated that 42% and 53% of the antibodies induced by 
immunization with heat-killed bacteria recognize RGP-1 and KGP, 
respectively (FIG. 5). However, even at very high concentrations, RGP-2 
did not hinder IgG binding to P. gingivalis. These observations were also 
confirmed by Western blot analysis (FIG. 6D) and indicate that the 
non-catalytic hemagglutinin domains of RGP-1 and KGP are responsible for 
approximately 50% of the induced IgG response, and as such, constitute 
major antigens of P. gingivalis. Chamber fluid from mice immunized with 
the N-terminal peptide of the catalytic domain of RGPs reacted with the 50 
kDa RGP-1, the catalytic domain of HMW RGP, with HMW RGP, with RGPs 
present in vesicles and bacterial membrane fractions, and with RGP-2 (FIG. 
6A). A similar pattern was observed when chamber fluid from animals 
immunized with whole RGP-2 was utilized (FIG. 6E). The lack of reactivity 
with KGP is in agreement with antibody-specificity results (Table 2). 
Although the adhesin domain-derived peptide induced a poor IgG response as 
detected by ELISA, we found reactivity to several proteins by Western blot 
analysis (FIG. 6C). RGP-2 was not recognized by this antibody due to the 
lack of an adhesin domain. However, reactivity could be detected with the 
27 kDa domains of RGP-1 and KGP and proteins migrating in the range of 
60-70 kDa in vesicle and membrane preparations. Significantly, the adhesin 
domains present in the 44 kDa and 17 kDa subunits (FIG. 4) did not bind 
antibody. 
Immunization with RGP-1 resulted in antibodies with specificity 
predominantly directed against the 44 kDa adhesin/hemagglutinin domain of 
RGP-1 and the 43 kDa domain of KGP (FIG. 6B). These domains were also 
recognized in vesicle and membrane preparations. Additional protein bands 
recognized by this antiserum included the 32 and 17 kDa proteins in KGP, 
as well as the equivalents in vesicles and membranes. However, the RGP-1 
catalytic domain was only weakly recognized, and RGP-2 not at all. These 
results are in agreement with previous studies in which the catalytic 
domains of RGPs were poorly recognized in antisera obtained from rabbits 
or chickens immunized with the entire RGP-1 molecule. Immunization with 
heat-killed bacteria results in antibodies (FIG. 6D) with specificities 
astonishingly similar to those induced by immunization with RGP-1. In 
addition to polypeptides composing the RGP-1 complex, high molecular 
weight proteins were also detected in vesicles and membranes. No 
reactivity was detected (Western blot analysis) for the catalytic domain 
of RGP-1 or RGP-2, results in agreement with those obtained with mice 
immunized with RGP-1 (FIG. 6B) and consistent with data obtained by ELISA 
in which antibodies generated following immunization with heat-killed P. 
gingivalis exhibited a very low titer against RGP-2. 
This study indicates that in mice the major IgG response is targeted to the 
adhesin/hemagglutinin domain of RGP-1. This is consistent with analysis of 
sera from patients with severe, untreated periodontitis. Such a specific 
response to the adhesin/hemagglutinin domain of gingipains mounted in 
human periodontitis patients appears to divert the immune response away 
from other protective antigens. In the mouse model, antibodies with this 
specificity can limit colonization and invasion of P. gingivalis. However, 
in human subjects where the local inflammatory response leads to bone loss 
and destruction of the periodontal ligament, such antibodies can aggravate 
local tissue damage within the periodontal ligament. In this study, 
immunization of mice with a peptide corresponding to the N-terminus of 
RGPs generated a protective antibody response, but those antibodies did 
not recognize either RGP-1 or RGP-2 in cell preparations, indicating that 
this epitope (FIG. 4) is not exposed in whole cells. Rabbit antisera 
generated to the N-terminal portion of the catalytic domain of RGP-1 and 
RGP-2 also did not recognize RGP-1 in membranes or vesicle preparations 
unless samples were denatured by boiling, again suggesting that this 
epitope is not exposed in whole cells or vesicles. Inhibition of the 
maturation and/or catalytic activity of RGPs can inhibit invasion and 
colonization of P. gingivalis in mice and man. Such enzymes contribute to 
virulence in a multifactorial manner by influencing adherence to host 
tissues, activating cascade systems, degrading host proteins, and 
disturbing host defenses. RGPs can act as processing proteinases 
responsible for self maturation and the maturation of KGP, fimbrillin, and 
a 75 kDa major cell surface protein. These latter proteins are required 
for full virulence of P. gingivalis [Malek et al. (1994) J. Bacteriol. 
176:1052; Goulbourne and Ellen (1991) J. Bacteriol. 173:5266; Lamont et 
al. (1994) Oral Microbiol. Immunol. 8:272; Lamont et al. (1992) Oral 
Microbiol. Immunol. 7:1993; Hamada et al. (1994) Infect. Immun.62:1696; 
Tokuda et al. (1996) Infect. Immun. 64:4067]. 
Except as noted hereafter, standard techniques for peptide synthesis, 
cloning, DNA isolation, amplification and purification, for enzymatic 
reactions involving DNA ligase, DNA polymerase, restriction endonucleases 
and the like, and various separation techniques are those known and 
commonly employed by those skilled in the art. A number of standard 
techniques are described in Ausubel et al. (1994) Current Protocols in 
Molecular Biology, Green Publishing, Inc., Sambrook et al. (1989) 
Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, 
Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring 
Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, 
Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. 
Enzymol. 100 and 101; Grossman and Moldave (eds.) 1980 Meth. Enzymol. 65; 
Miller (ed.) (1972) Experiments in Molecular Genetics, Cold spring Harbor 
Laboratory, Cold Spring Harbor, N.Y., Old Primrose (1981) Principles of 
Gene Manipulation, University of California Press, Berkeley; Schleif and 
Wensink (1981) Practical Methods in Molecular Biology; Glover (ed.) (1985) 
DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) 
(1985) Nucleic Acid Hybridisation, IRL Press, Oxford, UK; Setlow and 
Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, 
Plenum Press, New York. Abbreviations and nomenclature, where employed, 
are deemed standard in the field and commonly used in professional 
journals such as those cited herein. All references cited in this 
application are incorporated by reference in their entirety. 
The foregoing discussion and the following examples illustrate but are not 
intended to limit the invention. The skilled artisan will understand that 
alternative methods can be used to implement the invention. 
EXAMPLE 1 
Purification of Arg-Gingipains and Lys-Gingipains 
Bacterial Cultivation 
P. gingivalis strains HG66 (W83) and W50 (virulent) were used in these 
studies. Cells were grown in 500 ml of broth containing 15.0 g Trypticase 
Soy Broth (Difco, Detroit, Mich.), 2.5 g yeast extract, 2.5 mg hemin, 0.25 
g cysteine, 0.05 g dithiothreitol, 0.5 mg menadione (all from Sigma 
Chemical Company, St. Louis, Mo.) anaerobically at 37.degree. C. for 48 hr 
in an atmosphere of 85% N.sub.2, 10% CO.sub.2, 5% H.sub.2. The entire 500 
ml culture was used to inoculate 20 liters of the same medium, and the 
latter was incubated in a fermentation tank at 37.degree. C. for 48 hr (to 
a final optical density of 1.8 at 650 nm). RGP-1 can also be purified as 
described for RGP-2. 
Proteinase Purification (RGP-1) 
1200 ml cell-free supernatant was obtained from the 48 hr culture by 
centrifugation at 18,000.times.g for 30 min. at 4.degree. C. Proteins in 
the supernatant were precipitated out by 90% saturation with ammonium 
sulfate. After 2 hr at 4.degree. C., the suspension was centrifuged at 
18,000.times.g for 30 min. The resulting pellet was dissolved in 0.05 M 
sodium acetate buffer, pH 4.5, 0.15 NaCl, 5 mM CaCl.sub.2 ; the solution 
was dialyzed against the same buffer overnight at 4.degree. C., with three 
changes with a buffer:protein solution larger than 150:1. The dialysate 
was then centrifuged at 25,000.times.g for 30 min and the dark brown 
supernatant (26 ml) was then chromatographed over an agarose gel 
filtration column (5.0.times.150 cm; Sephadex G-150, Pharmacia, 
Piscataway, N.J.) which had been pre-equilibrated with the same buffer. 
The column was developed with said buffer at a flow rate of 36 ml/hr. 6 ml 
fractions were collected and assayed for both amidolytic and proteolytic 
activities, using Bz-L-Arg-pNA and azocasein as substrates. Four peaks 
containing amidolytic activity were identified. The fractions 
corresponding to peak 4 were combined, concentrated by ultrafiltration 
(Amicon PM-10 membrane; Amicon, Beverly, Mass.) and then dialyzed 
overnight against 0.05 Bis-Tris, 5 mM CaCl.sub.2, pH 6.0. The volume of 
the dialysate was 14 ml. 
The 14 ml dialysate from the previous step was then applied to a 
DEAE-cellulose (Whatman, Maidstone, England) column (1.times.10 cm) 
equilibrated with 0.05 mM Bis-Tris, 5 mM CaCl.sub.2, pH 6.0. The column 
was then washed with an additional 100 ml of the same buffer. About 75% of 
the amidolytic activity, but only about 50% of the protein, passed through 
the column. The column wash fluid was dialyzed against 0.05 M sodium 
acetate buffer containing 5 mM CaCl.sub.2 (pH 4.5). This 19 ml dialysate 
was applied to a Mono S FPLC column (Pharmacia LKB Biotechnology Inc., 
Piscataway, N.J.) equilibrated with the same buffer. The column was washed 
with the starting buffer at a flow rate of 1.0 ml/min for 20 min. Bound 
proteins were eluted first with a linear NaCl gradient (0 to 0.1 M) 
followed by a second linear NaCl gradient (0.1 to 0.25 M), each gradient 
applied over a 25 min time period. Fractions were assayed for amidolytic 
activity using Bz-L-Arg-pNA. Fractions with activity were pooled and 
re-chromatographed using the same conditions. Although not detectable by 
gel electrophoresis, trace contamination by a proteinase capable of 
cleaving after lysyl residues was sometimes observed. This contaminating 
activity was readily removed by applying the sample to an arginine-agarose 
affinity column (L-Arginine-SEPHAROSE 4B) equilibrated with 0.025 M 
Tris-HCl, 5 mM CaCl.sub.2, 0.15 M NaCl, pH 7.5. After washing with the 
same buffer, purified enzyme was eluted with 0.05 M sodium acetate buffer, 
5 mM CaCl.sub.2, pH 4.5. Yields of gingipain-1 were markedly reduced by 
this step (about 60%). 
RGP-1 can also be purified as described for RGP-2 with such appropriate 
modifications as are readily apparent to one of ordinary skill in the art. 
Proteinase Purification (HMW RGP) 
The culture supernatant (2,900 ml) was obtained by centrifugation of the 
whole culture (6,000.times.g, 30 min, 4.degree. C.). Chilled acetone 
(4,350 ml) was added to this fraction over a period of 15 min, with the 
temperature of the solution maintained below 0.degree. C. at all times, 
using an ice/salt bath and this mixture was centrifuged (6,000.times.g, 30 
min, -15.degree. C.). The precipitate was dissolved in 290 ml of 20 mM 
Bis-Tris-HCl, 150 mM NaCl, 5 mM CaCl.sub.2, 0.02% (w/v) NaN.sub.3, pH 6.8 
(Buffer A), and dialyzed against Buffer A containing 1.5 mM 
4,4'-Dithiodipyridine disulfide for 4 h, followed by 2 changes of buffer A 
overnight. The dialyzed fraction was centrifuged (27,000.times.g, 30 min, 
4.degree. C.), following which it was concentrated to 40 ml by 
ultrafiltration using an Amicon PM-10 membrane. This concentrated fraction 
was applied to a Sephadex G-150 column (5.times.115 cm=2260 ml; Pharmacia, 
Piscataway, N.J.) which had previously been equilibrated with Buffer A, 
and the fractionation was carried out at 30 ml/h (1.5 cm/h). Fractions (9 
ml) were assayed for activity against Bz-L-Arg-pNa and Z-L-Lys-pNa 
(Novabiochem; 0.5 mM). Amidolytic activities for Bz-L-Arg-pNa (0.5 mM) or 
Z-L-Lys-pNa were measured in 0.2 M Tris.Hcl, 1 mM CaCl.sub.2, 0.02% (w/v) 
NaN.sub.3, 10 mM L-cysteine, pH 7.6. General proteolytic activity was 
measured with azocasein (2% w/v) as described by Barrett and Kirschke 
(1981) Meth. Enzymol. 80:535-561 for cathepsin L. Three peaks with 
activity against the two substrates were found. The first (highest 
molecular weight) peak of activity was pooled, concentrated to 60 ml using 
ultrafiltration and dialyzed overnight against two changes of 50 mM 
Tris-HCl, 1 mM CaCl.sub.2, 0.02% NaN.sub.3, pH 7.4 (Buffer B). 
This high MW fraction was applied to an L-Arginine-Sepharose column 
(1.5.times.30 cm=50 ml), which had previously been equilibrated with 
Buffer B at a flow rate of 20 ml/hr (11.3 cm/h), following which the 
column was washed with two column volumes of Buffer B. Following this, a 
step gradient of 500 mM NaCl was applied in Buffer B and the column was 
washed with this concentration of NaCl until the A.sub.280 baseline fell 
to zero. After re-equilibration of the column in Buffer B, a gradient from 
0-750 mM L-Lysine was applied in a total volume of 300 ml, followed by 100 
ml of 750 mM L-Lysine. The column was once again re-equilibrated with 
Buffer B and a further gradient to 100 mM L-arginine in 300 ml was applied 
in the same way. Fractions (6 ml) from the Arg wash were assayed for 
activity against the two substrates as described previously. The arginine 
gradient eluted a major peak for an enzyme degrading Bz-L-Arg-pNa. The 
active fractions were pooled and dialyzed against two changes of 20 mM 
Bis-Tris-HCl, 1 mM CaCl.sub.2, 0.02% (v/w) NaN.sub.3, pH 6.4 (Buffer C) 
and concentrated down to 10 ml using an Amicon PM-10 membrane. 
The concentrate with activity for cleaving Bz-L-Arg-pNa was applied to a 
Mono Q FPLC column (Pharmacia LKB Biotechnology Inc, Piscataway, N.J.) 
equilibrated in Buffer C, the column was washed with 5 column volumes of 
Buffer C at 1.0 ml/min, following which bound protein was eluted with a 3 
step gradient [0-200 mM NaCl (10 min), followed by 200-250 mM NaCl (15 
min) and 250-500 mM NaCl (5 min)]. The active fractions from Mono Q were 
pooled and used for further analyses. 
RGP-2 Purification 
Cells of P. gingivalis (H66) were grown in 200 ml of broth containing 6.0 g 
of Trypticase Soy broth (Difco), 2.0 g of yeast extract, 1 mg of hemin, 
200 mg of cysteine, 20 mg dithiothreitol and 0.5 mg of menadione (all from 
Sigma Chemical Co., St. Louis, Mo.) anaerobically, at 37.degree. C. for 48 
h in an atmosphere of 85% N2, 10% CO2, 5% H2. The culture was used to 
inoculate 5 liters of the same broth, and incubated anaerobically, at 
37.degree. C. for about 48-60 h until the late stationary phase of 
bacteria growth (final optical density&gt;2.0). 
For purification of RGP-2, the initial steps of purification were performed 
according to the method design for 94 kDa HMW RGP and high molecular 
weight lysine-specific gingipain (KGP) purification [Pike et al. (1994) J. 
Biol. Chem. 269:406-411]. Briefly, the cell-free culture fluid was 
obtained by centrifugation of the whole culture and chilled to -20.degree. 
C. Acetone was slowly added to the chilled culture supernatant, with the 
temperature being maintained below 0.degree. C. The precipitated protein 
was collected by centrifugation, and the pellet was dissolved in 20 mM 
Bis-Tris, 150 mM NaCl, 0.02% NaN3 buffer (pH 6.8) containing 1.5 mM 
4,4'-dithiodipyridine disulfide (in a total volume equal to 1/20 of 
original culture supernatant subjected precipitation) and dialyzed first 
against the above buffer (one change) followed by two changes of the 
Bis-Tris/NaCl buffer supplemented with 5 mM CaCl2 but lacking 
4,4'-dithiodipyridine disulfide. The dialyzed protein solution was 
clarified by high speed centrifugation (40,000.times.g, 2 h), concentrated 
by ultrafiltration using an Amicon PM-10 membrane (Amicon, Danvers, 
Mass.), and the clarified solution was then applied to a gel filtration 
column (Sephadex G-150, Pharmacia, Piscataway N.J.) equilibrated with 
Bis-Tris buffer. The column was developed at a flow rate of 30 ml/h, and 
three peaks with activity against Bz-L-Arg-pNA and Z-L-Lys-pNA were found. 
The highest molecular mass peak of activity against 
Bz-L-Arg-pNA/Z-L-Lys-pNA was used for the purification of 95 kDa HMW RGP 
exactly as described by Pike et al. (1994) supra, while the lowest 
molecular mass peak having the majority of the activity against 
Bz-L-Arg-pNA was pooled, concentrated by ultrafiltration, and extensively 
dialyzed against several changes of 50 mM Bis-Tris, 1 mM CaCl2, pH 6.5 and 
loaded at a flow rate 20 ml/h on anion exchange resin DE-52 Cellulose 
(Whatman) column (1.5.times.20 cm) equilibrated with Bis-Tris/CaCl2 
buffer. This column was washed until the A.sub.280nm base line fell to 
zero; then a gradient of 0-200 mM NaCl was applied in a total volume of 
250 ml. Fractions (4 ml each) were assayed for activity against 
Bz-L-Arg-pNA. Some of this activity was found in the void volume (Vo) of 
the column, but the major peak was eluted at 100 mM NaCl concentration. 
Fractions from both peaks of activity were pooled, concentrated and 
dialyzed extensively either versus 50 mM sodium acetate buffer, 5 mM 
CaCl2, pH 4.5 (Vo) or against 50 mM Tris, 1 mM CaCl2, pH 7.4 with 0.02% 
NaN3 (NaCl elute). 
From the Vo (run-through) of the DE-52 column, RGP-1 was purified by means 
of HPLC on a Mono S column, followed by affinity chromatography over 
arginine-Sepharose 4B as described previously [Chen et al. (1991) supra]. 
