Inhibitor of C5a-mediated chemotaxis

Polypeptides are disclosed which inhibit the binding of the C5a chemotaxin to polymorphonuclear leukocytes by cleaving a six amino acid peptide from the carboxy-terminus of the C5a chemotaxin. The polypeptides can be isolated from virulent strains of group A streptococci, s. pyogenes, by enzymatic or detergent extraction.

The acute inflammatory response depends on the attraction of phagocytic 
polymorphonuclear leukocytes (PMN) to the site of microbial invasion by a 
chemotactic stimulus. The C5a chemotaxin is pivotal to the effectuation of 
this response in humans. C5a is a peptide derived from the fifth component 
of serum complement during complement activation by an invading microbial 
pathogen. 
C5a is known to mediate various pathological conditions, chronic 
inflammation, acute pulmonary disorders and even the metastatic spread of 
cancerous tumors. C5a is chemotactic for neutrophils, monocytes, 
macrophages, eosinophils and basophils. Thus, this serum factor is an 
important attractant to leukocytes and is crucial to their accumulation in 
vivo at sites of immunologic injury. Leukocytes accumulated at a site of 
inflammation release granular contents, various hydrolytic enzymes and 
other toxic components into the extracellular spaces. As a result, the 
surrounding tissue is damaged. Numerous chronic inflammatory diseases are 
thought to involve the abberant presence of C5a in tissue. Rheumatoid 
arthritis, osteoarthritis and psoriasis are a few examples. The lung is 
particularly vulnerable; excess c5a in circulation or in the lung can 
result in aggregation and migration of leukocytes into this organ. This 
can lead to microvascular occlusion, endothelial damage and subsequent 
edema. In J. Amer. Med. Soc., 244, 199 (1980), Hammerschmidt suggested 
that noncardiac pulmonary edema associated with transfusion and 
hemodialysis depend on an abberant increase in circulating C5a. 
The in vivo or pharmacologic control of inflammation is presumed to be 
highly dependent on the modulation of chemotaxis. There are three levels 
at which chemotactic inhibition can occur: (1) suppression of the 
leukocytic response to chemotactic stimuli, (2) prevention of chemotaxin 
generation, and (3) inactivation of the chemotaxins. Most therapeutically 
useful drugs act on leukocytes to alter their responsiveness to 
chemotactic stimuli. Likewise, a number of bacterial products are known to 
suppress the directed and random migration of polymorphonuclear 
leukocytes. These compounds include staphylococcal cell wall mucopeptide, 
C. perfringens O toxin, and lipopolysaccharides. Various group A 
streptococcal components also exert cell-directed inhibitory effects. 
These include streptolysin O, streptokinasestreptodornase, and 
lipoteichoic acid. However, these bacterial products are highly toxic and 
lack specificity. 
Studies concerning the inactivation of chemotactic factors have primarily 
concentrated on host mechanisms of self-regulation. Leukocytes release 
several enzymes which inactivate chemotaxins. These include the 
myeloperoxidase-hydrogen peroxide-halide system as well as certain 
azurophilic granular enzymes. Ward et al., in J. Immunol., 111, 177 (1973) 
have described a serum factor (CFI) which irreversibly inactivates C5a. 
Johnson et al., in J. Clin. Invest., 59, 951 (1977) have demonstrated that 
partially-purified CFI is a potent inhibitor of inflammatory reactions 
triggered by immune complexes in rat models, and have suggested that CFI 
is an important regulator of inflammation. However, the mechanism of 
action of CFI remains unknown, and the potential use of CFI as an 
anti-inflammatory agent is limited by its resistance to purification and 
the minuscule quantities present in serum. 
Other than non-specific proteolytic enzymes, there are no known chemical 
agents which can specifically alter the chemotactic properties of C5a. 
Therefore, the only drugs of therapeutic value which have thus far been 
developed are those which alter the chemotactic responsiveness of 
inflammatory cells. For example, see S. Klebanoff and R. Clark, in The 
Neutrophil: Function and Clinical Disorder, Elsevier, North-Holland 
Biochemical Press (1978) at pages 73-160. 
In 1983, D. E. Wexler et al., in Infection and Immunity, 39, 239 (1983) 
reported that the incubation of human serum with M.sup.+ group A 
streptococci did not elicit serum chemotactic activity, even though 
complement was activated to completion. Treatment of the bacteria with 
trypsin resulted in the release of the inhibitory molecule from the cell 
surface. The resistance of the chemotactic factor inactivator to pepsin 
and trypsin indicated that the protease-sensitive M protein was not 
involved, although the M protein contributes to the ability of group A 
streptococci to resist host phagocytic defenses. The inhibitory species 
was not structurally characterized and the chemotactic target as well as 
the mechanism of inactivation were not elucidated. 