The major activity peak eluted from DE-52 cellulose column with NaCl was 
applied to the arginine-Sepharose column (1.5.times.30 cm, 50 ml) 
equilibrated with Tris/CaCl.sub.2 buffer pH 7.4 at the flow rate of 20 
ml/h, following which the column was washed with buffer until activity 
against Bz-L-Arg-pNA fell below 20 mOD/min/ml, then a gradient to 100 mM 
L-arginine was applied in a volume of 300 ml. Three distinct peaks of 
activity obtained in this step, nonadsorbed, retarded and eluted with 
L-arginine, were concentrated, dialyzed against 3 changes of 50 mM sodium 
acetate buffer, 1 mM CaCl.sub.2, pH 4.5 and applied to a Mono S FPLC 
column equilibrated with the same buffer at a flow rate of 1 ml/min. The 
column was washed with starting buffer and bound protein eluted using a 
linear NaCl gradient (0-0.15 M NaCl over 30 min time period). Fractions in 
peaks containing activity were combined, dialyzed against 20 mM Bis-Tris, 
150 mM NaCl, 5 mM CaCl.sub.2, pH 6.8 with NaN.sub.3 and used for further 
analysis. 
Purification of Lys-Gingipain 
P. gingivalis strain HG66 (W83) was obtained from Roland Arnold (Emory 
University, Atlanta, Ga.). Cells were grown in 500 ml of broth containing 
15.0 g Trypticase Soy Broth (Difco, Detroit, Mich.), 2.5 g yeast extract, 
2.5 mg hemin, 0.25 g cysteine, 0.05 g dithiothreitol, 0.5 mg menadione 
(all from Sigma Chemical Company, St. Louis, Mo.) anaerobically at 
37.degree. C. for 48 hr in an atmosphere of 85% N.sub.2, 10% CO.sub.2, 5% 
H.sub.2. The entire 500 ml culture was used to inoculate 20 liters of the 
same medium, and the latter was incubated in a fermentation tank at 
37.degree. C. for 48 hr (to a final optical density of 1.8 at 650 nm). 
The culture supernatant (2,900 ml) was obtained by centrifugation of the 
whole culture (6,000.times.g, 30 min, 4.degree. C.). Chilled acetone 
(4,350 ml) was added to this fraction over a period of 15 min, with the 
temperature of the solution maintained below 0.degree. C. at all times, 
using an ice/salt bath to precipitate proteins. This mixture was 
centrifuged (6,000.times.g, 30 min, -15.degree. C.). The precipitate was 
dissolved in 290 ml of 20 mM Bis-Tris-HCl, 150 mM NaCl, 5 mM CaCl.sub.2, 
0.02% (w/v) NaN.sub.3, pH 6.8 (Buffer A), and dialyzed against Buffer A 
containing 1.5 mM 4,4'-Dithiodipyridine disulfide for 4 h, followed by 2 
changes of Buffer A overnight. The dialyzed fraction was centrifuged 
(27,000.times.g, 30 min, 4.degree. C.), following which the supernatant 
was concentrated to 40 ml by ultrafiltration using an Amicon PM-10 
membrane. This concentrated fraction was applied to a Sephadex G-150 
column (5.times.115 cm=2260 ml; Pharmacia, Piscataway, N.J.) which had 
previously been equilibrated with Buffer A, and the fractionation was 
carried out at 30 ml/h (1.5 cm/h). Fractions (9 ml) were assayed for 
activity against Bz-L-Arg-pNa and Z-L-Lys-pNa (Novabiochem; 0.5 mM). 
Amidolytic activities for Bz-L-Arg-pNa (0.5 mM) or Z-L-Lys-pNa were 
measured in 0.2 M Tris-HCl, 1 mM CaCl.sub.2, 0.02% (w/v) NaN.sub.3, 10 mM 
L-cysteine, pH 7.6. Three peaks with activity against both pNA substrates 
were found. The highest molecular weight peak of activity contained most 
of the Z-L-Lys-pNA amidolytic activity. The fractions of the highest 
molecular weight peak of activity were pooled, concentrated to 60 ml using 
ultrafiltration and dialyzed overnight against two changes of 50 mM 
Tris-HCl, 1 mM CaCl.sub.2, 0.02% NaN.sub.3, pH 7.4 (Buffer B). 
This high MW fraction concentrate was applied to an L-Arginine-Sepharose 
column (1.5.times.30 cm=50 ml), which had previously been equilibrated 
with Buffer B at a flow rate of 20 ml/hr (11.3 cm/h), following which the 
column was washed with two column volumes of Buffer B. Following this, a 
step gradient of 500 mM NaCl was applied in Buffer B and the column was 
washed with this concentration of NaCl until the A.sub.280 baseline fell 
to zero. After re-equilibration of the column with Buffer B, a linear 
gradient from 0-750 mM L-Lysine in Buffer B was applied in a total volume 
of 300 ml, followed by 100 ml of Buffer B containing 750 mM L-Lysine. The 
column was once again re-equilibrated with Buffer B and a further gradient 
to 100 mM L-arginine in 300 ml was applied in the same way. Fractions (6 
ml) from the Lys wash and from the Arg wash were assayed for activity 
against the two pNA substrates as described previously. The lysine 
gradient eluted a major peak of activity against Z-L-Lys-pNa only and the 
arginine gradient did the same for an enzyme degrading Bz-L-Arg-pNa. The 
active (for Z-L-Lys-pNA) fractions were pooled and dialyzed against two 
changes of 20 mM Bis-Tris-HCl, 1 mM CaCl.sub.2, 0.02% (w/v) NaN.sub.3, pH 
6.4 (Buffer C) and the dialyzate was concentrated to 10 ml using Amicon 
PM-10 membranes. 
The dialyzate was applied to an anion exchange FPLC column (Mono Q FPLC 
column, Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) equilibrated 
in Buffer C, the column was washed with 5 column volumes of Buffer C at a 
flow rate of 1.0 ml/min, following which bound protein was eluted with a 3 
step gradient [0-200 mM NaCl (10 min), followed by 200-275 mM NaCl (15 
min) and 275-500 mM NaCl (5 min), each in Buffer C. The active fractions 
from Mono Q chromatography were pooled. 
EXAMPLE 2 
Molecular Weight Determination 
The molecular weights of the purified Arg-gingipains and Lys-gingipains 
were estimated by gel filtration on a Superose 12 column (Pharmacia, 
Piscataway, N.J.) and by Tricine-SDS polyacrylamide gel electrophoresis. 
In the latter case, 1 mM TLCK was used to inactivate the protease prior to 
boiling, thus preventing autoproteolytic digestion. 
EXAMPLE 3 
Enzyme Assays 
Amidolytic activities of P. gingivalis proteinases were measured with the 
substrates MeO-Suc-Ala-Ala-Pro-Val-pNA at a concentration of 0.5 mM, 
Suc-Ala-Ala-Ala-pNA (0.5 mM), Suc-Ala-Ala-Pro-Phe-pNA (0.5 mM), Bz-Arg-pNA 
(1.0 mM), Cbz-Phe-Leu-Glu-pNA) (0.2 mM); S-2238, S-2222, S-2288 and S-2251 
each at a concentration of 0.05 mM; in 1.0 ml of 0.2 M Tris-HCl, 5 mM 
CaCl.sub.2, pH 7.5. In some cases either 5 mM cysteine and/or 50 mM 
glycyl-glycine (Gly-Gly) was also added to the reaction mixture. 
Z-L-Lys-pNa (0.5 mM) in 0.2 M Tris-HCl, 0.02% (w/v) NaN.sub.3, 10 mM 
L-cysteine, was used for assay oaf Lys-gingipain. 
General proteolytic activity was assayed using the same buffer system as 
described for detecting amidolytic activity, but using azocoll or 
azocasein (2% w/v) as substrate as described for Cathepsin L by Barrett 
and Kirschke (1981), Meth. Enzymol. 80, 535-561. 
For routine assays, pH optimum determination and measurement of the effect 
of stimulating agents and inhibitors on Arg-gingipains, only Bz-L-Arg-pNA 
was used as substrate. Potential inhibitory or stimulatory compounds were 
preincubated with enzyme for up to 20 min at room temperature at pH 7.5, 
in the presence of 5 mM CaCl.sub.2 (except when testing the effects of 
chelating agents) prior to the assay for enzyme activity. 
General proteolytic activity was assayed using the same buffer system as 
described for detecting amidolytic activity, but using azocoll or 
azocasein (1% w/v) as substrate. 
A unit of RGP enzymatic activity is based on the spectroscopic assay using 
benzoyl-Arg-p-nitroanilide as substrate and recording .DELTA. absorbance 
units at 405 nm/min/absorbance unit at 280 nm according to the method of 
Chen et al. (1992) supra. 
EXAMPLE 4 
Amino Acid Sequence Analysis 
Amino-terminal amino acid sequence analyses were carried out using an 
Applied Biosystems 4760A gas-phase sequenator, using the program designed 
by the manufacturer. Alternatively, amino acid sequences were deduced from 
the coding sequences of the corresponding coding sequences (see SEQ ID 
NO:1 and SEQ ID NO:3). The amino acid sequences of the COOH terminus of 
SDS-denatured RGP-1 and of the 50 kDa subunit of HMW RGP were determined. 
10 nmol aliquots of gingipain-1 were digested in 0.2 M N-ethylmorpholine 
acetate buffer, pH 8.0, with carboxypeptidase A and B at room temperature, 
using 1:100 and 1:50 molar ratios, respectively. Samples were removed at 
intervals spanning 0 to 12 hours, boiled to inactivate the 
carboxypeptidase, and protein was precipitated with 20% trichloracetic 
acid. Amino acid analyses were performed on the supernatants. 
EXAMPLE 5 
Materials 
MeO-Suc-Ala-Ala-Pro-Val-pNA, Suc-Ala-Ala-Pro-Phe-pNA, Gly-Pro-pNA, 
Suc-Ala-Ala-Ala-pNA, Bz-Arg-pNA, diisopropylfluorophosphate, 
phenylmethylsulfonyl fluoride, tosyl-L-lysine chloromethyl ketone (TLCK), 
tosyl-L-phenylalanine chloromethyl ketone (TPCK), 
trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane), an inhibitor of 
cysteine proteinases, leupeptin, antipain and azocasein were obtained from 
Sigma Chemical Co., St. Louis, Mo. 3,4-Dichloroisocoumarin was obtained 
from Boehringer, Indianapolis, Ind. and CBz-Phe-Leu-Glu-pNA and azocoll 
were obtained from Calbiochem, La Jolla, Calif. S-2238 
(D-Phe-Pip-Arg-pNA), S-2222 (Bz-Ile-Glu-(.gamma.-OR)-Gly-Arg-pNA), S-2288 
(D-Ile-Pro-Arg-pNA), and S-2251 (D-Val-Leu-Lys-pNA) were from Kabi-Vitrum, 
(Beaumont, Tex.). 
EXAMPLE 6 
Electrophoresis 
SDS-PAGE was performed as in Laemmli (1970) Nature 227:680-685. Prior to 
electrophoresis the samples were boiled in a buffer containing 20% 
glycerol, 4% SDS, and 0.1% bromophenol blue. The samples were run under 
reducing conditions by adding 2% .beta.-mercaptoethanol unless otherwise 
noted. Samples were heated for 5 min at 100.degree. C. prior to loading 
onto gels. A 5-15% gradient gel was used for the initial digests of C3 and 
C5, and the gels were subsequently stained with Coomassie Brilliant Blue 
R. The C5 digest used to visualize breakdown products before and after 
reduction of the disulfide bonds were electrophoresed in a 8% gel. 
Attempts to visualize C5a in the C5 digest were carried out using 13% gels 
that were developed with silver stain according to the method of Merril et 
al. (1979) Proc. Natl. Acad. Sci USA 76:4335-4339. In some experiments 
(with HMW RGP) SDS-PAGE using Tris-HCl/Tricine buffer was carried out per 
Shagger and Van Jagow (1987) Analyt. Biochem. 166:368-379. 
EXAMPLE 7 
Coding Sequences for Arg-gingipains and Lys-gingipains 
.lambda.DASH DNA libraries were constructed according to the protocols of 
Stratagene, using the lambda DASH.TM. II/BamHI cloning kit and DNA 
preparations from P. gingivalis strains HG66 (W83) and W50. A library of 
3.times.10.sup.5 independent recombinant clones was obtained using P. 
gingivalis H66 DNA, and 1.5.times.10.sup.5 independent recombinant clones 
were obtained from virulent P. gingivalis W50 DNA. The coding and amino 
acid sequences of the polyprotein precursor of the HMW RGP is given in SEQ 
ID NO:5. SEQ ID NO:7 provides the Lys-gingipain coding sequence and SEQ ID 
NO:8 the amino acid sequence. 
EXAMPLE 8 
Animal Model Studies 
A mouse animal model [described in Genco et al. (1991) Infect. Immun. 
59:1255-1263] was used to study the protective effects of immunogenic 
compositions comprising P. gingivalis proteinases and/or peptides derived 
therefrom. 
Peptides for use as immunogens were synthesized using an Applied Biosystems 
automated solid state process and the multi-lysine base according to the 
method of Tam, J. P. (1988) Proc. Natl. Acad. Sci. USA 85:5409-5413 and 
Posnett et al. (1988) J. Biol. Chem. 263:1719-1725. After purification, 
the peptides were suspended as described below. The multiple lysine base 
provides a framework for the simultaneous synthesis of multiple identical 
peptides and results in an "octopus"-like molecule which is antigenic 
without the need for conjugation to a carrier peptide. The multiple lysine 
base is not itself antigenic. Thus, this technique offers some advantages 
over the previous peptide immunizations which required conjugation to 
carrier proteins such as keyhole limpet hemocyanin and bovine serum 
albumin. RGP-related peptide sequences used in these experiments are 
provided below. 
Whole cell antigens for immunization were prepared by centrifugation of P. 
gingivalis cultures for 10 min at 10,000.times.g at room temperature and 
resuspension in 1/10 the original volume of anaerobic broth. Bacterial 
cells were heated to 95.degree. C. for 10 min, and heat-treated 
preparations were plated on anaerobic blood agar and incubated for 7 days 
under anaerobic conditions to confirm effective killing. RGPs were 
purified from strain HG66 as described hereinabove. 
Mice were immunized by injection of each immunogen (50 .mu.g/mouse in 
Freund's complete adjuvant) in subcutaneous chambers implanted in mice 
[Genco et al. (1992) Infect. Immun. 60:1447]. Animals immunized with 
heat-killed P. gingivalis received an initial immunization corresponding 
to 10.sup.8 CFU. Control mice were immunized with Freund's adjuvant only. 
Female BALB/c mice about 8 weeks old are obtained from Sasco (Omaha, Nebr.) 
or Charles River Laboratory (Wilmington, Mass.). Coil-shaped subcutaneous 
(SC) chambers were prepared from 0.5 mm stainless steel wire and 
surgically implanted in the SC tissue of the dorsolumbar region of each 
mouse, with anaesthesia. A recovery period of at least 10 days is allowed 
before further treatment. During the 10 day period, the outer incision 
heals completely and the chambers become encapsulated by a thin 
vascularized layer of fibrous connective tissue and gradually filled with 
approximately 0.5 ml of light-colored transudate. 
After the 10 day recovery period, the mice are immunized according to the 
scheme in Table 1: 
TABLE 1 
______________________________________ 
Group Immunogen Number of Mice 
______________________________________ 
A None 6 
B 50 kDa RGP-2 
6 
C 8 
D 8 
E 8 
F 95 kDa HMW RGP 
8 
G Heat-killed 
8 
P. gingivalis 
A7436 whole cells 
______________________________________ 
Stock solutions of immunogens were as follows: RGP-2, 1.65 mg/ml in 20 mM 
Bis-Tris, 150 mM NaCl, 5 mM CaCl.sub.2, 0.02% NaN.sub.3, pH 6.8 and 
diluted to 1 mg/ml for use in immunizations; Peptide B (SEQ ID NO:11, 
QLPFIFDVACVNGDFLFSMPCFAEALMRAQ, catalytic domain of HMW RGP), 1 mg/ml in 
cold NH.sub.4 HCO.sub.3 made fresh; Peptide C (SEQ ID NO:12, 
GEPNPYQPVSNLTATTQGQKVTLKWDAPSTK, hemagglutinin domain of HMW RGP) 1 mg/ml 
in 10 mM acetic acid; Peptide A (SEQ ID NO:10, YTPVEEKQNGRMIVIVAKKY, 
N-terminus of the HMW RGP catalytic subunit, 1 mg/ml in 10 mM acetic acid; 
RGP-2, 0.96 mg/ml in 20 mM Bis-Tris, 150 mM NaCl, 5 mM CaCl.sub.2, 0.02% 
NaN.sub.3, pH 6.8; and heat-killed whole P. gingivalis A7436 bacterial 
cells, 10.sup.9 /ml. Group A mice (unimmunized controls) were inoculated 
with only Freund's complete adjuvant. Groups B-F were immunized with 50 
.mu.g of MAP-peptides or protein in Freund's complete adjuvant per mouse 
in the primary immunizations injected into the chambers or SC. Groups B-F 
mice were given booster immunizations of 50 .mu.g MAP-peptide twice a week 
for 5 weeks in Preund's incomplete adjuvant. Group G mice were immunized 
by injecting the heat-killed whole bacterial cells into the chambers 
(without adjuvant). 10.sup.8 cells were injected into the chambers 
directly in the primary immunization; 10.sup.2 cells were injected in all 
booster immunizations. 
Mice are challenged with live P. gingivalis A7436 (2.times.10.sup.10 colony 
forming units) five weeks after the initial immunization. The mice are 
observed daily for general appearance, primary and/or secondary abscess 
formation and health status. Chamber fluid is removed daily with a 
hypodermic needle and syringe for bacteriologic culture and microscopic 
examination. Fluid is also examined for the presence and activity of 
antibodies to the respective peptides. All surviving animals are 
sacrificed 30 days after inoculation, and the sera are separated from 
blood obtained by cardiac puncture. 
During the 10 day period the outer incision heals completely and the 
chambers become encapsulated by a thin vascularized layer of fibrous 
connective tissue and gradually filled with approximately 0.5 ml of 
light-colored transudate. Ten days after implantation, chambers are 
inoculated with 0.1 ml of a suspension of P. gingivalis cells in 
prereduced Anaerobic Broth MIC (Difco Laboratories, Detroit, Mich.). 
Control SC chambers were injected with Schaedler broth lacking bacterial 
cells. Mice were examined daily for size and consistency of primary or 
secondary lesions and for general appearance, primary and/or secondary 
abscess formation and health status. Severe cachexia is characterized by 
ruffled hair, hunched bodies and weight loss. Chamber fluid is aseptically 
removed from each implanted chamber with a 25 gauge hypodermic needle and 
syringe at 1 to 7, and 14 days after inoculation for bacteriological 
culture and microscopic examination. All surviving animals are sacrificed 
at 30 days postinoculation and serum is separated from blood obtained by 
cardiac puncture. 
Aliquots of chamber fluid are streaked after live bacterial challenge for 
isolated microbial colonies on anaerobic blood agar plates (Remel, Lenexa, 
Kans.) and incubated for 7 days at 37.degree. C. under anaerobic 
conditions. P. gingivalis is then identified by standard techniques as 
described in Holdeman et al. (1984) "Anaerobic gram-negative straight, 
curved and helical rods. Family 1. Bacteroidaceae, Pribram," In N. R. 
Krieg and J. G. Holt (ed.) Bergey's Manual of Determinative Bacteriology, 
The Williams & Wilkins Co., Baltimore, Md., p. 602-631. Cultivable 
bacterial counts are obtained by serially diluting chamber fluid in 
Schaedler broth and spin plating onto anaerobic blood agar plates. 
Table 2 provides the results for recovery of P. gingivalis from the SC 
chambers at various times after challenges. 
TABLE 2 
______________________________________ 
P. gingivalis cultured from chamber fluid 
% of mouse SC chambers from which P. gingivalis was 
cultured on given day postinoculation and CFU 
obtained from chambers 
Group 
1 2 4 7 
______________________________________ 
A 83% 66% 83% 100% 
(1.8 .times. 10.sup.12) 
(1.6 .times. 10.sup.12) 
(1.1 .times. 10.sup.12) 
(7.2 .times. 10.sup.12) 
B 33% 
16% 
0%16% 
(7.6 .times. 10.sup.11) 
(4.7 .times. 10.sup.11) 
(1.5 .times. 10.sup.10) 
C 38% 
38% 
29% 
(1.4 .times. 10.sup.12)) 
(1.1 .times. 10.sup.10) 
(1.9 .times. 10.sup.11) 
D 63% 
75% 
63% 
(7.3 .times. 10.sup.11) 
(1.7 .times. 10.sup.11) 
(6.8 .times. 10.sup.10) 
(2.2 .times. 10.sup.11) 
E 38% 
50% 
0 
(1.4 .times. 10.sup.10) 
(4.7 .times. 10.sup.9) 
(4.0 .times. 10.sup.8) 
(ND) 
F 38% 
25% 
0 
(ND) 
(ND) 
(ND) 
G 13% 
0 
(ND) 
(ND) 
(ND) 
______________________________________ 
* ND means not detectable 
Table 3 summarizes the results of the analysis of the pathological course 
of the P. gingivalis challenge in control and immunized animals. 
TABLE 3 
______________________________________ 
Pathological course of P. gingivalis infection. 
% abdominal lesion 
% death 
______________________________________ 
A 50% 50% 
B 0 
C 13% 13% 
D 0 
E 0 
F 0 
G 0 
______________________________________ 
Specific immunoglobulin G (IgG) to P. gingivalis whole cells is quantitated 
from both chamber fluids and sera for each group of mice. IgG specific for 
P. gingivalis whole cells is assayed by a modification of an enzyme-linked 
immunosorbent assay (ELISA) described by Ebersole et al. (1989) J. Dent. 
Res. 68:286, abstract 837. The results are read with a V.sub.max kinetic 
photometer (Molecular Devices Corp., Menlo Park, Calif.) at 450 nm. An 
aliquot of serum from each group of mice (inoculated with different 
strains of P. gingivalis) is pooled and used as a positive standard and 
run on each plate. 
Further protection experiments are performed to test the following 
peptides: RGP Catalytic domain Peptide B, QLPFIFDVACVNGDFLFSMPCFAEALMRAQ, 
SEQ ID NO:11, MAP form; Scrambled catalytic domain, in both MAP and acid 
forms, DQANFLQCVGSLMCRLDFFFEAVMPIFPAA, SEQ ID NO:13; N-terminal sequence 
of catalytic subunit of HMW RGP, Peptide A, MAP form, YTPVEEKQNGRMIVIAKKY, 
MAP form, SEQ ID NO:10; Adhesin domain peptide (Peptide D) from 
adhesin/hemagglutinin domain of HMW RGP, in MAP and acid forms, 
GNHEYCVEVKYTAGVSPKVCKDVTV, SEQ ID NO:14; "Scrambled" adhesin domain 
peptide from HMW RGP, in MAP and acid forms, AHEKTYPVEDVNCSYVKTVCVGGKV, 
SEQ ID NO:15. 
Peptides equivalent in amino acid sequence to portions of Arg-gingipains, 
including adhesin/hemagglutinin domains and/or catalytic proteins, have 
protective effects when used to immunize mice in the animal model 
described herein. "Scrambled" peptides do not confer protective immunity 
to subsequent challenge by live, infectious P. gingivalis. 
Additional peptides within the scope of the present invention include 
RMFMNYEPGRYTPVEEKQNG (SEQ ID NO:16) which overlaps the activation site, 
TFAGFEDTYKRMFMNYEPGR (SEQ ID NO:17) which is located some twenty amino 
acids upstream of the activation site, 
DYTYTVYRDGTKIKEGLTATTFEEDGVATGNMEYCVCVKYTAGVSPKVC (SEQ ID NO:18), 
YTYTVYRDGTKIKEGLTATTFEEDG (SEQ ID NO:19), RDGTKIKEGLTATTFEEDGVATGN (SEQ ID 
NO:20) and KIKEGLTATTFEEDGVATGNHEY (SEQ ID NO:21), all of which contain 
the FEED (SEQ ID NO:22) sequence which participates in fibronectin 
binding. Peptide KWDAPNGTPNPNPNPNPNPNPGTTTLSE (SEQ ID NO:23) also can 
result in protective immunity after vaccination of a human or animal. 
A second immunization/challenge was carried out using Balb/C mice in the 
subcutaneous chamber model described above. Groups of eight mice per group 
were immunized by injection into the implanted subcutaneous chambers as 
set forth in Table 4: 
TABLE 4 
______________________________________ 
Group 
Immunogen Number of Mice 
______________________________________ 
A None 8 
B 50 kDa RGP-2 
8 
E Peptide D 
8 
F "Scrambled" Peptide D 
8 
G Peptide A 
8 
H Peptide A 
8 
I 95 kDa RGP-1 
8 
J heat-killed 
8 
P. gingivalis 
A7436 whole cells 
______________________________________ 
Group A mice (negative controls) were injected with Freund's complete 
adjuvant only. Mice in groups E-H were each first injected with 50 .mu.g 
MAP-peptide in Freund's complete adjuvant; eight boosts each contained 50 
.mu.g MAP-peptide in Freund's incomplete adjuvant. For groups E and F, 
boosts #3 and #6 were with free peptide. Groups B and F were treated as in 
the first experiment with eight boosts. Group J mice received heat-killed 
P. gingivalis A7436 cells without adjuvant (10.sup.8 cells in primary 
injection, 10.sup.2 cells per boost) 
Each mouse was challenged by injection of 3.9.times.10.sup.10 P. gingivalis 
A7436 into the subcutaneous chambers on the 32nd day after primary 
immunization. 
Table 5 presents the results for recovery of viable P. gingivalis cells 
from the subcutaneous chambers at days 1, 2, 3, 5 and 7 after challenge. 
TABLE 5 
______________________________________ 
Recovery of P. gingivalis from chambers 
following challenge 
% of mice from which P. gingivalis was cultured 
and (CFU) Day Following Challenge 
2 3 5 7 
______________________________________ 
100%A 100% 88% 88% 88% 
(1.6 .times. 10.sup.12).sup.12) 
(1.1 .times. 
(6 .times. 10.sup.12) 
(2.6 .times. 
10.sup.12) 10.sup.12) 
B 88% 75% 63% 75% 75% 
(1.0 .times. 10.sup.12) 
(2.1 .times. 10.sup.10) 
(2.8 .times. 
(2 .times. 10.sup.10) 
(2 .times. 
10.sup.10) 10.sup.10) 
C 75% 
50% 
(1.6 .times. 10.sup.12) 
(1.2 .times. 10.sup.10) 
(6 .times. 
(1.2 .times.0 10.sup.9) 
(1.6 .times. 
10.sup.9) 10.sup.8) 
D 75% 
75% 
(NF*)1 .times. 10.sup.10) 
(NF) 
(NF) 
E 75% 
63% 
(2.4 .times. 10.sup.10) 
(1 .times. 10.sup.10) 
(4.5 .times. 
(2 .times. 10.sup.8) 
(NF) 
10.sup.8) 
F 63% 
50% 
(NF)F) 
(NF) 
(NF) 
G 75% 
63% 
(1.5 .times. 10.sup.10)sup.11) 
(8 .times. 10.sup.9) 
(5 .times. 10.sup.8) 
(5 .times. 
10.sup.8) 
H 75% 
63% 
(NF) (1.4 .times. 10.sup.10) 
(NF) 
(NF) 
I 88% 
38% 
(NF)(6 .times. 10.sup.12) 
(NF) 
(NF) 
J 100% 
88% 
88% 
88% 
(1.4 .times. 10.sup.12) 
(1.7 .times. 10.sup.12) 
(NF) 
(NF) 
(NF) 
______________________________________ 
Table 6 summarizes the observations for pathological effects at 7 days 
after challenge. 
TABLE 6 
______________________________________ 
Pathology observed following 
challenge with P. gingivalis 
Group % Lesions % Deaths Cachexia 
______________________________________ 
A 38% 38% +++++ 
B + 
C ++ 
D ++++ 
E ++% 
F ++++ 
G + 
H + 
I -0 
J ++ 
______________________________________ 
Cachexia scored on a scale from +++++ to -, with +++++ as severe 
and - as no cachexia. 
In further animal experiments, seven days post primary immunization mice 
were boosted (10.times.) at 3 day intervals with RGP-1, RGP-2, or 
MAP-conjugated peptides (50 .mu.g/mouse in Freund's incomplete adjuvant). 
Animals immunized with heat-killed P. gingivalis were boosted (10.times.) 
at 3 day intervals with heat-killed P. gingivalis corresponding to 
10.sup.2 CFU. At 14, 21, and 28 days postimmunization, chamber fluid was 
removed with a hypodermic needle and syringe, and IgG specific for RGP-1, 
RGP-2, KGP, and whole cells quantitated by an immunosorbent assay 
[Ebersole et al. (1984) J. Clin. Microbiol. 19:639]. Mice were challenged 
by inoculation of 10.sup.9 CFU of P. gingivalis A7436 directly into 
chambers 49 days postimmunization and examined daily for size and 
consistency of lesions and health status. Severe cachexia was defined as 
ruffled hair, hunched bodies, and weight loss. Chamber fluid was removed 
from each implanted chamber at 1 to 7 days postchallenge for 
bacteriological culturing and immunological analysis. All surviving 
animals were sacrificed 30 days postchallenge, and sera were separated 
from blood obtained by cardiac puncture. 
TABLE 7 
______________________________________ 
Recovery of P. gingivalis from chamber fluid 
following challenge 
Number of mice from which P. gingivalis 
was cultured and/total number of mice 
sampled on the 
following day 
Total 
postinoculation.sup.a 
Group Mice 1 2 5 7 
______________________________________ 
22- 
21/22 20/22 20/22 D.sup.b 
immun- 
(1.4 .times. 10.sup.12).sup.c 
(1.1 .times. 10.sup.12) 
(2.4 .times. 10.sup.12) 
ized 
Pep- 32 23/32 
21/21 
19/32 
19/32 
tide A (1.9 .times. 10.sup.10)up.11) 
(9.8 .times. 10.sup.8) 
(&lt; 10.sup.6) 
Scram- 
8 8/8 
8/8 
7/8 
7/8 
bled (6.7 .times. 10.sup.10) 
(4.8 .times. 10.sup.10) 
(2.0 .times. 10.sup.10) 
5.6 .times. 10.sup.8) 
peptide 
Whole 24 
17/24 
11/27 
9/24 
6/24 
cells (7.4 .times. 10.sup.11) 
(8.8 .times. 10.sup.11) 
(4.6 .times. 10.sup.8) 
(&lt;10.sup.6) 
RGP-1 24 
12/24 
9/24 
4/24 
3/24 
(8 .times. 10.sup.9). 10.sup.12) 
(&lt;10.sup.6) 
(&lt;10.sup.6) 
RGP-2 22 
15/22 
9/22 
7/22 
6/22 
(1.8 .times. 10.sup.11).sup.11) 
(1.2 .times. 10.sup.10) 
(&lt;10.sup.6) 
______________________________________ 
.sup.a Aliquots of fluid from each chamber were streaked for 
isolation onto anaerobic blood agar plates and cultured at 37.degree. C. 
for 7 days under anerobic conditions. 
.sup.b All animals in this group had died by day 7. 
.sup.c Colony forming units obtained from chamber fluid. 
TABLE 8 
______________________________________ 
Pathological course of P. gingivalis infection in 
immunized mice 
Group Total Mice Lesions.sup.a 
Deaths Cachexia.sup.c 
______________________________________ 
Non-immunized 
22 14/22 14/22 +++++ 
Peptide A 1/32 
0/32 
+ 
Scrambled peptide 
8 5/8 
5/8 
++++ 
Whole cells 
0/24 
0/24 
+ 
RGP-1 0/22 
0/22 
++ 
RGP-2 0/24 
0/22 
+ 
______________________________________ 
.sup.a Number of mice with secondary lesion on the ventral 
abdomen/total of mice tested as detected on day 7. 
.sup.b Number of dead mice/total number of mice tested by day 7. 
.sup.c Cachexia scored on a scale from +++++ to -, with +++++ as severe 
cachexia and "-" as no cachexia. 
Additional animal experiments are carried out in a mouse periodontitis 
model. Oral infection is with P. gingivalis cells in 
carboxymethylcellulose by gavage. Where there is infection and resulting 
periodontal disease, there is measurable bone loss by the end of 6 weeks, 
P. gingivalis can be cultured from infected sites, and damage within the 
periodontal ligament can be assessed. 
TABLE 9 
__________________________________________________________________________ 
Enzyme linked immonosorbent assay (ELISA) analysis of chamber fluid and 
serum 
from mice immunized with gingipains Rs, peptide fragment of gingipains, 
and whole bacteria 
Antibodies titer.sup.a) against 
RGP-1 RGP-2 KGP whole P. gingivalis 
chamberA itgen used 
chamber 
chamber 
chamber 
for immunization 
serum 
fluid 
serum 
fluid 
serum 
fluld 
serum 
__________________________________________________________________________ 
RGP-1 200,000 .+-. 
724,000 .+-. 
6,600 .+-. 
55,000 .+-. 
105,000 .+-. 
676,000 .+-. 
13,000 .+-. 
282,000 .+-. 
38,200 28,000 
1,440 
3,600 
13,500 
41,250 
1,100 
27,000 
RGP-2 426,000 .+-. 3,600 .+-. 
2,800 .+-. 
100,000 .+-. 
--.sup.b) 
100,000 .+-. 
400 .+-. 
126,000 .+-. 
32,500 510 
415 15,200 
28 16,000 
20,800 
The N-terminal 
100,000 .+-. 
145 .+-. 
3,600 .+-. 
-- 
--,000 .+-. 
190,000 .+-. 
peptide of RGPs 
8,800 
12,100 
21,300 
Scrambled 
120,000 .+-..r..sup.c) 
n.r. 
8,700 .+-. 
n.r. 
93,000 .+-. 
n.r. 
195,000 .+-. 
N-terminal peptide 
19,400 
722 
10,000 
20,300 
Adhesive domain 
120,000 .+-. 
n.r. 
7,600 .+-. 
290 .+-. 
50 .+-. 
100,000 .+-. 
peptide 21,000 21 
17 
4 
18,000 
Scrambled adhesive 
145,000 .+-. 
n.r. 
4,100 .+-. 
n.r. 
109,000 .+-. 
n.r. 
155,000 .+-. 
domain peptide 
23,600 
650 
0,500 
20,300 
Heat killed 
331,000 .+-.0 .+-. 
760 .+-. 
49,000 .+-. 
20,000 .+-. 
234,000 .+-. 
12,000 .+-. 
178,000 .+-. 
P. gingivolis 
29,400 2,500 
48 7,800 
2,600 
24,000 
980 
21,000 
__________________________________________________________________________ 
Microplates were coated with purified gingipains (1 .mu. g/ml) or whole 
P. gingivalis cells (13), nonspecific binding sites blocked with bovine 
serum albumin, then incubated with serial dilutions of chamber fluid or 
serum. Quantity of antibodies bound to immobilized antigen was determine 
with peroxidaselabeled goat antimouse IgG. 
.sup.a) Expressed as a dilution factor of chamber fluid or serum at which 
there was 50% of maximal O.D..sub.540 reading calculated from sigmoidal 
curve obtained in ELISA assay. 