Therefore, the need exists for compositions which can inhibit C5a-mediated 
chemotaxis. Compositions which can transiently regulate the systemic or 
localized reactivity of C5a have the potential to limit pathophysiologics 
such as arthritis and other C5a-mediated inflammatory reactions. 
BRIEF DESCRIPTION OF THE INVENTION 
The present invention is directed to a composition of matter which is a 
potent inhibitor of the biologic activity of the C5a chemotaxin. The 
present invention is also directed to a method for the inhibition of the 
binding of the C5a chemotaxin to leukocytes comprising contacting the 
chemotaxin with an effective amount of the composition of the invention. 
The present composition comprises a substantially-pure polypeptide which 
incorporates all or a portion of the amino acid sequence of a protein 
which is expressed in surface-bound form by virulent strains of group A 
streptococci (streptococcus pyogenes), wherein said polypeptide blocks the 
binding of C5a chemotaxin to polymorphonuclear leukocytes by cleaving a 
six amino acid peptide from the carboxy-terminus of the C5a chemotaxin. 
This streptococcal chemotactic factor inactivator polypeptide (hereinafter 
"SCFI") can be extracted from virulent strains of group A streptococci by 
chemical or enzymatic methods. Therefore, as used herein, the term 
"substantially pure" is intended to mean that SCFI has been isolated from 
its natural association with the bacterial cellular surface and the other 
factors associated therewith, such as protein M, streptococcal proteinase 
and the like. 
Although the molecular weight of the native SCFI protein has not been 
established, the procedures of the present invention readily afford a 
number of polypeptides which attack the carboxy-terminal region of C5a to 
produce a molecule that lacks the capacity for functional interaction with 
receptors on polymorphonuclear leukocytes. It is believed that these 
biologically-active polypeptides are fragments of the native SCFI protein. 
Specific embodiments of SCFI include the 135 and 137 Kd (Kilodalton) 
molecular weight polypeptides derived by the controlled extraction of 
group A streptococci with trypsin or by the extraction of group A 
streptococci with a nonionic detergent, and the 120 Kd and 124 Kd 
polypeptides derived by the extraction of group A streptococci with a 
zwitterionic detergent. 
The biological and chemical properties of SCFI are summarized in Table I, 
below. 
TABLE 1 
______________________________________ 
Biological and Biochemical Properties of SCFI 
______________________________________ 
Location: Outer surface of Group A strepto- 
cocci (S. pyogenes).sup.1 
Distribution: Specific to S. pyogenes, not 
significantly associated with 
group B, C, D, F, or G strepto- 
cocci 
Activity: Specific endopeptidolytic cleavage 
of complement C5a protein 
Chemical nature: 
protein 
Adsorption maximum: 
276 nm 
Identifiable: Polyclonal hyperimmune rabbit 
serum 
Extraction: Mild trypsin, muralytic enzyme and 
detergents 
______________________________________ 
.sup.1 Production by M6, M49, M12, M11 and M2 serotypes confirmed (P. 
Cleary et al., in Recent Advances in Streptococci and Streptococcal 
Diseases, Y. Kinura et al., eds., Reed Book Ltd., England (1985) at pages 
179-180). 
Based on ELISA assays, immunodiffusion analysis and neutralization studies 
with hyperimmune sera, it is believed that SCFI is antigenically identical 
for all strains and serotypes of group A streptococci.

DETAILED DESCRIPTION OF THE INVENTION 
The isolation and characterization of SCFI will be further described by 
reference to the following detailed examples. 
EXAMPLE I - Trypsin Extraction 
A. Materials and Methods 
1. Streptococcal strain and growth. Strain CS101, virulent M49, T14 group A 
streptococci were used as the source of SCFI. Bacteria were grown in 
Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplemented with 
2% neopeptone (Difco). One liter of exponential-phase culture was 
inoculated into 25 liters of growth medium and incubated overnight at 
37.degree. C. Bacteria were harvested by continuous-flow centrifugation, 
and the packed pellet was stored at -20.degree. C. until use. 
2. Trypsin extraction. Frozen pellets from four batch cultures of 
streptococci, totaling 160 g (wet weight), were thawed and washed twice in 
Sorenson 0.067M phosphate buffer (pH 8.0). Washed bacteria were suspended 
in 770 ml of the same buffer and incubated with trypsin (type II-0); Sigma 
Chemical Co., St. Louis, MO, hereinafter "Sigma")(100 .mu.g/ml final 
concentration) at 37.degree. C. for 90 min with gentle stirring. The 
bacteria were then pelleted by centrifugation at 13,000.times.g for 10 min 
at 4.degree. C. After discarding the pellet, the supernatant was 
sterilized with a 0.45-.mu.m nitrocellulose membrane (Millipore Corp., 
Bedford. Mass.). 