.sup.b) Detectable IgG binding but too low to be quantitated 
.sup.c) No IgG binding at the lowest (5 fold) chamber fluid or serum 
dilution. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 24 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 34 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not R - #elevant 
(D) TOPOLOGY: unknown 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (v) FRAGMENT TYPE: N-terminal 
- (ix) FEATURE: 
(A) NAME/KEY: Region 
(B) LOCATION: 38..43 
#/product= "Xaa"HER INFORMATION: 
#Xaa /label= 
#couldis used to denote an amino acid which 
#identified with certainty." 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Ar - #g Met Ile Val Ile Val 
# 15 
- Ala Lys Lys Tyr Glu Gly Asp Ile Lys Asp Ph - #e Val Asp Trp Lys Asn 
# 30 
- Gln Arg 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 4 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not R - #elevant 
(D) TOPOLOGY: unknown 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (v) FRAGMENT TYPE: C-terminal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Glu Leu Leu Arg 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 3159 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Porphryomona - #s gingivalis 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 949..3159 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- CTGCAGAGGG CTGGTAAAGA CCGCCTCGGG ATCGAGGCCT TTGAGACGGG CA - #CAAGCCGC 
60 
- CGCAGCCTCC TCTTCGAAGG TGTCTCGAAC GTCCACATCG GTGAATCCGT AG - #CAGTGCTC 
120 
- ATTGCCATTG AGCAGCACCG AGGTGTGGCG CATCAGATAT ATTTTCATCA GT - #GGATTATT 
180 
- AGGGTATCGG TCAGAAAAAG CCTTCCGAAT CCGACAAAGA TAGTAGAAAG AG - #AGTGCATC 
240 
- TGAAAACAGA TCATTCGAGG ATTATCGATC AACTGAAAAG GCAGGAGTTG TT - #TTGCGTTT 
300 
- TGGTTCGGAA AATTACCTGA TCAGCATTCG TAAAAACGTG GCGCGAGAAT TT - #TTTCGTTT 
360 
- TGGCGCGAGA ATTAAAAATT TTTGGAACCA CAGCGAAAAA AATCTCGCGC CG - #TTTTCTCA 
420 
- GGATTTACAG ACCACAATCC GAGCATTTTC GGTTCGTAAT TCATCGAAGA GA - #CAGGTTTT 
480 
- ACCGCATTGA AATCAGAGAG AGAATATCCG TAGTCCAACG GTTCATCCTT AT - #ATCAGAGG 
540 
- TTAAAAGATA TGGTACGCTC ATCGAGGAGC TGATTGGCTT AGTAGGTGAG AC - #TTTCTTAA 
600 
- GAGACTATCG GCACCTACAG GAAGTTCATG GCACACAAGG CAAAGGAGGC AA - #TCTTCGCA 
660 
- GACCGGACTC ATATCAAAAG GATGAAACGA CTTTTCCATA CGACAACCAA AT - #AGCCGTCT 
720 
- ACGGTAGACG AATGCAAACC CAATATGAGG CCATCAATCA ATCCGAATGA CA - #GCTTTTGG 
780 
- GCAATATATT ATGCATATTT TGATTCGCGT TTAAAGGAAA AGTGCATATA TT - #TGCGATTG 
840 
- TGGTATTTCT TTCGGTTTCT ATGTGAATTT TGTCTCCCAA GAAGACTTTA TA - #ATGCATAA 
900 
#AAA AAC 957ACTACAC AGTAAAATCA TATTCTAATT TCATCAAA ATG 
# Met - # Lys Asn 
# 1 
- TTG AAC AAG TTT GTT TCG ATT GCT CTT TGC TC - #T TCC TTA TTA GGA GGA 
1005 
Leu Asn Lys Phe Val Ser Ile Ala Leu Cys Se - #r Ser Leu Leu Gly Gly 
# 15 
- ATG GCA TTT GCG CAG CAG ACA GAG TTG GGA CG - #C AAT CCG AAT GTC AGA 
1053 
Met Ala Phe Ala Gln Gln Thr Glu Leu Gly Ar - #g Asn Pro Asn Val Arg 
# 35 
- TTG CTC GAA TCC ACT CAG CAA TCG GTG ACA AA - #G GTT CAG TTC CGT ATG 
1101 
Leu Leu Glu Ser Thr Gln Gln Ser Val Thr Ly - #s Val Gln Phe Arg Met 
# 50 
- GAC AAC CTC AAG TTC ACC GAA GTT CAA ACC CC - #T AAG GGA ATC GGA CAA 
1149 
Asp Asn Leu Lys Phe Thr Glu Val Gln Thr Pr - #o Lys Gly Ile Gly Gln 
# 65 
- GTG CCG ACC TAT ACA GAA GGG GTT AAT CTT TC - #C GAA AAA GGG ATG CCT 
1197 
Val Pro Thr Tyr Thr Glu Gly Val Asn Leu Se - #r Glu Lys Gly Met Pro 
# 80 
- ACG CTT CCC ATT CTA TCA CGC TCT TTG GCG GT - #T TCA GAC ACT CGT GAG 
1245 
Thr Leu Pro Ile Leu Ser Arg Ser Leu Ala Va - #l Ser Asp Thr Arg Glu 
# 95 
- ATG AAG GTA GAG GTT GTT TCC TCA AAG TTC AT - #C GAA AAG AAA AAT GTC 
1293 
Met Lys Val Glu Val Val Ser Ser Lys Phe Il - #e Glu Lys Lys Asn Val 
100 1 - #05 1 - #10 1 - 
#15 
- CTG ATT GCA CCC TCC AAG GGC ATG ATT ATG CG - #T AAC GAA GAT CCG AAA 
1341 
Leu Ile Ala Pro Ser Lys Gly Met Ile Met Ar - #g Asn Glu Asp Pro Lys 
# 130 
- AAG ATC CCT TAC GTT TAT GGA AAG AGC TAC TC - #G CAA AAC AAA TTC TTC 
1389 
Lys Ile Pro Tyr Val Tyr Gly Lys Ser Tyr Se - #r Gln Asn Lys Phe Phe 
# 145 
- CCG GGA GAG ATC GCC ACG CTT GAT GAT CCT TT - #T ATC CTT CGT GAT GTG 
1437 
Pro Gly Glu Ile Ala Thr Leu Asp Asp Pro Ph - #e Ile Leu Arg Asp Val 
# 160 
- CGT GGA CAG GTT GTA AAC TTT GCG CCT TTG CA - #G TAT AAC CCT GTG ACA 
1485 
Arg Gly Gln Val Val Asn Phe Ala Pro Leu Gl - #n Tyr Asn Pro Val Thr 
# 175 
- AAG ACG TTG CGC ATC TAT ACG GAA ATC ACT GT - #G GCA GTG AGC GAA ACT 
1533 
Lys Thr Leu Arg Ile Tyr Thr Glu Ile Thr Va - #l Ala Val Ser Glu Thr 
180 1 - #85 1 - #90 1 - 
#95 
- TCG GAA CAA GGC AAA AAT ATT CTG AAC AAG AA - #A GGT ACA TTT GCC GGC 
1581 
Ser Glu Gln Gly Lys Asn Ile Leu Asn Lys Ly - #s Gly Thr Phe Ala Gly 
# 210 
- TTT GAA GAC ACA TAC AAG CGC ATG TTC ATG AA - #C TAC GAG CCG GGG CGT 
1629 
Phe Glu Asp Thr Tyr Lys Arg Met Phe Met As - #n Tyr Glu Pro Gly Arg 
# 225 
- TAC ACA CCG GTA GAG GAA AAA CAA AAT GGT CG - #T ATG ATC GTC ATC GTA 
1677 
Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Ar - #g Met Ile Val Ile Val 
# 240 
- GCC AAA AAG TAT GAG GGA GAT ATT AAA GAT TT - #C GTT GAT TGG AAA AAC 
1725 
Ala Lys Lys Tyr Glu Gly Asp Ile Lys Asp Ph - #e Val Asp Trp Lys Asn 
# 255 
- CAA CGC GGT CTC CGT ACC GAG GTG AAA GTG GC - #A GAA GAT ATT GCT TCT 
1773 
Gln Arg Gly Leu Arg Thr Glu Val Lys Val Al - #a Glu Asp Ile Ala Ser 
260 2 - #65 2 - #70 2 - 
#75 
- CCC GTT ACA GCT AAT GCT ATT CAG CAG TTC GT - #T AAG CAA GAA TAC GAG 
1821 
Pro Val Thr Ala Asn Ala Ile Gln Gln Phe Va - #l Lys Gln Glu Tyr Glu 
# 290 
- AAA GAA GGT AAT GAT TTG ACC TAT GTT CTT TT - #G GTT GGC GAT CAC AAA 
1869 
Lys Glu Gly Asn Asp Leu Thr Tyr Val Leu Le - #u Val Gly Asp His Lys 
# 305 
- GAT ATT CCT GCC AAA ATT ACT CCG GGG ATC AA - #A TCC GAC CAG GTA TAT 
1917 
Asp Ile Pro Ala Lys Ile Thr Pro Gly Ile Ly - #s Ser Asp Gln Val Tyr 
# 320 
- GGA CAA ATA GTA GGT AAT GAC CAC TAC AAC GA - #A GTC TTC ATC GGT CGT 
1965 
Gly Gln Ile Val Gly Asn Asp His Tyr Asn Gl - #u Val Phe Ile Gly Arg 
# 335 
- TTC TCA TGT GAG AGC AAA GAG GAT CTG AAG AC - #A CAA ATC GAT CGG ACT 
2013 
Phe Ser Cys Glu Ser Lys Glu Asp Leu Lys Th - #r Gln Ile Asp Arg Thr 
340 3 - #45 3 - #50 3 - 
#55 
- ATT CAC TAT GAG CGC AAT ATA ACC ACG GAA GA - #C AAA TGG CTC GGT CAG 
2061 
Ile His Tyr Glu Arg Asn Ile Thr Thr Glu As - #p Lys Trp Leu Gly Gln 
# 370 
- GCT CTT TGT ATT GCT TCG GCT GAA GGA GGC CC - #A TCC GCA GAC AAT GGT 
2109 
Ala Leu Cys Ile Ala Ser Ala Glu Gly Gly Pr - #o Ser Ala Asp Asn Gly 
# 385 
- GAA AGT GAT ATC CAG CAT GAG AAT GTA ATC GC - #C AAT CTG CTT ACC CAG 
2157 
Glu Ser Asp Ile Gln His Glu Asn Val Ile Al - #a Asn Leu Leu Thr Gln 
# 400 
- TAT GGC TAT ACC AAG ATT ATC AAA TGT TAT GA - #T CCG GGA GTA ACT CCT 
2205 
Tyr Gly Tyr Thr Lys Ile Ile Lys Cys Tyr As - #p Pro Gly Val Thr Pro 
# 415 
- AAA AAC ATT ATT GAT GCT TTC AAC GGA GGA AT - #C TCG TTG GTC AAC TAT 
2253 
Lys Asn Ile Ile Asp Ala Phe Asn Gly Gly Il - #e Ser Leu Val Asn Tyr 
420 4 - #25 4 - #30 4 - 
#35 
- ACG GGC CAC GGT AGC GAA ACA GCT TGG GGT AC - #G TCT CAC TTC GGC ACC 
2301 
Thr Gly His Gly Ser Glu Thr Ala Trp Gly Th - #r Ser His Phe Gly Thr 
# 450 
- ACT CAT GTG AAG CAG CTT ACC AAC AGC AAC CA - #G CTA CCG TTT ATT TTC 
2349 
Thr His Val Lys Gln Leu Thr Asn Ser Asn Gl - #n Leu Pro Phe Ile Phe 
# 465 
- GAC GTA GCT TGT GTG AAT GGC GAT TTC CTA TT - #C AGC ATG CCT TGC TTC 
2397 
Asp Val Ala Cys Val Asn Gly Asp Phe Leu Ph - #e Ser Met Pro Cys Phe 
# 480 
- GCA GAA GCC CTG ATG CGT GCA CAA AAA GAT GG - #T AAG CCG ACA GGT ACT 
2445 
Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gl - #y Lys Pro Thr Gly Thr 
# 495 
- GTT GCT ATC ATA GCG TCT ACG ATC AAC CAG TC - #T TGG GCT TCT CCT ATG 
2493 
Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Se - #r Trp Ala Ser Pro Met 
500 5 - #05 5 - #10 5 - 
#15 
- CGC GGG CAG GAT GAG ATG AAC GAA ATT CTG TG - #C GAA AAA CAC CCG AAC 
2541 
Arg Gly Gln Asp Glu Met Asn Glu Ile Leu Cy - #s Glu Lys His Pro Asn 
# 530 
- AAC ATC AAG CGT ACT TTC GGT GGT GTC ACC AT - #G AAC GGT ATG TTT GCT 
2589 
Asn Ile Lys Arg Thr Phe Gly Gly Val Thr Me - #t Asn Gly Met Phe Ala 
# 545 
- ATG GTG GAA AAG TAT AAA AAG GAT GGT GAG AA - #G ATG CTC GAC ACA TGG 
2637 
Met Val Glu Lys Tyr Lys Lys Asp Gly Glu Ly - #s Met Leu Asp Thr Trp 
# 560 
- ACT GTT TTC GGC GAC CCC TCG CTG CTC GTT CG - #T ACA CTT GTC CCG ACC 
2685 
Thr Val Phe Gly Asp Pro Ser Leu Leu Val Ar - #g Thr Leu Val Pro Thr 
# 575 
- AAA ATG CAG GTT ACG GCT CCG GCT CAG ATT AA - #T TTG ACG GAT GCT TCA 
2733 
Lys Met Gln Val Thr Ala Pro Ala Gln Ile As - #n Leu Thr Asp Ala Ser 
580 5 - #85 5 - #90 5 - 
#95 
- GTC AAC GTA TCT TGC GAT TAT AAT GGT GCT AT - #T GCT ACC ATT TCA GCC 
2781 
Val Asn Val Ser Cys Asp Tyr Asn Gly Ala Il - #e Ala Thr Ile Ser Ala 
# 610 
- AAT GGA AAG ATG TTC GGT TCT GCA GTT GTC GA - #A AAT GGA ACA GCT ACA 
2829 
Asn Gly Lys Met Phe Gly Ser Ala Val Val Gl - #u Asn Gly Thr Ala Thr 
# 625 
- ATC AAT CTG ACA GGT CTG ACA AAT GAA AGC AC - #G CTT ACC CTT ACA GTA 
2877 
Ile Asn Leu Thr Gly Leu Thr Asn Glu Ser Th - #r Leu Thr Leu Thr Val 
# 640 
- GTT GGT TAC AAC AAA GAG ACG GTT ATT AAG AC - #C ATC AAC ACT AAT GGT 
2925 
Val Gly Tyr Asn Lys Glu Thr Val Ile Lys Th - #r Ile Asn Thr Asn Gly 
# 655 
- GAG CCT AAC CCC TAC CAG CCC GTT TCC AAC TT - #G ACA GCT ACA ACG CAG 
2973 
Glu Pro Asn Pro Tyr Gln Pro Val Ser Asn Le - #u Thr Ala Thr Thr Gln 
660 6 - #65 6 - #70 6 - 
#75 
- GGT CAG AAA GTA ACG CTC AAG TGG GAT GCA CC - #G AGC ACG AAA ACC AAT 
3021 
Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pr - #o Ser Thr Lys Thr Asn 
# 690 
- GCA ACC ACT AAT ACC GCT CGC AGC GTG GAT GG - #C ATA CGA GAA TTG GTT 
3069 
Ala Thr Thr Asn Thr Ala Arg Ser Val Asp Gl - #y Ile Arg Glu Leu Val 
# 705 
- CTT CTG TCA GTC AGC GAT GCC CCC GAA CTT CT - #T CGC AGC GGT CAG GCC 
3117 
Leu Leu Ser Val Ser Asp Ala Pro Glu Leu Le - #u Arg Ser Gly Gln Ala 
# 720 
- GAG ATT GTT CTT GAA GCT CAC GAT GTT TGG AA - #T GAT GGA TCC 
#3159 
Glu Ile Val Leu Glu Ala His Asp Val Trp As - #n Asp Gly Ser 
# 735 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 737 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- Met Lys Asn Leu Asn Lys Phe Val Ser Ile Al - #a Leu Cys Ser Ser Leu 
# 15 
- Leu Gly Gly Met Ala Phe Ala Gln Gln Thr Gl - #u Leu Gly Arg Asn Pro 
# 30 
- Asn Val Arg Leu Leu Glu Ser Thr Gln Gln Se - #r Val Thr Lys Val Gln 
# 45 
- Phe Arg Met Asp Asn Leu Lys Phe Thr Glu Va - #l Gln Thr Pro Lys Gly 
# 60 
- Ile Gly Gln Val Pro Thr Tyr Thr Glu Gly Va - #l Asn Leu Ser Glu Lys 
# 80 
- Gly Met Pro Thr Leu Pro Ile Leu Ser Arg Se - #r Leu Ala Val Ser Asp 
# 95 
- Thr Arg Glu Met Lys Val Glu Val Val Ser Se - #r Lys Phe Ile Glu Lys 
# 110 
- Lys Asn Val Leu Ile Ala Pro Ser Lys Gly Me - #t Ile Met Arg Asn Glu 
# 125 
- Asp Pro Lys Lys Ile Pro Tyr Val Tyr Gly Ly - #s Ser Tyr Ser Gln Asn 
# 140 
- Lys Phe Phe Pro Gly Glu Ile Ala Thr Leu As - #p Asp Pro Phe Ile Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Arg Asp Val Arg Gly Gln Val Val Asn Phe Al - #a Pro Leu Gln Tyr Asn 
# 175 
- Pro Val Thr Lys Thr Leu Arg Ile Tyr Thr Gl - #u Ile Thr Val Ala Val 
# 190 
- Ser Glu Thr Ser Glu Gln Gly Lys Asn Ile Le - #u Asn Lys Lys Gly Thr 
# 205 
- Phe Ala Gly Phe Glu Asp Thr Tyr Lys Arg Me - #t Phe Met Asn Tyr Glu 
# 220 
- Pro Gly Arg Tyr Thr Pro Val Glu Glu Lys Gl - #n Asn Gly Arg Met Ile 
225 2 - #30 2 - #35 2 - 
#40 
- Val Ile Val Ala Lys Lys Tyr Glu Gly Asp Il - #e Lys Asp Phe Val Asp 
# 255 
- Trp Lys Asn Gln Arg Gly Leu Arg Thr Glu Va - #l Lys Val Ala Glu Asp 
# 270 
- Ile Ala Ser Pro Val Thr Ala Asn Ala Ile Gl - #n Gln Phe Val Lys Gln 
# 285 
- Glu Tyr Glu Lys Glu Gly Asn Asp Leu Thr Ty - #r Val Leu Leu Val Gly 