3. Ammonium sulfate fractionation. Trypsin extract was precipitated at 
4.degree. C. by addition of crystalline ammonium sulfate to 55% 
saturation. After 30 min, the mixture was centrifuged at 13,000.times.g 
for 10 min, and the pellet was discarded. The supernatant was adjusted to 
90% saturation with ammonium sulfate, and after 30 min, the resulting 
precipitate was collected by centrifugation. The pellet was suspended in 
50 ml of 0.01M sodium phosphate buffer (ph 6.7) containing 30% ammonium 
sulfate. 
4. Hydrophobic interaction chromatography. The salt-fractionated trypsin 
extract was loaded onto a column (2.1 by 29 cm) of phenylsepharose-CL 
(Pharmacia Inc., Piscataway, NJ) equilibrated with 0.01M sodium phosphate 
(ph 6.7)-30% ammonium sulfate. The column was washed with one column 
volume of equilibration buffer and eluted with a 480 ml linear ammonium 
sulfate gradient (30 to 0%) at 50 ml/hr. Fractions (4 ml) were analyzed 
for optical density (at 280 nm) and chemotactic inhibitory activity. SCFI 
activity (expressed as the reciprocal of the chemotactic differential) was 
scored at a 1/100 final dilution in 50% ZAS. Salt concentrations were 
determined from the refractive index. 
5. Anion-exchange chromatography. SCFI-containing fractions from the 
phenylsepharose hydrophobic column were pooled and dialyzed exhaustively 
against 0.05M Tris.multidot. hydrochloride buffer (pH 7.0). The dialyzed 
pool was loaded onto a DEAE-cellulose anion-exchange column (1.1 by 22 cm; 
Cellex-D; Bio-Rad Laboratories, Richmond, Calif.) equilibrated in the same 
buffer as the sample, and the column was eluted with a 200-ml linear 
gradient of NaCl (0 to 0.5M). The column was run at 11 ml/hr, and 
fractions (1.0 ml) were analyzed as described above, except that SCFI 
activity was scored at 1/200 final dilution in 50% ZAS. 
6. Gel permeation chromatography. SCFI-containing fractions from the 
anion-exchange column were pooled and concentrated to 0.8 ml by 
ultrafiltration with a PM-10 membrane (Amicon Corp., Lexington, Mass.). 
This material was applied to a Sephacryl-300 column (1.6 by 90 cm; 
Pharmacia) equilibrated with 0.05M Tris.multidot.hydrochloride (pH 
7.4)-0.14M NaCl (TBS) and eluted at 7.0 ml/h with the same buffer. 
Fractions (0.8 ml) were analyzed for optical density (at 280 mn), and SCFI 
activity was quantitated by serial dilution. Fractions containing 
.gtoreq.200 U of SCFI activity per ml were pooled and stored at 
-70.degree. C. This material was designated S300 SCFI. 
7. PAGGE. Discontinuous sodium dodecyl sulfatepolyacrylamide gradient gel 
electrophoresis (SDS-PAGGE) was performed by a modification of the Laemmli 
system [Nature (London), 227, 680 (1970)] with an exponential gradient of 
5 to 20% acrylamide. Nondenaturing gels for molecular weight comparisons 
were performed under the same conditions except for the exclusion of SDS 
and an extended electrophoretic run time of 5.5 hr for pore limit 
separations [J. Margolis, Nature (London), 214, 680 (1967)]. Proteins were 
visualized in the gels by the silver staining technique of Oakley et al., 
Anal. Biochem., 105, 361 (1980). Molecular weight standards for SDS gels 
were cytochrome c, carbonic anhydrase, ovalbumin, bovine serum albumin, 
.beta.-galactosidase (Sigma), and apoferritin (Schwarz/Mann, Orangeburg, 
N.Y.). 
8. Antiserum preparation. Antisera to S300 SCFI was produced in New Zealand 
White rabbits by subcutaneous injection of 75 .mu.g of column-purified 
protein elumsified in Freund complete adjuvant. Subsequent booster 
injections of 40 .mu.g of protein in incomplete Freund adjuvant were made 
4 weeks apart, and rabbits were bled 1 week after each booster. The 
content of SCFI-specific antibody was assessed by neutralization of SCFI 
activity and by immunodiffusion analysis. 
9. Western blot analysis. SDS-PAGGE gels were electroblotted onto a 
0.45-.mu.m (pore size) nitrocellulose membrane (SS BA85; Schleicher and 
Schuell, Inc., Keene, N.H.) by the method described by Burnette et al., 
Anal. Biochem., 112, 195 (1981), with the Hoefer Transphor, model TE-50. 