# 300 
- Asp His Lys Asp Ile Pro Ala Lys Ile Thr Pr - #o Gly Ile Lys Ser Asp 
305 3 - #10 3 - #15 3 - 
#20 
- Gln Val Tyr Gly Gln Ile Val Gly Asn Asp Hi - #s Tyr Asn Glu Val Phe 
# 335 
- Ile Gly Arg Phe Ser Cys Glu Ser Lys Glu As - #p Leu Lys Thr Gln Ile 
# 350 
- Asp Arg Thr Ile His Tyr Glu Arg Asn Ile Th - #r Thr Glu Asp Lys Trp 
# 365 
- Leu Gly Gln Ala Leu Cys Ile Ala Ser Ala Gl - #u Gly Gly Pro Ser Ala 
# 380 
- Asp Asn Gly Glu Ser Asp Ile Gln His Glu As - #n Val Ile Ala Asn Leu 
385 3 - #90 3 - #95 4 - 
#00 
- Leu Thr Gln Tyr Gly Tyr Thr Lys Ile Ile Ly - #s Cys Tyr Asp Pro Gly 
# 415 
- Val Thr Pro Lys Asn Ile Ile Asp Ala Phe As - #n Gly Gly Ile Ser Leu 
# 430 
- Val Asn Tyr Thr Gly His Gly Ser Glu Thr Al - #a Trp Gly Thr Ser His 
# 445 
- Phe Gly Thr Thr His Val Lys Gln Leu Thr As - #n Ser Asn Gln Leu Pro 
# 460 
- Phe Ile Phe Asp Val Ala Cys Val Asn Gly As - #p Phe Leu Phe Ser Met 
465 4 - #70 4 - #75 4 - 
#80 
- Pro Cys Phe Ala Glu Ala Leu Met Arg Ala Gl - #n Lys Asp Gly Lys Pro 
# 495 
- Thr Gly Thr Val Ala Ile Ile Ala Ser Thr Il - #e Asn Gln Ser Trp Ala 
# 510 
- Ser Pro Met Arg Gly Gln Asp Glu Met Asn Gl - #u Ile Leu Cys Glu Lys 
# 525 
- His Pro Asn Asn Ile Lys Arg Thr Phe Gly Gl - #y Val Thr Met Asn Gly 
# 540 
- Met Phe Ala Met Val Glu Lys Tyr Lys Lys As - #p Gly Glu Lys Met Leu 
545 5 - #50 5 - #55 5 - 
#60 
- Asp Thr Trp Thr Val Phe Gly Asp Pro Ser Le - #u Leu Val Arg Thr Leu 
# 575 
- Val Pro Thr Lys Met Gln Val Thr Ala Pro Al - #a Gln Ile Asn Leu Thr 
# 590 
- Asp Ala Ser Val Asn Val Ser Cys Asp Tyr As - #n Gly Ala Ile Ala Thr 
# 605 
- Ile Ser Ala Asn Gly Lys Met Phe Gly Ser Al - #a Val Val Glu Asn Gly 
# 620 
- Thr Ala Thr Ile Asn Leu Thr Gly Leu Thr As - #n Glu Ser Thr Leu Thr 
625 6 - #30 6 - #35 6 - 
#40 
- Leu Thr Val Val Gly Tyr Asn Lys Glu Thr Va - #l Ile Lys Thr Ile Asn 
# 655 
- Thr Asn Gly Glu Pro Asn Pro Tyr Gln Pro Va - #l Ser Asn Leu Thr Ala 
# 670 
- Thr Thr Gln Gly Gln Lys Val Thr Leu Lys Tr - #p Asp Ala Pro Ser Thr 
# 685 
- Lys Thr Asn Ala Thr Thr Asn Thr Ala Arg Se - #r Val Asp Gly Ile Arg 
# 700 
- Glu Leu Val Leu Leu Ser Val Ser Asp Ala Pr - #o Glu Leu Leu Arg Ser 
705 7 - #10 7 - #15 7 - 
#20 
- Gly Gln Ala Glu Ile Val Leu Glu Ala His As - #p Val Trp Asn Asp Gly 
# 735 
- Ser 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 7266 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Porphyromona - #s gingivalis 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 949..6063 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- CTGCAGAGGG CTGGTAAAGA CCGCCTCGGG ATCGAGGCCT TTGAGACGGG CA - #CAAGCCGC 
60 
- CGCAGCCTCC TCTTCGAAGG TGTCTCGAAC GTCCACATCG GTGAATCCGT AG - #CAGTGCTC 
120 
- ATTGCCATTG AGCAGCACCG AGGTGTGGCG CATCAGATAT ATTTTCATCA GT - #GGATTATT 
180 
- AGGGTATCGG TCAGAAAAAG CCTTCCGAAT CCGACAAAGA TAGTAGAAAG AG - #AGTGCATC 
240 
- TGAAAACAGA TCATTCGAGG ATTATCGATC AACTGAAAAG GCAGGAGTTG TT - #TTGCGTTT 
300 
- TGGTTCGGAA AATTACCTGA TCAGCATTCG TAAAAACGTG GCGCGAGAAT TT - #TTTCGTTT 
360 
- TGGCGCGAGA ATTAAAAATT TTTGGAACCA CAGCGAAAAA AATCTCGCGC CG - #TTTTCTCA 
420 
- GGATTTACAG ACCACAATCC GAGCATTTTC GGTTCGTAAT TCATCGAAGA GA - #CAGGTTTT 
480 
- ACCGCATTGA AATCAGAGAG AGAATATCCG TAGTCCAACG GTTCATCCTT AT - #ATCAGAGG 
540 
- TTAAAAGATA TGGTACGCTC ATCGAGGAGC TGATTGGCTT AGTAGGTGAG AC - #TTTCTTAA 
600 
- GAGACTATCG GCACCTACAG GAAGTTCATG GCACACAAGG CAAAGGAGGC AA - #TCTTCGCA 
660 
- GACCGGACTC ATATCAAAAG GATGAAACGA CTTTTCCATA CGACAACCAA AT - #AGCCGTCT 
720 
- ACGGTAGACG AATGCAAACC CAATATGAGG CCATCAATCA ATCCGAATGA CA - #GCTTTTGG 
780 
- GCAATATATT ATGCATATTT TGATTCGCGT TTAAAGGAAA AGTGCATATA TT - #TGCGATTG 
840 
- TGGTATTTCT TTCGGTTTCT ATGTGAATTT TGTCTCCCAA GAAGACTTTA TA - #ATGCATAA 
900 
#AAA AAC 957ACTACAC AGTAAAATCA TATTCTAATT TCATCAAA ATG 
# Met - # Lys Asn 
# 1 
- TTG AAC AAG TTT GTT TCG ATT GCT CTT TGC TC - #T TCC TTA TTA GGA GGA 
1005 
Leu Asn Lys Phe Val Ser Ile Ala Leu Cys Se - #r Ser Leu Leu Gly Gly 
# 15 
- ATG GCA TTT GCG CAG CAG ACA GAG TTG GGA CG - #C AAT CCG AAT GTC AGA 
1053 
Met Ala Phe Ala Gln Gln Thr Glu Leu Gly Ar - #g Asn Pro Asn Val Arg 
# 35 
- TTG CTC GAA TCC ACT CAG CAA TCG GTG ACA AA - #G GTT CAG TTC CGT ATG 
1101 
Leu Leu Glu Ser Thr Gln Gln Ser Val Thr Ly - #s Val Gln Phe Arg Met 
# 50 
- GAC AAC CTC AAG TTC ACC GAA GTT CAA ACC CC - #T AAG GGA ATC GGA CAA 
1149 
Asp Asn Leu Lys Phe Thr Glu Val Gln Thr Pr - #o Lys Gly Ile Gly Gln 
# 65 
- GTG CCG ACC TAT ACA GAA GGG GTT AAT CTT TC - #C GAA AAA GGG ATG CCT 
1197 
Val Pro Thr Tyr Thr Glu Gly Val Asn Leu Se - #r Glu Lys Gly Met Pro 
# 80 
- ACG CTT CCC ATT CTA TCA CGC TCT TTG GCG GT - #T TCA GAC ACT CGT GAG 
1245 
Thr Leu Pro Ile Leu Ser Arg Ser Leu Ala Va - #l Ser Asp Thr Arg Glu 
# 95 
- ATG AAG GTA GAG GTT GTT TCC TCA AAG TTC AT - #C GAA AAG AAA AAT GTC 
1293 
Met Lys Val Glu Val Val Ser Ser Lys Phe Il - #e Glu Lys Lys Asn Val 
100 1 - #05 1 - #10 1 - 
#15 
- CTG ATT GCA CCC TCC AAG GGC ATG ATT ATG CG - #T AAC GAA GAT CCG AAA 
1341 
Leu Ile Ala Pro Ser Lys Gly Met Ile Met Ar - #g Asn Glu Asp Pro Lys 
# 130 
- AAG ATC CCT TAC GTT TAT GGA AAG AGC TAC TC - #G CAA AAC AAA TTC TTC 
1389 
Lys Ile Pro Tyr Val Tyr Gly Lys Ser Tyr Se - #r Gln Asn Lys Phe Phe 
# 145 
- CCG GGA GAG ATC GCC ACG CTT GAT GAT CCT TT - #T ATC CTT CGT GAT GTG 
1437 
Pro Gly Glu Ile Ala Thr Leu Asp Asp Pro Ph - #e Ile Leu Arg Asp Val 
# 160 
- CGT GGA CAG GTT GTA AAC TTT GCG CCT TTG CA - #G TAT AAC CCT GTG ACA 
1485 
Arg Gly Gln Val Val Asn Phe Ala Pro Leu Gl - #n Tyr Asn Pro Val Thr 
# 175 
- AAG ACG TTG CGC ATC TAT ACG GAA ATC ACT GT - #G GCA GTG AGC GAA ACT 
1533 
Lys Thr Leu Arg Ile Tyr Thr Glu Ile Thr Va - #l Ala Val Ser Glu Thr 
180 1 - #85 1 - #90 1 - 
#95 
- TCG GAA CAA GGC AAA AAT ATT CTG AAC AAG AA - #A GGT ACA TTT GCC GGC 
1581 
Ser Glu Gln Gly Lys Asn Ile Leu Asn Lys Ly - #s Gly Thr Phe Ala Gly 
# 210 
- TTT GAA GAC ACA TAC AAG CGC ATG TTC ATG AA - #C TAC GAG CCG GGG CGT 
1629 
Phe Glu Asp Thr Tyr Lys Arg Met Phe Met As - #n Tyr Glu Pro Gly Arg 
# 225 
- TAC ACA CCG GTA GAG GAA AAA CAA AAT GGT CG - #T ATG ATC GTC ATC GTA 
1677 
Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Ar - #g Met Ile Val Ile Val 
# 240 
- GCC AAA AAG TAT GAG GGA GAT ATT AAA GAT TT - #C GTT GAT TGG AAA AAC 
1725 
Ala Lys Lys Tyr Glu Gly Asp Ile Lys Asp Ph - #e Val Asp Trp Lys Asn 
# 255 
- CAA CGC GGT CTC CGT ACC GAG GTG AAA GTG GC - #A GAA GAT ATT GCT TCT 
1773 
Gln Arg Gly Leu Arg Thr Glu Val Lys Val Al - #a Glu Asp Ile Ala Ser 
260 2 - #65 2 - #70 2 - 
#75 
- CCC GTT ACA GCT AAT GCT ATT CAG CAG TTC GT - #T AAG CAA GAA TAC GAG 
1821 
Pro Val Thr Ala Asn Ala Ile Gln Gln Phe Va - #l Lys Gln Glu Tyr Glu 
# 290 
- AAA GAA GGT AAT GAT TTG ACC TAT GTT CTT TT - #G GTT GGC GAT CAC AAA 
1869 
Lys Glu Gly Asn Asp Leu Thr Tyr Val Leu Le - #u Val Gly Asp His Lys 
# 305 
- GAT ATT CCT GCC AAA ATT ACT CCG GGG ATC AA - #A TCC GAC CAG GTA TAT 
1917 
Asp Ile Pro Ala Lys Ile Thr Pro Gly Ile Ly - #s Ser Asp Gln Val Tyr 
# 320 
- GGA CAA ATA GTA GGT AAT GAC CAC TAC AAC GA - #A GTC TTC ATC GGT CGT 
1965 
Gly Gln Ile Val Gly Asn Asp His Tyr Asn Gl - #u Val Phe Ile Gly Arg 
# 335 
- TTC TCA TGT GAG AGC AAA GAG GAT CTG AAG AC - #A CAA ATC GAT CGG ACT 
2013 
Phe Ser Cys Glu Ser Lys Glu Asp Leu Lys Th - #r Gln Ile Asp Arg Thr 
340 3 - #45 3 - #50 3 - 
#55 
- ATT CAC TAT GAG CGC AAT ATA ACC ACG GAA GA - #C AAA TGG CTC GGT CAG 
2061 
Ile His Tyr Glu Arg Asn Ile Thr Thr Glu As - #p Lys Trp Leu Gly Gln 
# 370 
- GCT CTT TGT ATT GCT TCG GCT GAA GGA GGC CC - #A TCC GCA GAC AAT GGT 
2109 
Ala Leu Cys Ile Ala Ser Ala Glu Gly Gly Pr - #o Ser Ala Asp Asn Gly 
# 385 
- GAA AGT GAT ATC CAG CAT GAG AAT GTA ATC GC - #C AAT CTG CTT ACC CAG 
2157 
Glu Ser Asp Ile Gln His Glu Asn Val Ile Al - #a Asn Leu Leu Thr Gln 
# 400 
- TAT GGC TAT ACC AAG ATT ATC AAA TGT TAT GA - #T CCG GGA GTA ACT CCT 
2205 
Tyr Gly Tyr Thr Lys Ile Ile Lys Cys Tyr As - #p Pro Gly Val Thr Pro 
# 415 
- AAA AAC ATT ATT GAT GCT TTC AAC GGA GGA AT - #C TCG TTG GTC AAC TAT 
2253 
Lys Asn Ile Ile Asp Ala Phe Asn Gly Gly Il - #e Ser Leu Val Asn Tyr 
420 4 - #25 4 - #30 4 - 
#35 
- ACG GGC CAC GGT AGC GAA ACA GCT TGG GGT AC - #G TCT CAC TTC GGC ACC 
2301 
Thr Gly His Gly Ser Glu Thr Ala Trp Gly Th - #r Ser His Phe Gly Thr 
# 450 
- ACT CAT GTG AAG CAG CTT ACC AAC AGC AAC CA - #G CTA CCG TTT ATT TTC 
2349 
Thr His Val Lys Gln Leu Thr Asn Ser Asn Gl - #n Leu Pro Phe Ile Phe 
# 465 
- GAC GTA GCT TGT GTG AAT GGC GAT TTC CTA TT - #C AGC ATG CCT TGC TTC 
2397 
Asp Val Ala Cys Val Asn Gly Asp Phe Leu Ph - #e Ser Met Pro Cys Phe 
# 480 
- GCA GAA GCC CTG ATG CGT GCA CAA AAA GAT GG - #T AAG CCG ACA GGT ACT 
2445 
Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gl - #y Lys Pro Thr Gly Thr 
# 495 
- GTT GCT ATC ATA GCG TCT ACG ATC AAC CAG TC - #T TGG GCT TCT CCT ATG 
2493 
Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Se - #r Trp Ala Ser Pro Met 
500 5 - #05 5 - #10 5 - 
#15 
- CGC GGG CAG GAT GAG ATG AAC GAA ATT CTG TG - #C GAA AAA CAC CCG AAC 
2541 
Arg Gly Gln Asp Glu Met Asn Glu Ile Leu Cy - #s Glu Lys His Pro Asn 
# 530 
- AAC ATC AAG CGT ACT TTC GGT GGT GTC ACC AT - #G AAC GGT ATG TTT GCT 
2589 
Asn Ile Lys Arg Thr Phe Gly Gly Val Thr Me - #t Asn Gly Met Phe Ala 
# 545 
- ATG GTG GAA AAG TAT AAA AAG GAT GGT GAG AA - #G ATG CTC GAC ACA TGG 
2637 
Met Val Glu Lys Tyr Lys Lys Asp Gly Glu Ly - #s Met Leu Asp Thr Trp 
# 560 
- ACT GTT TTC GGC GAC CCC TCG CTG CTC GTT CG - #T ACA CTT GTC CCG ACC 
2685 
Thr Val Phe Gly Asp Pro Ser Leu Leu Val Ar - #g Thr Leu Val Pro Thr 
# 575 
- AAA ATG CAG GTT ACG GCT CCG GCT CAG ATT AA - #T TTG ACG GAT GCT TCA 
2733 
Lys Met Gln Val Thr Ala Pro Ala Gln Ile As - #n Leu Thr Asp Ala Ser 
580 5 - #85 5 - #90 5 - 
#95 
- GTC AAC GTA TCT TGC GAT TAT AAT GGT GCT AT - #T GCT ACC ATT TCA GCC 
2781 
Val Asn Val Ser Cys Asp Tyr Asn Gly Ala Il - #e Ala Thr Ile Ser Ala 
# 610 
- AAT GGA AAG ATG TTC GGT TCT GCA GTT GTC GA - #A AAT GGA ACA GCT ACA 
2829 
Asn Gly Lys Met Phe Gly Ser Ala Val Val Gl - #u Asn Gly Thr Ala Thr 
# 625 
- ATC AAT CTG ACA GGT CTG ACA AAT GAA AGC AC - #G CTT ACC CTT ACA GTA 
2877 
Ile Asn Leu Thr Gly Leu Thr Asn Glu Ser Th - #r Leu Thr Leu Thr Val 
# 640 
- GTT GGT TAC AAC AAA GAG ACG GTT ATT AAG AC - #C ATC AAC ACT AAT GGT 
2925 
Val Gly Tyr Asn Lys Glu Thr Val Ile Lys Th - #r Ile Asn Thr Asn Gly 
# 655 
- GAG CCT AAC CCC TAC CAG CCC GTT TCC AAC TT - #G ACA GCT ACA ACG CAG 
2973 
Glu Pro Asn Pro Tyr Gln Pro Val Ser Asn Le - #u Thr Ala Thr Thr Gln 
660 6 - #65 6 - #70 6 - 
#75 
- GGT CAG AAA GTA ACG CTC AAG TGG GAT GCA CC - #G AGC ACG AAA ACC AAT 
3021 
Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pr - #o Ser Thr Lys Thr Asn 
# 690 
- GCA ACC ACT AAT ACC GCT CGC AGC GTG GAT GG - #C ATA CGA GAA TTG GTT 
3069 
Ala Thr Thr Asn Thr Ala Arg Ser Val Asp Gl - #y Ile Arg Glu Leu Val 
# 705 
- CTT CTG TCA GTC AGC GAT GCC CCC GAA CTT CT - #T CGC AGC GGT CAG GCC 
3117 
Leu Leu Ser Val Ser Asp Ala Pro Glu Leu Le - #u Arg Ser Gly Gln Ala 
# 720 
- GAG ATT GTT CTT GAA GCT CAC GAT GTT TGG AA - #T GAT GGA TCC GGT TAT 
3165 
Glu Ile Val Leu Glu Ala His Asp Val Trp As - #n Asp Gly Ser Gly Tyr 
# 735 
- CAG ATT CTT TTG GAT GCA GAC CAT GAT CAA TA - #T GGA CAG GTT ATA CCC 
3213 
Gln Ile Leu Leu Asp Ala Asp His Asp Gln Ty - #r Gly Gln Val Ile Pro 
740 7 - #45 7 - #50 7 - 
#55 
- AGT GAT ACC CAT ACT CTT TGG CCG AAC TGT AG - #T GTC CCG GCC AAT CTG 
3261 
Ser Asp Thr His Thr Leu Trp Pro Asn Cys Se - #r Val Pro Ala Asn Leu 
# 770 
- TTC GCT CCG TTC GAA TAT ACT GTT CCG GAA AA - #T GCA GAT CCT TCT TGT 