Protein transfer was carried out at 500 V for 4 hr with cooling in an 
electrode buffer consisting of 20 mM Tris base, 150 mM glycine, and 20% 
methanol. After transfer, the nitrocellulose was treated by a modification 
of the procedure of Blake et al., [Anal. Biochem., 175, 175, (1984)], and 
all steps were performed at 25.degree. C. The protein blot was immersed in 
a solution of 3% gelatin-TBS for 45 min, then washed in 0.05% Tween 20-TBS 
(TTBS) for 45 min. The blot was then probed with a 1:100 dilution of 
rabbit SCFI antiserum in TTBS for 2 hr and washed three times in TTBS (5 
min per wash). The blot was subsequently exposed for 2 hr to alkaline 
phosphataseconjugated goat anti-rabbit antibody (1:800 in TTBS) (Sigma) 
and washed sequentially with TTBS, TBS, and 0.015M Veronal acetate buffer 
(pH 9.6) (twice each). Development of the blot was carried out by 
immersion for 2 to 5 min at 25.degree. C. in 9 ml of 0.015M Veronal 
acetate buffer (pH 9.6) containing 1 mg of 5-bromo-4-chloroindoxyl 
phosphate (Sigma), 1 mg of nitroblue tetrazolium (Sigma), and 4.4 mM 
MgCl.sub.2. Staining was stopped at the desired band intensity by 
immersion in 10% acetic acid. 
10. Chemotaxis inhibition assay for SCFI activity. Chemotactic activity of 
zymosan-activated serum (ZAS) was measured by the underagarose chemotaxis 
assay system of Nelson et al. as described in D. E. Wexler et al., Infect. 
Immunology, 39, 239 (1983), the disclosure of which is incorporated by 
reference herein. Mixed human leukocytes were prepared from heparinized 
blood by dextran sedimentation as previously described by Wexler et al., 
supra. Soluble SCFI activity was quantitated by incubating serial twofold 
dilutions of samples in phosphate-buffered saline (ph 7.4) with an equal 
volume of ZAS for 60 min at 37.degree. C. ZAS was prepared from serum of a 
healthy donor who lacked SCFI-specific antibody. The SCFI titer represents 
the reciprocal of the final dilution which inhibits the directed migration 
of PMNs to this chemotaxin by 50%. This 50% effective dose for inhibition 
is expressed as units per milliliter of SCFI activity. Alternatively, SCFI 
activity was scored by the less quantitative but more rapid method of 
measuring the decrease in the chemotactic activity of 50% ZAS induced 
after incubation with a single dilution of a sample within an experiment. 
In this case, SCFI activity was expressed as the reciprocal of the 
chemotactic differential (spontaneous distance subtracted from the 
chemotactic distance). 
B. Results 
1. Extraction and Purification. Extraction of SCFI (11,000 U of activity) 
was obtained by incubating bacteria (160 g [wet weight]) at 
6.times.10.sup.10 cells per ml with 0.1 mg of trypsin per ml. After a 
two-step ammonium sulfate fractionation in which SCFI was precipitated 
between 55 and 90% salt, most of SCFI activity and 67% of total protein 
were recovered. 
The 55 to 90% ammonium sulfate fraction was chromatographed on a 
phenylsepharose hydrophobic interaction column, eluting bound material 
with a decreasingconcentration gradient ammonium sulfate. SCFI activity 
appeared in fractions having 18 to 10% salt, and the amount of protein in 
the SCFI pool was reduced approximately fivefold, from 297 to 56 mg. At 
this point in purification, only trace amounts of phosphate (&lt;0.05 mg) 
were detectable. The phenylsepharose SCFI pool was dialyzed and 
fractionated further by ion-exchange chromatography in a DEAE-cellulose 
column equilibrated at pH 7.0. The anionic nature of SCFI had been 
previously determined by its ability to bind DEAE-cellulose resin at pH 
5.0. Most of the SCFI activity eluted between 0.12 and 0.14M NaCl in a 
single peak that exhibited a slight trailing edge. The final yield of SCFI 
activity after this step was 38% of the extracted activity, and this 
material contained 1.8 mg of protein for 250-fold protein purification. 
The results of these purification steps are summarized in Table II, below. 
TABLE II 
______________________________________ 
Analysis of SCFI Purification Steps 
Protein SCFI Sp act (U mg 
Purification Step 
(mg).sup.a (U) of protein) 
______________________________________ 
Trypsin extraction 
441 110,600 251 
Salt Fractionation 
297 100,875 340 
Hydrophobic chroma- 
56 62,100 1,109 
tography 
Anion-exchange 
1.8 42,240 23,467 
chromatography 
______________________________________ 
.sup.a Measured by the Lowry method with bovine serum albumin as the 
standard. 
2. Characterization of purified SCFI. Gel permeation analysis was performed 
to provide an estimate of the degree of homogeneity of the SCFI-associated 
material. The column elution profile demonstrated that the SCFI activity 
was heterogeneous, since it segregated into two closely spaced peaks. This 
heterogeneity was reproducible, indicating that the SCFI activity was 
associated with a minimum of two molecular species that differed in size. 
A single major absorbance peak was detected, which corresponded with the 
SCFI-containing fractions. 