3309 
Phe Ala Pro Phe Glu Tyr Thr Val Pro Glu As - #n Ala Asp Pro Ser Cys 
# 785 
- TCC CCT ACC AAT ATG ATA ATG GAT GGT ACT GC - #A TCC GTT AAT ATA CCG 
3357 
Ser Pro Thr Asn Met Ile Met Asp Gly Thr Al - #a Ser Val Asn Ile Pro 
# 800 
- GCC GGA ACT TAT GAC TTT GCA ATT GCT GCT CC - #T CAA GCA AAT GCA AAG 
3405 
Ala Gly Thr Tyr Asp Phe Ala Ile Ala Ala Pr - #o Gln Ala Asn Ala Lys 
# 815 
- ATT TGG ATT GCC GGA CAA GGA CCG ACG AAA GA - #A GAT GAT TAT GTA TTT 
3453 
Ile Trp Ile Ala Gly Gln Gly Pro Thr Lys Gl - #u Asp Asp Tyr Val Phe 
820 8 - #25 8 - #30 8 - 
#35 
- GAA GCC GGT AAA AAA TAC CAT TTC CTT ATG AA - #G AAG ATG GGT AGC GGT 
3501 
Glu Ala Gly Lys Lys Tyr His Phe Leu Met Ly - #s Lys Met Gly Ser Gly 
# 850 
- GAT GGA ACT GAA TTG ACT ATA AGC GAA GGT GG - #T GGA AGC GAT TAC ACC 
3549 
Asp Gly Thr Glu Leu Thr Ile Ser Glu Gly Gl - #y Gly Ser Asp Tyr Thr 
# 865 
- TAT ACT GTC TAT CGT GAC GGC ACG AAG ATC AA - #G GAA GGT CTG ACG GCT 
3597 
Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Ly - #s Glu Gly Leu Thr Ala 
# 880 
- ACG ACA TTC GAA GAA GAC GGT GTA GCT ACG GG - #C AAT CAT GAG TAT TGC 
3645 
Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gl - #y Asn His Glu Tyr Cys 
# 895 
- GTG GAA GTT AAG TAC ACA GCC GGC GTA TCT CC - #G AAG GTA TGT AAA GAC 
3693 
Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pr - #o Lys Val Cys Lys Asp 
900 9 - #05 9 - #10 9 - 
#15 
- GTT ACG GTA GAA GGA TCC AAT GAA TTT GCT CC - #T GTA CAG AAC CTG ACC 
3741 
Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pr - #o Val Gln Asn Leu Thr 
# 930 
- GGT AGT GCA GTC GGC CAG AAA GTA ACG CTC AA - #G TGG GAT GCA CCT AAT 
3789 
Gly Ser Ala Val Gly Gln Lys Val Thr Leu Ly - #s Trp Asp Ala Pro Asn 
# 945 
- GGT ACC CCG AAT CCA AAT CCG AAT CCG AAT CC - #G AAT CCC GGA ACA ACA 
3837 
Gly Thr Pro Asn Pro Asn Pro Asn Pro Asn Pr - #o Asn Pro Gly Thr Thr 
# 960 
- ACA CTT TCC GAA TCA TTC GAA AAT GGT ATT CC - #T GCC TCA TGG AAG ACG 
3885 
Thr Leu Ser Glu Ser Phe Glu Asn Gly Ile Pr - #o Ala Ser Trp Lys Thr 
# 975 
- ATC GAT GCA GAC GGT GAC GGG CAT GGC TGG AA - #G CCT GGA AAT GCT CCC 
3933 
Ile Asp Ala Asp Gly Asp Gly His Gly Trp Ly - #s Pro Gly Asn Ala Pro 
980 9 - #85 9 - #90 9 - 
#95 
- GGA ATC GCT GGC TAC AAT AGC AAT GGT TGT GT - #A TAT TCA GAG TCA TTC 
3981 
Gly Ile Ala Gly Tyr Asn Ser Asn Gly Cys Va - #l Tyr Ser Glu Ser Phe 
# 10105 
- GGT CTT GGT GGT ATA GGA GTT CTT ACC CCT GA - #C AAC TAT CTG ATA ACA 
4029 
Gly Leu Gly Gly Ile Gly Val Leu Thr Pro As - #p Asn Tyr Leu Ile Thr 
# 10250 
- CCG GCA TTG GAT TTG CCT AAC GGA GGT AAG TT - #G ACT TTC TGG GTA TGC 
4077 
Pro Ala Leu Asp Leu Pro Asn Gly Gly Lys Le - #u Thr Phe Trp Val Cys 
# 10405 
- GCA CAG GAT GCT AAT TAT GCA TCC GAG CAC TA - #T GCG GTG TAT GCA TCT 
4125 
Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Ty - #r Ala Val Tyr Ala Ser 
# 10550 
- TCG ACC GGT AAC GAT GCA TCC AAC TTC ACG AA - #T GCT TTG TTG GAA GAG 
4173 
Ser Thr Gly Asn Asp Ala Ser Asn Phe Thr As - #n Ala Leu Leu Glu Glu 
# 10751065 - # 1070 
- ACG ATT ACG GCA AAA GGT GTT CGC TCG CCG GA - #A GCT ATT CGT GGT CGT 
4221 
Thr Ile Thr Ala Lys Gly Val Arg Ser Pro Gl - #u Ala Ile Arg Gly Arg 
# 10905 
- ATA CAG GGT ACT TGG CGC CAG AAG ACG GTA GA - #C CTT CCC GCA GGT ACG 
4269 
Ile Gln Gly Thr Trp Arg Gln Lys Thr Val As - #p Leu Pro Ala Gly Thr 
# 11050 
- AAA TAT GTT GCT TTC CGT CAC TTC CAA AGC AC - #G GAT ATG TTC TAC ATC 
4317 
Lys Tyr Val Ala Phe Arg His Phe Gln Ser Th - #r Asp Met Phe Tyr Ile 
# 11205 
- GAC CTT GAT GAG GTT GAG ATC AAG GCC AAC GG - #C AAG CGC GCA GAC TTC 
4365 
Asp Leu Asp Glu Val Glu Ile Lys Ala Asn Gl - #y Lys Arg Ala Asp Phe 
# 11350 
- ACG GAA ACG TTC GAG TCT TCT ACT CAT GGA GA - #G GCA CCG GCG GAA TGG 
4413 
Thr Glu Thr Phe Glu Ser Ser Thr His Gly Gl - #u Ala Pro Ala Glu Trp 
# 11551145 - # 1150 
- ACT ACT ATC GAT GCC GAT GGC GAT GGT CAG GG - #T TGG CTC TGT CTG TCT 
4461 
Thr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gl - #y Trp Leu Cys Leu Ser 
# 11705 
- TCC GGA CAA TTG GAC TGG CTG ACA GCT CAT GG - #C GGC ACC AAC GTA GTA 
4509 
Ser Gly Gln Leu Asp Trp Leu Thr Ala His Gl - #y Gly Thr Asn Val Val 
# 11850 
- GCC TCT TTC TCA TGG AAT GGA ATG GCT TTG AA - #T CCT GAT AAC TAT CTC 
4557 
Ala Ser Phe Ser Trp Asn Gly Met Ala Leu As - #n Pro Asp Asn Tyr Leu 
# 12005 
- ATC TCA AAG GAT GTT ACA GGC GCA ACG AAG GT - #A AAG TAC TAC TAT GCA 
4605 
Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Va - #l Lys Tyr Tyr Tyr Ala 
# 12150 
- GTC AAC GAC GGT TTT CCC GGG GAT CAC TAT GC - #G GTG ATG ATC TCC AAG 
4653 
Val Asn Asp Gly Phe Pro Gly Asp His Tyr Al - #a Val Met Ile Ser Lys 
# 12351225 - # 1230 
- ACG GGC ACG AAC GCC GGA GAC TTC ACG GTT GT - #T TTC GAA GAA ACG CCT 
4701 
Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Va - #l Phe Glu Glu Thr Pro 
# 12505 
- AAC GGA ATA AAT AAG GGC GGA GCA AGA TTC GG - #T CTT TCC ACG GAA GCC 
4749 
Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe Gl - #y Leu Ser Thr Glu Ala 
# 12650 
- AAT GGC GCC AAA CCT CAA AGT GTA TGG ATC GA - #G CGT ACG GTA GAT TTG 
4797 
Asn Gly Ala Lys Pro Gln Ser Val Trp Ile Gl - #u Arg Thr Val Asp Leu 
# 12805 
- CCT GCG GGC ACG AAG TAT GTT GCT TTC CGT CA - #C TAC AAT TGC TCG GAT 
4845 
Pro Ala Gly Thr Lys Tyr Val Ala Phe Arg Hi - #s Tyr Asn Cys Ser Asp 
# 12950 
- TTG AAC TAC ATT CTT TTG GAT GAT ATT CAG TT - #C ACC ATG GGT GGC AGC 
4893 
Leu Asn Tyr Ile Leu Leu Asp Asp Ile Gln Ph - #e Thr Met Gly Gly Ser 
# 13151305 - # 1310 
- CCC ACC CCG ACC GAT TAT ACC TAC ACG GTG TA - #T CGT GAC GGT ACG AAG 
4941 
Pro Thr Pro Thr Asp Tyr Thr Tyr Thr Val Ty - #r Arg Asp Gly Thr Lys 
# 13305 
- ATC AAG GAA GGT CTG ACC GAA ACG ACC TTC GA - #A GAA GAC GGC GTA GCT 
4989 
Ile Lys Glu Gly Leu Thr Glu Thr Thr Phe Gl - #u Glu Asp Gly Val Ala 
# 13450 
- ACA GGC AAT CAT GAG TAT TGC GTG GAA GTG AA - #G TAC ACA GCC GGC GTA 
5037 
Thr Gly Asn His Glu Tyr Cys Val Glu Val Ly - #s Tyr Thr Ala Gly Val 
# 13605 
- TCT CCG AAA GAG TGC GTA AAC GTA ACT ATT AA - #T CCG ACT CAG TTC AAT 
5085 
Ser Pro Lys Glu Cys Val Asn Val Thr Ile As - #n Pro Thr Gln Phe Asn 
# 13750 
- CCT GTA AAG AAC CTG AAG GCA CAA CCG GAT GG - #C GGC GAC GTG GTT CTC 
5133 
Pro Val Lys Asn Leu Lys Ala Gln Pro Asp Gl - #y Gly Asp Val Val Leu 
# 13951385 - # 1390 
- AAG TGG GAA GCC CCG AGC GCA AAA AAG ACA GA - #A GGT TCT CGT GAA GTA 
5181 
Lys Trp Glu Ala Pro Ser Ala Lys Lys Thr Gl - #u Gly Ser Arg Glu Val 
# 14105 
- AAA CGG ATC GGA GAC GGT CTT TTC GTT ACG AT - #C GAA CCT GCA AAC GAT 
5229 
Lys Arg Ile Gly Asp Gly Leu Phe Val Thr Il - #e Glu Pro Ala Asn Asp 
# 14250 
- GTA CGT GCC AAC GAA GCC AAG GTT GTG CTC GC - #A GCA GAC AAC GTA TGG 
5277 
Val Arg Ala Asn Glu Ala Lys Val Val Leu Al - #a Ala Asp Asn Val Trp 
# 14405 
- GGA GAC AAT ACG GGT TAC CAG TTC TTG TTG GA - #T GCC GAT CAC AAT ACA 
5325 
Gly Asp Asn Thr Gly Tyr Gln Phe Leu Leu As - #p Ala Asp His Asn Thr 
# 14550 
- TTC GGA AGT GTC ATT CCG GCA ACC GGT CCT CT - #C TTT ACC GGA ACA GCT 
5373 
Phe Gly Ser Val Ile Pro Ala Thr Gly Pro Le - #u Phe Thr Gly Thr Ala 
# 14751465 - # 1470 
- TCT TCC AAT CTT TAC AGT GCG AAC TTC GAG TA - #T TTG ATC CCG GCC AAT 
5421 
Ser Ser Asn Leu Tyr Ser Ala Asn Phe Glu Ty - #r Leu Ile Pro Ala Asn 
# 14905 
- GCC GAT CCT GTT GTT ACT ACA CAG AAT ATT AT - #C GTT ACA GGA CAG GGT 
5469 
Ala Asp Pro Val Val Thr Thr Gln Asn Ile Il - #e Val Thr Gly Gln Gly 
# 15050 
- GAA GTT GTA ATC CCC GGT GGT GTT TAC GAC TA - #T TGC ATT ACG AAC CCG 
5517 
Glu Val Val Ile Pro Gly Gly Val Tyr Asp Ty - #r Cys Ile Thr Asn Pro 
# 15205 
- GAA CCT GCA TCC GGA AAG ATG TGG ATC GCA GG - #A GAT GGA GGC AAC CAG 
5565 
Glu Pro Ala Ser Gly Lys Met Trp Ile Ala Gl - #y Asp Gly Gly Asn Gln 
# 15350 
- CCT GCA CGT TAT GAC GAT TTC ACA TTC GAA GC - #A GGC AAG AAG TAC ACC 
5613 
Pro Ala Arg Tyr Asp Asp Phe Thr Phe Glu Al - #a Gly Lys Lys Tyr Thr 
# 15551545 - # 1550 
- TTC ACG ATG CGT CGC GCC GGA ATG GGA GAT GG - #A ACT GAT ATG GAA GTC 
5661 
Phe Thr Met Arg Arg Ala Gly Met Gly Asp Gl - #y Thr Asp Met Glu Val 
# 15705 
- GAA GAC GAT TCA CCT GCA AGC TAT ACC TAT AC - #A GTC TAT CGT GAC GGC 
5709 
Glu Asp Asp Ser Pro Ala Ser Tyr Thr Tyr Th - #r Val Tyr Arg Asp Gly 
# 15850 
- ACG AAG ATC AAG GAA GGT CTG ACC GAA ACG AC - #C TAC CGC GAT GCA GGA 
5757 
Thr Lys Ile Lys Glu Gly Leu Thr Glu Thr Th - #r Tyr Arg Asp Ala Gly 
# 16005 
- ATG AGT GCA CAA TCT CAT GAG TAT TGC GTA GA - #G GTT AAG TAC GCA GCC 
5805 
Met Ser Ala Gln Ser His Glu Tyr Cys Val Gl - #u Val Lys Tyr Ala Ala 
# 16150 
- GGC GTA TCT CCG AAG GTT TGT GTG GAT TAT AT - #T CCT GAC GGA GTG GCA 
5853 
Gly Val Ser Pro Lys Val Cys Val Asp Tyr Il - #e Pro Asp Gly Val Ala 
# 16351625 - # 1630 
- GAC GTA ACG GCT CAG AAG CCT TAC ACG CTG AC - #A GTT GTT GGA AAG ACG 
5901 
Asp Val Thr Ala Gln Lys Pro Tyr Thr Leu Th - #r Val Val Gly Lys Thr 
# 16505 
- ATC ACG GTA ACT TGC CAA GGC GAA GCT ATG AT - #C TAC GAC ATG AAC GGT 
5949 
Ile Thr Val Thr Cys Gln Gly Glu Ala Met Il - #e Tyr Asp Met Asn Gly 
# 16650 
- CGT CGT CTG GCA GCC GGT CGC AAC ACA GTT GT - #T TAC ACG GCT CAG GGC 
5997 
Arg Arg Leu Ala Ala Gly Arg Asn Thr Val Va - #l Tyr Thr Ala Gln Gly 
# 16805 
- GGC TAC TAT GCA GTC ATG GTT GTC GTT GAC GG - #C AAG TCT TAC GTA GAG 
6045 
Gly Tyr Tyr Ala Val Met Val Val Val Asp Gl - #y Lys Ser Tyr Val Glu 
# 16950 
- AAA CTC GCT GTA AAG TAA TTCTGTCTTG GACTCGGAGA CT - #TTGTGCAG 
6093 
Lys Leu Ala Val Lys * 
1700 1705 
- ACACTTTTAA TATAGGTCTG TAATTGTCTC AGAGTATGAA TCGATCGCCC GA - #CCTCCTTT 
6153 
- TAAGGAAGTC TGGGCGACTT CGTTTTTATG CCTATTATTC TAATATACTT CT - #GAAACAAT 
6213 
- TTGTTCCAAA AAGTTGCATG AAAAGATTAT CTTACTATCT TTGCACTGCA AA - #AGGGGAGT 
6273 
- TTCCTAAGGT TTTCCCCGGA GTAGTACGGT AATAACGGTG TGGTAGTTCA GC - #TGGTTAGA 
6333 
- ATACCTGCCT GTCACGCAGG GGGTCGCGGG TTCGAGTCCC GTCCATACCG CT - #AAATAGCT 
6393 
- GAAAGATAGG CTATAGGTCA TCTGAAGCAA TTTTAGAAAC GAATCCAAAA GC - #GTCTTAAT 
6453 
- TCCAACGAAT TAAGGCGCTT TTTCTTTGTC GCCACCCCAC ACGTCGGATG AG - #GTTCGGAA 
6513 
- TAGGCGTATA TTCCGTAAAT ATGCCTCCGG TGGTTCCATT TTGGTTACAA AA - #AACAAAGG 
6573 
- GGCTGAAAAT TGTAACCACA GACGACGTTA AGACGATGTT TAGACGATTG AC - #AAATTACT 
6633 
- CTGTTTCAAA ATCATATGTC GAACTTTGTA GCCGTATGGT TACACTAATT TT - #GGAGCAAA 
6693 
- ATGAAGAGTC AATTTCGTTC AGTTTTTTAC TTGCGCAGCA ATTACATCAA CA - #AAGAAGGT 
6753 
- AAAACTCCTG TCCTTATTCG TATTTATCTG AATAAGGAAC GCCTGTCGTT GG - #GTTCGACA 
6813 
- GGGCTGGCTG TTAATCCCAT ACAATGGGAT TCAGAAAAAG AGAAAGTCAA AG - #GACATAGT 
6873 
- GCAGAAGCAC TTGAAGTCAA TCGAAAGATC GAAGAAATCA GGGCTGATAT TC - #TGACCATT 
6933 
- TACAAACGTT TGGAAGTAAC AGTAGATGAT TTGACGCCGG AGAGGATCAA AT - #CGGAATAC 
6993 
- TGCGGACAGA CGGATACATT AAACAGTATA GTGGAACTTT TCGATAAACA TA - #ACGAGGAT 
7053 
- GTCCGGGCCC AGGTGGGAAT CAATAAAACG GCTGCCACTT TACAAAAATA CG - #AAAACAGC 
7113 
- AAACGGCATT TTACCCGATT CCTCAAAGCG AAGTACAACA GAACGGATCT CA - #AATTCTCA 
7173 
- GAGCTTACCC CGTTGGTCAT TCATAACTTT GAGATATATC TGCTGACTGT AG - #CCCATTGT 
7233 
# 7266 CCAA AATCTTGAAG CTT 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1704 am - #ino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- Met Lys Asn Leu Asn Lys Phe Val Ser Ile Al - #a Leu Cys Ser Ser Leu 
# 15 
- Leu Gly Gly Met Ala Phe Ala Gln Gln Thr Gl - #u Leu Gly Arg Asn Pro 
# 30 
- Asn Val Arg Leu Leu Glu Ser Thr Gln Gln Se - #r Val Thr Lys Val Gln 
# 45 
- Phe Arg Met Asp Asn Leu Lys Phe Thr Glu Va - #l Gln Thr Pro Lys Gly 
# 60 
- Ile Gly Gln Val Pro Thr Tyr Thr Glu Gly Va - #l Asn Leu Ser Glu Lys 
# 80 
- Gly Met Pro Thr Leu Pro Ile Leu Ser Arg Se - #r Leu Ala Val Ser Asp 
# 95 
- Thr Arg Glu Met Lys Val Glu Val Val Ser Se - #r Lys Phe Ile Glu Lys 