Spectrophotometric analysis of the S300 pooled material, designated S300 
SCFI, indicated an absorption maximum at 276 nm, confirming that this pool 
contained protein. To further assess the purity and molecular weight of 
this material, we analyzed S300 SCFI by PAGGE under nondenaturing (no SDS) 
and denaturing conditions. S300 SCFI electrophoresed in the absence of SDS 
produced a pattern markedly different from that in the SDS gel. 
Silver-stained gels of undenatured SCFI revealed three major proteins and 
no more than four minor components. These peptides most likely differed in 
size, since the gel was run under the pore limit conditions developed by 
J. Margolis et al., Nature (London), 214, 680 (1967). In contrast, the SDS 
gel exhibited seven major high-molecular-weight bands ranging from 103,000 
to 114,000 M.sub.r and over 50 minor species spanning a wide range of 
lower molecular weights. The SDS pattern was unaffected by 
2-mercaptoethanol, indicating the absence of disulfide bonds. 
3. Immunological properties. As described herein above, hyperimmune 
antiserum was prepared which was able to neutralize SCFI activity, since 
preincubation of S300 SCFI with the serum resulted in a dose-dependent 
reduction of antichemotactic activity. Preimmune serum had no effect on 
activity. The antiserum was highly specific for SCFI, since nondenaturing 
PAGGE Western blot patterns for crude trypsin extracts were identical to 
that of the purified material. 
The resistance of SCFI to mild pepsin digestion and its relative resistance 
to trypsin distinguish it from M protein, which is known to be sensitive 
to both proteases. To test relatedness further, the two antigens were 
compared by immunodiffusion. Rabbit antiserum specific for SCFI produced a 
single precipitin arc in reaction to the immunogen, S300 SCFI, or 
partially purified SCFI extracted from cells with group C streptococcal 
phage lysin (DEAE-SCFI). In contrast, this antiserum did not react with 
Lancefield acid extracts known to contain M49 antigen. Moreover, M49 
typing serum did not react with either preparation of SCFI but reacted 
strongly with an acid extract of M49 cells. In addition to immunodiffusion 
experiments, purified M24, M5, and M6 proteins at 16.0 .mu.g each, were 
unable to bind competitively to SCFI antibody in enzyme-linked 
immunosorbent inhibition assays which reproducibly detect 5 ng of SCFI 
antigen. 
EXAMPLE II - Nonionic Detergent Extraction 
A. Materials and Methods 
1. Extraction of streptococci. A 50-ml culture of M49 bacteria in early 
exponential phase was inoculated into 2 liters of Todd-Hewitt broth-2% 
neopeptone and grown to late exponential phase. Bacteria were centrifuged 
at 13,000.times.g for 10 min at 4.degree. C. and washed once in Hanks 
balanced salt solution containing 0.1% gelatin. The bacterial pellet was 
suspended at 5.times.10.sup.10 cells per ml in TBS containing 1% Nonidet 
P-40 (NP-40)(nonylphenoxy)(EtO).sub.9 H, Sigma), and the extraction 
mixture was stirred gently at either 4.degree. or 37.degree. C. for 60 
min. After detergent treatment, bacteria were sedimented at 13,000.times.g 
for 10 min at 4.degree. C., and the supernatants were filter-sterilized 
with a 0.45-.mu.m (pore size) nitrocellulose membrane (Millipore). This 
material is designated SCFI-det. 
2. Double immunodiffusion. Double-diffusion analysis in agar was performed 
by the method of O. Ouchterlony, Acta. Pathol. Microbiol. Scand., 32, 231 
(1953). Gels were cast in microscope slides and consisted of 1% noble agar 
in 0.02M phosphate buffer (pH 7.4). Samples (15 .mu.l) were added to 3 mm 
wells, and the plates were incubated for 2 hr at 37.degree. C., followed 
by 18 hr at 24.degree. C. Precipitin reactions were photographed directly 
without staining. 
B. Results 
1. Extraction and Purification. M.sup.+ bacteria were incubated in buffer 
containing 1% NP-40 for 1 hr at 4.degree. C. The bacterium-free 
supernatant, when examined by immunodiffusion with S300 SCFI-specific 
antiserum, gave a reaction of identity with S300 SCFI antigen. Crude NP-40 
extracts were examined by Western blot analysis of SDS-PAGGE gels to 
determine the size and degree of homogeneity of the SCFI antigen 
(SCFI-det). NP-40 extract, prepared at either 4.degree. or 37.degree. C., 
was electrophoresed in duplicate sets on the same gel; one set was Western 
blotted for detection of SCFI, and the other was silver stained for total 
extracted protein. The results demonstrated that two antigenic species of 
similar molecular weight, but of unequal band intensity, were present. The 
SCFI bands corresponded to two bands on the duplicate silver-stained gel 
when the blot and stained gel were physically aligned. The two 
corresponding silver-stained bands also stained unequally and were well 
separated from bands of lower molecular weight. Their molecular weights 
were estimated to be 135,000 and 137,000 when compared with protein 
standards. SCFI-det could be extracted equally well at 4.degree. and 
37.degree. C. over the 1 hr incubation period. 