# 110 
- Lys Asn Val Leu Ile Ala Pro Ser Lys Gly Me - #t Ile Met Arg Asn Glu 
# 125 
- Asp Pro Lys Lys Ile Pro Tyr Val Tyr Gly Ly - #s Ser Tyr Ser Gln Asn 
# 140 
- Lys Phe Phe Pro Gly Glu Ile Ala Thr Leu As - #p Asp Pro Phe Ile Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Arg Asp Val Arg Gly Gln Val Val Asn Phe Al - #a Pro Leu Gln Tyr Asn 
# 175 
- Pro Val Thr Lys Thr Leu Arg Ile Tyr Thr Gl - #u Ile Thr Val Ala Val 
# 190 
- Ser Glu Thr Ser Glu Gln Gly Lys Asn Ile Le - #u Asn Lys Lys Gly Thr 
# 205 
- Phe Ala Gly Phe Glu Asp Thr Tyr Lys Arg Me - #t Phe Met Asn Tyr Glu 
# 220 
- Pro Gly Arg Tyr Thr Pro Val Glu Glu Lys Gl - #n Asn Gly Arg Met Ile 
225 2 - #30 2 - #35 2 - 
#40 
- Val Ile Val Ala Lys Lys Tyr Glu Gly Asp Il - #e Lys Asp Phe Val Asp 
# 255 
- Trp Lys Asn Gln Arg Gly Leu Arg Thr Glu Va - #l Lys Val Ala Glu Asp 
# 270 
- Ile Ala Ser Pro Val Thr Ala Asn Ala Ile Gl - #n Gln Phe Val Lys Gln 
# 285 
- Glu Tyr Glu Lys Glu Gly Asn Asp Leu Thr Ty - #r Val Leu Leu Val Gly 
# 300 
- Asp His Lys Asp Ile Pro Ala Lys Ile Thr Pr - #o Gly Ile Lys Ser Asp 
305 3 - #10 3 - #15 3 - 
#20 
- Gln Val Tyr Gly Gln Ile Val Gly Asn Asp Hi - #s Tyr Asn Glu Val Phe 
# 335 
- Ile Gly Arg Phe Ser Cys Glu Ser Lys Glu As - #p Leu Lys Thr Gln Ile 
# 350 
- Asp Arg Thr Ile His Tyr Glu Arg Asn Ile Th - #r Thr Glu Asp Lys Trp 
# 365 
- Leu Gly Gln Ala Leu Cys Ile Ala Ser Ala Gl - #u Gly Gly Pro Ser Ala 
# 380 
- Asp Asn Gly Glu Ser Asp Ile Gln His Glu As - #n Val Ile Ala Asn Leu 
385 3 - #90 3 - #95 4 - 
#00 
- Leu Thr Gln Tyr Gly Tyr Thr Lys Ile Ile Ly - #s Cys Tyr Asp Pro Gly 
# 415 
- Val Thr Pro Lys Asn Ile Ile Asp Ala Phe As - #n Gly Gly Ile Ser Leu 
# 430 
- Val Asn Tyr Thr Gly His Gly Ser Glu Thr Al - #a Trp Gly Thr Ser His 
# 445 
- Phe Gly Thr Thr His Val Lys Gln Leu Thr As - #n Ser Asn Gln Leu Pro 
# 460 
- Phe Ile Phe Asp Val Ala Cys Val Asn Gly As - #p Phe Leu Phe Ser Met 
465 4 - #70 4 - #75 4 - 
#80 
- Pro Cys Phe Ala Glu Ala Leu Met Arg Ala Gl - #n Lys Asp Gly Lys Pro 
# 495 
- Thr Gly Thr Val Ala Ile Ile Ala Ser Thr Il - #e Asn Gln Ser Trp Ala 
# 510 
- Ser Pro Met Arg Gly Gln Asp Glu Met Asn Gl - #u Ile Leu Cys Glu Lys 
# 525 
- His Pro Asn Asn Ile Lys Arg Thr Phe Gly Gl - #y Val Thr Met Asn Gly 
# 540 
- Met Phe Ala Met Val Glu Lys Tyr Lys Lys As - #p Gly Glu Lys Met Leu 
545 5 - #50 5 - #55 5 - 
#60 
- Asp Thr Trp Thr Val Phe Gly Asp Pro Ser Le - #u Leu Val Arg Thr Leu 
# 575 
- Val Pro Thr Lys Met Gln Val Thr Ala Pro Al - #a Gln Ile Asn Leu Thr 
# 590 
- Asp Ala Ser Val Asn Val Ser Cys Asp Tyr As - #n Gly Ala Ile Ala Thr 
# 605 
- Ile Ser Ala Asn Gly Lys Met Phe Gly Ser Al - #a Val Val Glu Asn Gly 
# 620 
- Thr Ala Thr Ile Asn Leu Thr Gly Leu Thr As - #n Glu Ser Thr Leu Thr 
625 6 - #30 6 - #35 6 - 
#40 
- Leu Thr Val Val Gly Tyr Asn Lys Glu Thr Va - #l Ile Lys Thr Ile Asn 
# 655 
- Thr Asn Gly Glu Pro Asn Pro Tyr Gln Pro Va - #l Ser Asn Leu Thr Ala 
# 670 
- Thr Thr Gln Gly Gln Lys Val Thr Leu Lys Tr - #p Asp Ala Pro Ser Thr 
# 685 
- Lys Thr Asn Ala Thr Thr Asn Thr Ala Arg Se - #r Val Asp Gly Ile Arg 
# 700 
- Glu Leu Val Leu Leu Ser Val Ser Asp Ala Pr - #o Glu Leu Leu Arg Ser 
705 7 - #10 7 - #15 7 - 
#20 
- Gly Gln Ala Glu Ile Val Leu Glu Ala His As - #p Val Trp Asn Asp Gly 
# 735 
- Ser Gly Tyr Gln Ile Leu Leu Asp Ala Asp Hi - #s Asp Gln Tyr Gly Gln 
# 750 
- Val Ile Pro Ser Asp Thr His Thr Leu Trp Pr - #o Asn Cys Ser Val Pro 
# 765 
- Ala Asn Leu Phe Ala Pro Phe Glu Tyr Thr Va - #l Pro Glu Asn Ala Asp 
# 780 
- Pro Ser Cys Ser Pro Thr Asn Met Ile Met As - #p Gly Thr Ala Ser Val 
785 7 - #90 7 - #95 8 - 
#00 
- Asn Ile Pro Ala Gly Thr Tyr Asp Phe Ala Il - #e Ala Ala Pro Gln Ala 
# 815 
- Asn Ala Lys Ile Trp Ile Ala Gly Gln Gly Pr - #o Thr Lys Glu Asp Asp 
# 830 
- Tyr Val Phe Glu Ala Gly Lys Lys Tyr His Ph - #e Leu Met Lys Lys Met 
# 845 
- Gly Ser Gly Asp Gly Thr Glu Leu Thr Ile Se - #r Glu Gly Gly Gly Ser 
# 860 
- Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Th - #r Lys Ile Lys Glu Gly 
865 8 - #70 8 - #75 8 - 
#80 
- Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Va - #l Ala Thr Gly Asn His 
# 895 
- Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gl - #y Val Ser Pro Lys Val 
# 910 
- Cys Lys Asp Val Thr Val Glu Gly Ser Asn Gl - #u Phe Ala Pro Val Gln 
# 925 
- Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Va - #l Thr Leu Lys Trp Asp 
# 940 
- Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro As - #n Pro Asn Pro Asn Pro 
945 9 - #50 9 - #55 9 - 
#60 
- Gly Thr Thr Thr Leu Ser Glu Ser Phe Glu As - #n Gly Ile Pro Ala Ser 
# 975 
- Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly Hi - #s Gly Trp Lys Pro Gly 
# 990 
- Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser As - #n Gly Cys Val Tyr Ser 
# 10050 
- Glu Ser Phe Gly Leu Gly Gly Ile Gly Val Le - #u Thr Pro Asp Asn Tyr 
# 10205 
- Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn Gl - #y Gly Lys Leu Thr Phe 
# 10401030 - # 1035 
- Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Se - #r Glu His Tyr Ala Val 
# 10550 
- Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser As - #n Phe Thr Asn Ala Leu 
# 10705 
- Leu Glu Glu Thr Ile Thr Ala Lys Gly Val Ar - #g Ser Pro Glu Ala Ile 
# 10850 
- Arg Gly Arg Ile Gln Gly Thr Trp Arg Gln Ly - #s Thr Val Asp Leu Pro 
# 11005 
- Ala Gly Thr Lys Tyr Val Ala Phe Arg His Ph - #e Gln Ser Thr Asp Met 
# 11201110 - # 1115 
- Phe Tyr Ile Asp Leu Asp Glu Val Glu Ile Ly - #s Ala Asn Gly Lys Arg 
# 11350 
- Ala Asp Phe Thr Glu Thr Phe Glu Ser Ser Th - #r His Gly Glu Ala Pro 
# 11505 
- Ala Glu Trp Thr Thr Ile Asp Ala Asp Gly As - #p Gly Gln Gly Trp Leu 
# 11650 
- Cys Leu Ser Ser Gly Gln Leu Asp Trp Leu Th - #r Ala His Gly Gly Thr 
# 11805 
- Asn Val Val Ala Ser Phe Ser Trp Asn Gly Me - #t Ala Leu Asn Pro Asp 
# 12001190 - # 1195 
- Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Al - #a Thr Lys Val Lys Tyr 
# 12150 
- Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly As - #p His Tyr Ala Val Met 
# 12305 
- Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Ph - #e Thr Val Val Phe Glu 
# 12450 
- Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly Al - #a Arg Phe Gly Leu Ser 
# 12605 
- Thr Glu Ala Asn Gly Ala Lys Pro Gln Ser Va - #l Trp Ile Glu Arg Thr 
# 12801270 - # 1275 
- Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Al - #a Phe Arg His Tyr Asn 
# 12950 
- Cys Ser Asp Leu Asn Tyr Ile Leu Leu Asp As - #p Ile Gln Phe Thr Met 
# 13105 
- Gly Gly Ser Pro Thr Pro Thr Asp Tyr Thr Ty - #r Thr Val Tyr Arg Asp 
# 13250 
- Gly Thr Lys Ile Lys Glu Gly Leu Thr Glu Th - #r Thr Phe Glu Glu Asp 
# 13405 
- Gly Val Ala Thr Gly Asn His Glu Tyr Cys Va - #l Glu Val Lys Tyr Thr 
# 13601350 - # 1355 
- Ala Gly Val Ser Pro Lys Glu Cys Val Asn Va - #l Thr Ile Asn Pro Thr 
# 13750 
- Gln Phe Asn Pro Val Lys Asn Leu Lys Ala Gl - #n Pro Asp Gly Gly Asp 
# 13905 
- Val Val Leu Lys Trp Glu Ala Pro Ser Ala Ly - #s Lys Thr Glu Gly Ser 
# 14050 
- Arg Glu Val Lys Arg Ile Gly Asp Gly Leu Ph - #e Val Thr Ile Glu Pro 
# 14205 
- Ala Asn Asp Val Arg Ala Asn Glu Ala Lys Va - #l Val Leu Ala Ala Asp 
# 14401430 - # 1435 
- Asn Val Trp Gly Asp Asn Thr Gly Tyr Gln Ph - #e Leu Leu Asp Ala Asp 
# 14550 
- His Asn Thr Phe Gly Ser Val Ile Pro Ala Th - #r Gly Pro Leu Phe Thr 
# 14705 
- Gly Thr Ala Ser Ser Asn Leu Tyr Ser Ala As - #n Phe Glu Tyr Leu Ile 
# 14850 
- Pro Ala Asn Ala Asp Pro Val Val Thr Thr Gl - #n Asn Ile Ile Val Thr 
# 15005 
- Gly Gln Gly Glu Val Val Ile Pro Gly Gly Va - #l Tyr Asp Tyr Cys Ile 
# 15201510 - # 1515 
- Thr Asn Pro Glu Pro Ala Ser Gly Lys Met Tr - #p Ile Ala Gly Asp Gly 
# 15350 
- Gly Asn Gln Pro Ala Arg Tyr Asp Asp Phe Th - #r Phe Glu Ala Gly Lys 
# 15505 
- Lys Tyr Thr Phe Thr Met Arg Arg Ala Gly Me - #t Gly Asp Gly Thr Asp 
# 15650 
- Met Glu Val Glu Asp Asp Ser Pro Ala Ser Ty - #r Thr Tyr Thr Val Tyr 
# 15805 
- Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu Th - #r Glu Thr Thr Tyr Arg 
# 16001590 - # 1595 
- Asp Ala Gly Met Ser Ala Gln Ser His Glu Ty - #r Cys Val Glu Val Lys 
# 16150 
- Tyr Ala Ala Gly Val Ser Pro Lys Val Cys Va - #l Asp Tyr Ile Pro Asp 
# 16305 
- Gly Val Ala Asp Val Thr Ala Gln Lys Pro Ty - #r Thr Leu Thr Val Val 
# 16450 
- Gly Lys Thr Ile Thr Val Thr Cys Gln Gly Gl - #u Ala Met Ile Tyr Asp 
# 16605 
- Met Asn Gly Arg Arg Leu Ala Ala Gly Arg As - #n Thr Val Val Tyr Thr 
# 16801670 - # 1675 
- Ala Gln Gly Gly Tyr Tyr Ala Val Met Val Va - #l Val Asp Gly Lys Ser 
# 16950 
- Tyr Val Glu Lys Leu Ala Val Lys 
# 1705 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 3561 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1336..2862 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- CTGCAGAAGT TCACTCTTTC GCATATAGTG ACCCTCTTTT CTCTCAGCAT AA - #TGGCACCT 
60 
- ATCATATCAG TAAGGGGCGT ATTGTCTTTT CGAACAATGT ACAGCCCGAG AA - #CTCTTTAC 
120 
- TTCCACATCA CACCCCCGAC TCCTTAGTCA AGGATCTTTT TTCCGCTTTC CC - #CTCCGCTC 
180 
- TCTTCCTCAT GCTGGACTGA CTTAACCTTG GTCTGCTCTA CTTTTCGGTT GT - #AAATACAT 
240 
- GCAACACAAT AACTTTTTTA AGTGTTGTTA GACAACACTT TTACAAGACT CT - #GACTTTTA 
300 
- ATGAGGTGGA GCATGAACCT TTTCCTCTTT CATCTTCTCC TTCAGATTAC AG - #TCAATATT 
360 
- TTGGCAAAAG GCTAATTGAC AGCCTTTTAT AAGGGTTAAT CCCTTGTCGC TT - #ATATTGAA 
420 
- AACATGTTCT TTACGATCCG ATACTCTTCT TAAATCGAAA TTTTTCTCTA AA - #TTGCGCCG 
480 
- CAACAAAACT CCTTGAGAAA AGTACCAATA GAAATAGAAG GTAGCATTTT GC - #CTTTAAAT 
540 
- TCCTTTTCTT TTCTTGGATT GTTCTTGAAA TGAATCTTAT TTGTGGATCT TT - #TTTGTTTT 
600 
- TTTTAACCCG GCCGTGGTTC TCTGAATCAC GACCATAAAT TGTTTTAAAG TA - #TGAGGAAA 
660 
- TTATTATTGC TGATCGCGGC GTCCCTTTTG GGAGTTGGTC TTTACGCCCA AA - #ACGCCAAG 
720 
- ATTAAGCTTG ATGCTCCGAC TACTCGAACG ACATGCACGA ACAATAGCTT CA - #AGCAGTTC 
780 
- GATGCAAGCT TTTCGTTCAA TGAAGTCGAG CTGAGAAAGG TGGAGACCAA AG - #GTGGTACT 
840 
- TTCGCCTCAG TGTCAATTCC GGGTGCATTC CCGACCGGTG AGGTTGGTTC TC - #CCGAAGTG 
900 
- CCAGCAGTTA GGAAGTTGAT TGCTGTGCCT GTCGAAGCCA GACCTGTTGT TC - #GCGTGAAA 
960 
- AGTTTTACCG AGCAAGTTTA CTGTCTGAAC CAATACGGTT CCGAAAAGCT CA - #TGCCACAT 
1020 
- CAACCCTCTA TGAGCAAGAG TGATGATCCC GAAAAGCTTC CCTTCGCTTA CA - #ATGCTGCT 
1080 
- GCTTATGCAC GCAAAGGTTT TGTCGGACAA GAACTGACCC AAGTAGAAAT GT - #TGGGGACA 
1140 
- ATGCGTGGTG TTCGCATTGC AGCTCTTACC ATTAATCCTG TTCAGTATGA TG - #TAGTTGCA 
1200 
- AACCAATTGA AGGTTAGAAA CAACATCGAA ATTGAAGTAA GCTTTCAGGG AG - #CTGATGAA 
1260 
- GTAGCTACAC AACGTTTGTA TGATGCTTCT TTTAGCCCTT ATTTCGAAAC AG - #CTTATAAA 
1320 
#GAC TTG TAT AAT ACG 1371AT ACA GAT CAT GGC 
#Val Tyr Thr Asp His Gly Asp Leu Tyr Asn T - #hr 
# 10 
- CCG GTT CGT ATG CTT GTT GTT GCA GGT GCA AA - #A TTC AAA GAA GCT CTC 
1419 
Pro Val Arg Met Leu Val Val Ala Gly Ala Ly - #s Phe Lys Glu Ala Leu 
# 25 
- AAG CCT TGG CTC ACT TGG AAG GCT CAA AAG GG - #C TTC TAT CTG GAT GTG 
1467 
Lys Pro Trp Leu Thr Trp Lys Ala Gln Lys Gl - #y Phe Tyr Leu Asp Val 
# 40 
- CAT TAC ACA GAC GAA GCT GAA GTA GGA ACG AC - #A AAC GCC TCT ATC AAG 
1515 
His Tyr Thr Asp Glu Ala Glu Val Gly Thr Th - #r Asn Ala Ser Ile Lys 
# 60 
- GCA TTT ATT CAC AAG AAA TAC AAT GAT GGA TT - #G GCA GCT ACT GCT GCT 
1563 
Ala Phe Ile His Lys Lys Tyr Asn Asp Gly Le - #u Ala Ala Thr Ala Ala 
# 75 
- CCG GTC TTC TTG GCT TTG GTT GGT GAC ACT GA - #C GTT ATT AGC GGA GAA 
1611 
Pro Val Phe Leu Ala Leu Val Gly Asp Thr As - #p Val Ile Ser Gly Glu 
# 90 
- AAA GGA AAG AAA ACA AAA AAA GTT ACC GAC TT - #G TAT TAC ACT GCA GTC 
1659 
Lys Gly Lys Lys Thr Lys Lys Val Thr Asp Le - #u Tyr Tyr Thr Ala Val 
# 105 
- GAT GGC GAC TAT TTC CCT GAA ATG TAT ACT TT - #C CGT ATG TCT GCT TCT 