2. Characterization. The results of the trypsin extraction procedure for 
the preparation of SCFI indicated the need for a nonenzymatic method that 
would be less likely to result in peptide bond cleavage. The ability of 
the nonionic detergent NP-40 to solubilize SCFI antigen was assessed by 
immunological analysis of M.sup.+ bacterial extracts derived after a short 
incubation with the detergent at 4.degree. C. Immunodiffusion analysis of 
this extract indicated that a species was present having antigenic 
determinants in common with S300 SCFI. Western blots of NP-40 extracts 
identified a major band of 135,000 M.sub.r (molecular weight) and a less 
abundant protein of 137,000 M.sub.r. Thus, if trypsin and 
detergent-extracted antigen were derived from the same population of 
molecules on the bacterial surface, it is likely that the heterogeneity of 
S300 SCFI is largely an aspect of enzymatic hydrolysis during extraction. 
The detection of at least five peptide species retaining biological 
activity by HPLC analysis indicated that limited cleavage of the native 
protein does not eliminate activity in at least some of the products. 
Example III - Inhibition of C5a with S300 SCFI 
A. Materials and Methods 
1. Preparation of Human C5a and C5a.sub.desArg. C5a was generated in 
zymosan-activated human serum containing 1M .epsilon.-aminocaproic acid 
and it was purified according to the procedure of Fernandez and Hugli, J. 
Immunol., 120, 109 (1978). C5a.sub.desArg was purified by the same method 
except that serum was activated in the absence of .epsilon.-aminocaproic 
acid. Human C5a was radio-labeled with Na.sup.125 I (Amersham) by the 
solid-phase lactoperoxidase/glucose oxidase method as described by D. E. 
Chenoweth et al., J. Exp. Med., 156, 68 (1982). .sup.125 I-labeled 
C5a.sub.desArg for analysis by NaDodSO.sub.4 /PAGE analysis was obtained 
from Upjohn (Kalamazoo, MI). 
2. Preparation of Human Peripheral Polymorphonuclear Leukocytes (PMNs). 
Human PMNs were collected by venipuncture of healthy volunteers as 
described by Boyum, Scand. J. Clin. Lab. Invest., 21, Suppl. 97,77 (1968). 
After Ficoll-Hypaque centrifugation (J. Immunol. Methods, 24, 389 (1978)), 
the PMN-containing layer was subjected to hypotonic lysis in 0.87% 
NH.sub.4 Cl to remove contaminating erythrocytes by the method of 
Goldstein et al., J. Immunol., 111, 33 (1973). The PMNs were harvested by 
centrifugation at 150.times.g for 10 min, washed twice in 0.9% NaCl, and 
resuspended to the appropriate cell density in RPMI (GIBCO) tissue culture 
medium containing 1% bovine serum albumin. 
3. C5a Binding assay. Binding of .sup.125 I-labeled C5a to PMNs was 
assessed by mixing 4.times.10.sup.6 PMNs per ml in RPMI medium/1% bovine 
serum albumin with .sup.125 I-labeled C5a at a final concentration of 1 nM 
in a 0.1-ml vol. The mixtures were incubated for 15 min at 24.degree. C. 
in 1.5-ml conical polypropylene microfuge tubes followed by centrifugation 
at 11,000.times.g for 30 sec in a Beckman Microfuge B. The amount of 
cell-bound .sup.125 I-labeled C5a was determined by transferring one-half 
of the PMN-free supernatant to a separate tube and comparing the .gamma. 
radioactivity of the paired samples P (pelleted cells+one-half 
supernatant) and S (one-half supernatant). Cell-bound .sup.125 I-labeled 
C5a was calculated according to the following equation: 
##EQU1## 
NS, nonspecific binding to the assay tube, was cpm(P+S)/cpm(S). Of the 
total ligand, 30-50% was PMN-associated under these conditions. 
4. Amino acid analysis. For determination of free amino acids, unhydrolyzed 
samples or 2 nmol of hydrolyzed C5a standard were examined by using a 
Durrum amino acid analyzer. Hydrolysis of C5a was done in 5.6 M HCl by 
heating in a vacuum-sealed ampoule at 100.degree. C. for 18 hr. 
5. Carboxyl-terminal analysis. C5a.sub.desArg (1 nmol) or purified 
SCFI-inactivated C5a in 0.1 M NH.sub.4 HCO.sub.3 (pH 8.15) was digested 
with 0.1 nmol of carboxypeptidase A (phenylmethylsulfonyl 
fluoride-treated)(Worthington) and 0.1 nmol of carboxypeptidase B 
(Calbiochem) for 4.5 hr at 37.degree. C. These samples were lyophilized 
and analyzed for free amino acids in the absence of hydrolysis as 
described above. 