1707 
Asp Gly Asp Tyr Phe Pro Glu Met Tyr Thr Ph - #e Arg Met Ser Ala Ser 
# 120 
- TCC CCA GAA GAA CTG ACG AAC ATC ATT GAT AA - #G GTA TTG ATG TAT GAA 
1755 
Ser Pro Glu Glu Leu Thr Asn Ile Ile Asp Ly - #s Val Leu Met Tyr Glu 
125 1 - #30 1 - #35 1 - 
#40 
- AAG GCT ACT ATG CCG GAT AAG AGC TAT TTG GA - #A AAG GCC CTC TTG ATT 
1803 
Lys Ala Thr Met Pro Asp Lys Ser Tyr Leu Gl - #u Lys Ala Leu Leu Ile 
# 155 
- GCC GGT GCT GAC TCC TAC TGG AAT CCT AAG AT - #A GGC CAG CAA ACC ATC 
1851 
Ala Gly Ala Asp Ser Tyr Trp Asn Pro Lys Il - #e Gly Gln Gln Thr Ile 
# 170 
- AAA TAT GCT GTA CAG TAT TAC TAC AAT CAA GA - #T CAT GGC TAT ACA GAT 
1899 
Lys Tyr Ala Val Gln Tyr Tyr Tyr Asn Gln As - #p His Gly Tyr Thr Asp 
# 185 
- GTG TAC ACT TAC CCT AAA GCT CCT TAT ACA GG - #C TGC TAT AGT CAC TTG 
1947 
Val Tyr Thr Tyr Pro Lys Ala Pro Tyr Thr Gl - #y Cys Tyr Ser His Leu 
# 200 
- AAT ACC GGT GTC GGC TTT GCC AAC TAT ACA GT - #G CAT GGA TCT GAG ACA 
1995 
Asn Thr Gly Val Gly Phe Ala Asn Tyr Thr Va - #l His Gly Ser Glu Thr 
205 2 - #10 2 - #15 2 - 
#20 
- TCA TGG GCA GAT CCG TCC GTG ACC GCC ACT CA - #A GTG AAA GCA CTC ACA 
2043 
Ser Trp Ala Asp Pro Ser Val Thr Ala Thr Gl - #n Val Lys Ala Leu Thr 
# 235 
- AAT AAG AAC AAA TAC TTC TTA GCT ATT GGG AA - #C TGC TGT GTT ACA GCT 
2091 
Asn Lys Asn Lys Tyr Phe Leu Ala Ile Gly As - #n Cys Cys Val Thr Ala 
# 250 
- CAA TTC GAT TAT CCA CAG CCT TGC TTT GGA GA - #G GTA ATG ACT CGT GTC 
2139 
Gln Phe Asp Tyr Pro Gln Pro Cys Phe Gly Gl - #u Val Met Thr Arg Val 
# 265 
- AAG GAG AAA GGT GCT TAT GCC TAT ATC GGT TC - #A TCT CCA AAT TCT TAT 
2187 
Lys Glu Lys Gly Ala Tyr Ala Tyr Ile Gly Se - #r Ser Pro Asn Ser Tyr 
# 280 
- TGG GGC GAG GAC TAC TAT TGG AGT GTG GGT GC - #T AAT GCA GTA TTT GGT 
2235 
Trp Gly Glu Asp Tyr Tyr Trp Ser Val Gly Al - #a Asn Ala Val Phe Gly 
285 2 - #90 2 - #95 3 - 
#00 
- GTT CAG CCT ACT TTT GAA GGT ACG TCT ATG GG - #T TCT TAT GAT GCT ACA 
2283 
Val Gln Pro Thr Phe Glu Gly Thr Ser Met Gl - #y Ser Tyr Asp Ala Thr 
# 315 
- TTC TTG GAA GAT TCG TAC AAC ACA GTG AAC TC - #T ATT ATG TGG GCA GGT 
2331 
Phe Leu Glu Asp Ser Tyr Asn Thr Val Asn Se - #r Ile Met Trp Ala Gly 
# 330 
- AAT CTT GCT GCT ACT CAT GCC GAA AAT ATC GG - #C AAT GTT ACC CAT ATC 
2379 
Asn Leu Ala Ala Thr His Ala Glu Asn Ile Gl - #y Asn Val Thr His Ile 
# 345 
- GGT GCT CAT TAC TAT TGG GAA GCT TAT CAT GT - #C CTT GGC GAT GGT TCG 
2427 
Gly Ala His Tyr Tyr Trp Glu Ala Tyr His Va - #l Leu Gly Asp Gly Ser 
# 360 
- GTT ATG CCT TAT CGT GCA ATG CCT AAG ACC AA - #T ACT TAT ACG CTT CCT 
2475 
Val Met Pro Tyr Arg Ala Met Pro Lys Thr As - #n Thr Tyr Thr Leu Pro 
365 3 - #70 3 - #75 3 - 
#80 
- GCT TCT CTG CCT CAG AAT CAG GCT TCT TAT AG - #C ATT CAG GCT TCT GCC 
2523 
Ala Ser Leu Pro Gln Asn Gln Ala Ser Tyr Se - #r Ile Gln Ala Ser Ala 
# 395 
- GGT TCT TAC GTA GCT ATT TCT AAA GAT GGA GT - #T TTG TAT GGA ACA GGT 
2571 
Gly Ser Tyr Val Ala Ile Ser Lys Asp Gly Va - #l Leu Tyr Gly Thr Gly 
# 410 
- GTT GCT AAT GCC AGC GGT GTT GCG ACT GTG AA - #T ATG ACT AAG CAG ATT 
2619 
Val Ala Asn Ala Ser Gly Val Ala Thr Val As - #n Met Thr Lys Gln Ile 
# 425 
- ACG GAA AAT GGT AAT TAT GAT GTA GTT ATC AC - #T CGC TCT AAT TAT CTT 
2667 
Thr Glu Asn Gly Asn Tyr Asp Val Val Ile Th - #r Arg Ser Asn Tyr Leu 
# 440 
- CCT GTG ATC AAG GAA ATT CAG GCA GGA GAG CC - #T AGC CCC TAC CAG CCT 
2715 
Pro Val Ile Lys Glu Ile Gln Ala Gly Glu Pr - #o Ser Pro Tyr Gln Pro 
445 4 - #50 4 - #55 4 - 
#60 
- GTT TCC AAC TTG ACT GCT ACA ACG CAG GGT CA - #G AAA GTA ACG CTC AAG 
2763 
Val Ser Asn Leu Thr Ala Thr Thr Gln Gly Gl - #n Lys Val Thr Leu Lys 
# 475 
- TGG GAT GCC CCG AGC GCA AAG AAG GCA GAA GG - #T TCC CGT GAA GTA AAA 
2811 
Trp Asp Ala Pro Ser Ala Lys Lys Ala Glu Gl - #y Ser Arg Glu Val Lys 
# 490 
- CGG ATC GGA GAC GGT CTT TTC GTT ACG ATC GA - #A CCT GCA AAC GAT GTA 
2859 
Arg Ile Gly Asp Gly Leu Phe Val Thr Ile Gl - #u Pro Ala Asn Asp Val 
# 505 
- CGT GCCAACGAAG CCAAGGTTGT GCTCGCAGCA GACAACGTAT GGGGAGACA - #A 
2912 
Arg 
- TACGGGTTAC CAGTTCTTGT TGGATGCCGA TCACAATACA TTCGGAAGTG TC - #ATTCCGGC 
2972 
- AACCGGTCCT CTCTTTACCG GAAGAGCTTC TTCCAATCTT TACAGTGCGA AC - #TTCGAGTA 
3032 
- TTTGATCCCG GCCAATGCCG ATCCTGTTGT TACTACACAG AATATTATCG TT - #ACAGGACA 
3092 
- GGGTGAAGTT GTAATCCCCG GTGGTGTTTA CGACTATTGC ATTACGAAGC CG - #GAACCTGC 
3152 
- ATCCGGAAAG ATGTGGATCG CAGGAGATGG AGGCAACCAG CCTGCACGTT AT - #GACGATTT 
3212 
- CACATTCGAA GCAGGCAAGA AGTACACCTT CACGATGCGT CGCGCCGGAA TG - #GGAGATGG 
3272 
- AACTGATATG GAAGTCGAAG ACGATTCACC TGCAAGCTAT ACCTACACGG TG - #TATCGTGA 
3332 
- CGGCACGAAG ATCAAGGAAG GTCTGACGGC TACGACATTC GAAGAAGACG GT - #GTAGCTGC 
3392 
- AGGCAATCAT GAGTATTGCG TGGAAGTTAA GTACACAGCC GGCGTATCTC CG - #AAGGTATG 
3452 
- TAAAGACGTT ACGGTAGAAG GATCCAATGA ATTTGCTCCT GTACAGAACC TG - #ACCGGTAG 
3512 
# 3561AGTAA CGCTTAAGTG GGATGCACCT AATGGTACC 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 509 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- Asp Val Tyr Thr Asp His Gly Asp Leu Tyr As - #n Thr Pro Val Arg Met 
# 15 
- Leu Val Val Ala Gly Ala Lys Phe Lys Glu Al - #a Leu Lys Pro Trp Leu 
# 30 
- Thr Trp Lys Ala Gln Lys Gly Phe Tyr Leu As - #p Val His Tyr Thr Asp 
# 45 
- Glu Ala Glu Val Gly Thr Thr Asn Ala Ser Il - #e Lys Ala Phe Ile His 
# 60 
- Lys Lys Tyr Asn Asp Gly Leu Ala Ala Thr Al - #a Ala Pro Val Phe Leu 
# 80 
- Ala Leu Val Gly Asp Thr Asp Val Ile Ser Gl - #y Glu Lys Gly Lys Lys 
# 95 
- Thr Lys Lys Val Thr Asp Leu Tyr Tyr Thr Al - #a Val Asp Gly Asp Tyr 
# 110 
- Phe Pro Glu Met Tyr Thr Phe Arg Met Ser Al - #a Ser Ser Pro Glu Glu 
# 125 
- Leu Thr Asn Ile Ile Asp Lys Val Leu Met Ty - #r Glu Lys Ala Thr Met 
# 140 
- Pro Asp Lys Ser Tyr Leu Glu Lys Ala Leu Le - #u Ile Ala Gly Ala Asp 
145 1 - #50 1 - #55 1 - 
#60 
- Ser Tyr Trp Asn Pro Lys Ile Gly Gln Gln Th - #r Ile Lys Tyr Ala Val 
# 175 
- Gln Tyr Tyr Tyr Asn Gln Asp His Gly Tyr Th - #r Asp Val Tyr Thr Tyr 
# 190 
- Pro Lys Ala Pro Tyr Thr Gly Cys Tyr Ser Hi - #s Leu Asn Thr Gly Val 
# 205 
- Gly Phe Ala Asn Tyr Thr Val His Gly Ser Gl - #u Thr Ser Trp Ala Asp 
# 220 
- Pro Ser Val Thr Ala Thr Gln Val Lys Ala Le - #u Thr Asn Lys Asn Lys 
225 2 - #30 2 - #35 2 - 
#40 
- Tyr Phe Leu Ala Ile Gly Asn Cys Cys Val Th - #r Ala Gln Phe Asp Tyr 
# 255 
- Pro Gln Pro Cys Phe Gly Glu Val Met Thr Ar - #g Val Lys Glu Lys Gly 
# 270 
- Ala Tyr Ala Tyr Ile Gly Ser Ser Pro Asn Se - #r Tyr Trp Gly Glu Asp 
# 285 
- Tyr Tyr Trp Ser Val Gly Ala Asn Ala Val Ph - #e Gly Val Gln Pro Thr 
# 300 
- Phe Glu Gly Thr Ser Met Gly Ser Tyr Asp Al - #a Thr Phe Leu Glu Asp 
305 3 - #10 3 - #15 3 - 
#20 
- Ser Tyr Asn Thr Val Asn Ser Ile Met Trp Al - #a Gly Asn Leu Ala Ala 
# 335 
- Thr His Ala Glu Asn Ile Gly Asn Val Thr Hi - #s Ile Gly Ala His Tyr 
# 350 
- Tyr Trp Glu Ala Tyr His Val Leu Gly Asp Gl - #y Ser Val Met Pro Tyr 
# 365 
- Arg Ala Met Pro Lys Thr Asn Thr Tyr Thr Le - #u Pro Ala Ser Leu Pro 
# 380 
- Gln Asn Gln Ala Ser Tyr Ser Ile Gln Ala Se - #r Ala Gly Ser Tyr Val 
385 3 - #90 3 - #95 4 - 
#00 
- Ala Ile Ser Lys Asp Gly Val Leu Tyr Gly Th - #r Gly Val Ala Asn Ala 
# 415 
- Ser Gly Val Ala Thr Val Asn Met Thr Lys Gl - #n Ile Thr Glu Asn Gly 
# 430 
- Asn Tyr Asp Val Val Ile Thr Arg Ser Asn Ty - #r Leu Pro Val Ile Lys 
# 445 
- Glu Ile Gln Ala Gly Glu Pro Ser Pro Tyr Gl - #n Pro Val Ser Asn Leu 
# 460 
- Thr Ala Thr Thr Gln Gly Gln Lys Val Thr Le - #u Lys Trp Asp Ala Pro 
465 4 - #70 4 - #75 4 - 
#80 
- Ser Ala Lys Lys Ala Glu Gly Ser Arg Glu Va - #l Lys Arg Ile Gly Asp 
# 495 
- Gly Leu Phe Val Thr Ile Glu Pro Ala Asn As - #p Val Arg 
# 505 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 46 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Lys Il - #e Lys Glu Gly Leu Thr 
# 15 
- Ala Thr Thr Glu Asp Asp Gly Val Ala Thr Gl - #y Asn His Glu Tyr Cys 
# 30 
- Val Glu Lys Tyr Thr Ala Gly Ser Val Ser Pr - #o Lys Val Cys 
# 45 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 20 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (v) FRAGMENT TYPE: N-terminal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Ar - #g Met Ile Val Ile Val 
# 15 
- Ala Lys Lys Tyr 
20 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 30 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- Gln Leu Pro Phe Ile Phe Asp Val Ala Cys Va - #l Asn Gly Asp Phe Leu 
# 15 
- Phe Ser Met Pro Cys Phe Ala Glu Ala Leu Me - #t Arg Ala Gln 
# 30 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 31 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- Gly Glu Pro Asn Pro Tyr Gln Pro Val Ser As - #n Leu Thr Ala Thr Thr 
# 15 
- Gln Gly Gln Lys Val Thr Leu Lys Trp Asp Al - #a Pro Ser Thr Lys 
# 30 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 30 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- Asp Gln Ala Asn Phe Leu Gln Cys Val Gly Se - #r Leu Met Cys Arg Leu 
# 15 
- Asp Phe Phe Phe Glu Ala Val Met Pro Ile Ph - #e Pro Ala Ala 
# 30 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 25 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- Gly Asn His Glu Tyr Cys Val Glu Val Lys Ty - #r Thr Ala Gly Val Ser 
# 15 
- Pro Lys Val Cys Lys Asp Val Thr Val 
# 25 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 25 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- Ala His Glu Lys Thr Tyr Pro Val Glu Asp Va - #l Asn Cys Ser Tyr Val 
# 15 
- Lys Thr Val Cys Val Gly Gly Lys Val 
# 25 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 20 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- Arg Met Phe Met Asn Tyr Glu Pro Gly Arg Ty - #r Thr Pro Val Glu Glu 
# 15 
- Lys Gln Asn Gly 
20 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 20 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- Thr Phe Ala Gly Phe Glu Asp Thr Tyr Lys Ar - #g Met Phe Met Asn Tyr 
# 15 
- Glu Pro Gly Arg 
20 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 49 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Th - #r Lys Ile Lys Glu Gly 
# 15 
- Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Va - #l Ala Thr Gly Asn Met 
# 30 
- Glu Tyr Cys Val Cys Val Lys Tyr Thr Ala Gl - #y Val Ser Pro Lys Val 
# 45 
- Cys 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 25 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Ly - #s Ile Lys Glu Gly Leu 
# 15 
- Thr Ala Thr Thr Phe Glu Glu Asp Gly 
# 25 
- (2) INFORMATION FOR SEQ ID NO:20: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 24 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu Th - #r Ala Thr Thr Phe Glu 
# 15 
- Glu Asp Gly Val Ala Thr Gly Asn 
20 
- (2) INFORMATION FOR SEQ ID NO:21: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 23 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- Lys Ile Lys Glu Gly Leu Thr Ala Thr Thr Ph - #e Glu Glu Asp Gly Val 
# 15 
- Ala Thr Gly Asn His Glu Tyr 
20 
- (2) INFORMATION FOR SEQ ID NO:22: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 4 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- Phe Glu Glu Asp 
1 
- (2) INFORMATION FOR SEQ ID NO:23: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 28 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- Lys Trp Asp Ala Pro Asn Gly Thr Pro Asn Pr - #o Asn Pro Asn Pro Asn 
# 15 
- Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu Se - #r Glu 
# 25 
- (2) INFORMATION FOR SEQ ID NO:24: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 20 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- Tyr Thr Pro Val Glu Glu Lys Glu Asn Gly Ar - #g Met Ile Val Ile Val 
# 15 
- Ala Lys Lys Tyr 
20 
__________________________________________________________________________