6. NaDodSO.sub.4 /PAGE. Discontinuous polyacrylamide gel electrophoresis 
was carried out under reducing conditions by a modification of the 
procedure of Example I(A)(7), supra. For electrophoresis of .sup.125 
I-labeled C5a.sub.desArg, a separation gel was used consisting of an 
exponential gradient of 15-20% acrylamide (12-16% glycerol) and 
radiolabeled protein visualized by autoradiography with Kodak XAR-5 x-ray 
film. Denatured bovine serum albumin was prepared by a modification of the 
alkaline urea method of Anson, J. Gen. Physiol., 22, 79 (1938). Bovine 
serum albumin (50 .mu.g) in H.sub.2 O was first incubated with 50 mM 
dithiothreitol for 10 min at room temperature, then the protein was 
alkalinized with NaOH (0.08 M final NaOH concentration). Crystalline urea 
was added to 7.3 M, and the mixture was incubated at room temperature for 
1 hr. The denatured bovine serum albumin was diluted with an equal volume 
of 0.062 M Tris.HCl(pH 7.4) and dialyzed overnight against the same 
buffer. A 5-15% acrylamide gradient was used for electrophoresis of bovine 
serum albumin (Sigma), and silver staining was by the Oakley method (Anal. 
Biochem., 105, 61 (1980)). 
B. Results 
1. Inactivation of .sup.125 I-labeled C5a by SCFI. The 
concentration-dependent effect of S300 SCFI on the PMN receptor binding 
and the antigenic properties of .sup.125 I-labeled C5a were assessed. S300 
SCFI, ranging in concentration from 0 to 625 ng/ml, was incubated with 5 
nM .sup.125 I-labeled C5a (41 ng/ml) for 60 min at 37.degree. C., and the 
ability of treated ligand to bind PMNs was measured. Specific PMN binding 
of .sup.125 I-labeled C5a was completely inhibited by prior incubation of 
the C5a with SCFI (10 ng/ml), and as little as 0.07 ng of SCFI per ml 
produced a significant binding inhibition. This represents a weight ratio 
of C5a to SCFI of about 600-4:1. The possibility that this effect was due 
to the complete destruction of C5a by either SCFI or enzymatic 
contaminants of the SCFI preparation was highly unlikely because the 
antigenicity of the .sup.125 I-labeled C5a remained unaltered. 
2. Proteolytic Cleavage of C5a by SCFI. NaDodSO.sup.4 /PAGE analysis was 
performed to determine whether inactivation of .sup.125 I-labeled 
C5a.sub.desArg by SCFI was accompanied by a decrease in molecular weight, 
as would be expected from enzymatic cleavage of the molecule. .sup.125 
I-labeled C5a.sub.desArg incubated with SCFI exhibited a significantly 
greater electrophoretic mobility than untreated .sup.125 I-labeled 
C5a.sub.desArg. This difference in mobility was small, corresponding to a 
M.sub.r of about -500, suggesting that SCFI mediates the cleavage of 
.sup.125 I-labeled C5a.sub.desArg near the amino or carboxyl terminus. 
To locate the peptide bond in C5a that is cleaved by SCFI, the 
carboxyl-terminal sequence of immunoaffinity-purified SCFI-inactivated C5a 
was compared with that of native C5a.sub.desArg. Under partial digestion 
conditions, the relative amount of each amino acid released by 
carboxypeptidases A and B depends on its distance from the COOH terminus 
of the peptide in question. Because the sequence of C5a is known, this 
approach unequivocally identifies the COOH-terminal residue. The results 
presented in Table III demonstrate that SCFI-inactivated C5a lacks the end 
sequence -Asp.sup.69 -Met.sup.70 -Gln.sup.71 -Leu.sup.72 -Gly.sup.73 
-Arg.sup.74, and therefore, C5a cleavage occurs between Lys.sup.68 and 
Asp.sup.69. 
TABLE III 
______________________________________ 
Carboxyl-terminal analysis 
of SCFI-inactivated C5a 
Amino acid 
C5a.sub.desArg, nmol 
SCFI-inactivated C5a*, nmol 
______________________________________ 
Gly 1.06 0.11 
Leu 0.86 0.06 
Glx 0.85 0.02 
Met 0.37 0.00 
Asx 0.32 0.00 
Lys 0.32 1.00 
His 0.28 1.12 
Ser 0.00 0.67 
Ile 0.13 0.19 
Phe 0.12 0.26 
Tyr 0.15 0.78 
Thr 0.06 0.36 
______________________________________ 
##STR1## 
##STR2## 
*One nanomole of carboxypeptidasedigested C5a.sub.desArg or 
SCFIinactivated C5a was analyzed. SCFI inactivation was assessed by the 
reduction in ligandreceptor binding affinity. 
EXAMPLE IV - Zwitterionic Detergent Extraction 
1-10 1 batches of a group A streptococcal culture, strain CS101 serotype 
M49, T14, are grown to log phase in Todd-Hewitt broth supplemented with 
neopeptone at 37.degree. C. At log phase, cells are harvested, washed with 
0.1 M Na.sub.2 HPO.sub.4, pH 7.4 buffer and resuspended in an extraction 
buffer to a final concentration of 0.1 M Na.sub.2 HPO.sub.4, 0.5% 
((3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, "CHAPS," 
Sigma), with 10 .mu.g/ml DNAse, 10 .mu.g/ml RNAse, 75 .mu.g/ml 
phenylmethylsulfonyl fluoride, (PMSF) and 1.0 mM sodium iodoacetate. 
The suspension is stirred for 90 min at 42.degree. C., centrifuged and the 
pellet discarded. The supernatant (the crude extract) is concentrated and 
then applied to a Sephadex G-50-300 column. The column is equilibrated and 
eluted with 0.05M Tris.HCl, pH 7.0. Fractions containing SCFI are pooled 
and applied directly to a cellulose QAE anion exchange column. This column 
is eluted with an increasing linear salt gradient from 0.05M Tris.HCl, pH 
7.0 to 0.5M Tris.HCl/0.5M NaCl, pH 7.0. Fractions containing the SCFI are 
pooled, adjusted to a 30% saturation of (NH.sub.4).sub.2 SO.sub.4, applied 
to a Phenyl Sepharose CL-4B column and eluted with a decreasing linear 
salt gradient from 0.05M Tris.HCl/30% (NH.sub.4).sub.2 SO.sub.4, pH 7.0 to 
0.05M Tris.HCl, pH 7.0. Fractions containing SCFI are pooled, desalted by 
and concentrated to yield a pure preparation. 
SCFI isolated and purified by the above procedure comprises primarily two 
peptides when analyzed by SDS PAGGE electrophoresis, which were estimated 
to have molecular weights of 124 Kd and 120 Kd. This mixture of peptides 
also exhibits a spectrum of activity with respect to C5a which is 
essentially similar to the activity of S300 SCFI as determined 
hereinabove. 
DISCUSSION 
The results of the present study show that the streptococcal chemotactic 
factor inactivator polypeptide of the present invention (SCFI) is a 
protease that attacks the COOH-terminal region of C5a at the carboxyl side 
of a lysine residue to produce a molecule that lacks the capacity for 
functional interaction with PMN receptors. SCFI-inactivated C5a is similar 
to the reaction product of C5a with yeast carboxypeptidase Y, C5a-(1-69), 
in lacking COOH-terminal residues required for receptor stimulation. 
However, distinctions between SCFI-inactivated C5a and C5a-(1-69) exist on 
both structural and mechanistic levels. The product of SCFI hydrolysis 
lacks six COOH-terminal amino acid residues as the result of cleavage 
between Lys.sup.68 and Asp.sup.69 ; this contrasts with the five-residue 
loss in the carboxypeptidase Y C5a derivative. Moreover, on the basis of 
its activity against denatured bovine serum albumin, SCFI appears to 
cleave C5a by an endoprotease activity as opposed to the sequential 
hydrolysis of peptide bonds characteristic of carboxypeptidases. SCFI also 
appears to be highly site-selective because, aside from the loss of the 
six carboxyl-terminal residues, the C5a residue was largely intact. This 
latter conclusion is based on the detection in purified SCFI-inactivated 
C5a of a single carboxyl terminus and, therefore, one polypeptide chain, 
as well as NaDodSO.sub.4 /PAGE analysis indicating that the decrease in 
molecular weight of the C5a was quite small (M.sub.r, about 500). 
In summary, the potential of group A streptococci to inactivate serum 
chemotactic activity is mediated by a surface-bound protein that exhibits 
a high degree of specificity for the C5a chemotaxin. In accordance with 
the present invention, this protein or the polypeptide fragments thereof, 
can be extracted from group A streptococci in essentially pure form. The 
purified compounds retain the ability of the native protein to block the 
binding of C5a to polymorphonuclear leukocytes by the proteolytic 
degradation thereof. It is expected that these purified polypeptides will 
be useful for the investigation of the human immune response, and may 
provide the basis for therapeutic agents for the treatment of human and 
animal pathologies mediated by C5a. 
While certain representative embodiments of the invention have been 
described herein for purposes of illustration, it will be apparent to 
those skilled in the art that modifications therein may be made without 
departing from the spirit and scope of the invention. For example, 
although SCFI has been described primarily in terms of the polypeptides 
designated "S300 SCFI" and the 120 Kd and the 124 Kd polypeptides derived 
by trypsin and detergent extraction of group A streptococci, any 
polypeptide which includes a common amino acid sequence and which 
deactivates C5a by a similar biological pathway is intended to be included 
within the scope of the present invention.