Patent Application: US-13265202-A

Abstract:
a novel gene defining a novel enzyme in the udp - d - galactose : b - n - acetyl - glucosamine β - 1 , 4 - galactosyltransferase family , termed β4gal - t2 , with unique enzymatic properties is disclosed . the enzymatic activity of β4gal - t2 is shown to be distinct from that of previously identified enzymes of this gene family . the invention discloses isolated dna molecules and dna constructs encoding β4gal - t2 and derivatives thereof by way of amino acid deletion , substitution or insertion exhibiting β4gal - t2 activity , as well as cloning and expression vectors including such dna , cells transfected with the vectors , and recombinant methods for providing β4gal - t2 . the enzyme β4gal - t2 and β4gal - t2 - active derivatives thereof are disclosed , in particular soluble derivatives comprising the catalytically active domain of β4gal - t2 . further , the invention discloses methods of obtaining β - 1 , 4 - galactosyl glycosylated saccharides , glycopeptides or glycoproteins by use of an enzymically active β4gal - t2 protein or fusion protein thereof or by using cells stably transfected with a vector including dna encoding an enzymatically active β4gal - t2 protein as an expression system for recombinant production of such glycopeptides or glycoproteins . also a method for the identification of dna sequence variations in the β4gal - t2 gene by isolating dna from a patient , amplifying β4gal - t2 - coding exons by pcr , and detecting the presence of dna sequence variation , are disclosed .

Description:
all patent applications , patents , and literature references cited in this specification are hereby incorporated by reference in their entirety . in the case of conflict , the present description , including definitions , is intended to control . 1 . “ nucleic acid ” or “ polynucleotide ” as used herein refers to purine - and pyrimidine - containing polymers of any length , either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo - polydeoxyribo nucleotides . this includes single - and double - stranded molecules , i . e ., dna - dna , dna - rna and rna - rna hybrids , as well as “ protein nucleic acids ” ( pna ) formed by conjugating bases to an amino acid backbone . this also includes nucleic acids containing modified bases ( see below ). 2 . “ complementary dna or cdna ” as used herein refers to a dna molecule or sequence that has been enzymatically synthesized from the sequences present in an mrna template , or a clone of such a dna molecule . a “ dna construct ” is a dna molecule or a clone of such a molecule , either single - or double - stranded , which has been modified to contain segments of dna that are combined and juxtaposed in a manner that would not otherwise exist in nature . by way of non - limiting example , a cdna or dna which has no introns is inserted adjacent to , or within , exogenous dna sequences . 3 . a plasmid or , more generally , a vector , is a dna construct containing genetic information that may provide for its replication when inserted into a host cell . a plasmid generally contains at least one gene sequence to be expressed in the host cell , as well as sequences that facilitate such gene expression , including promoters and transcription initiation sites . it may be a linear or closed circular molecule . 4 . nucleic acids are “ hybridizable ” to each other when at least one strand of one nucleic acid can anneal to another nucleic acid under defined stringency conditions . stringency of hybridization is determined , e . g ., by a ) the temperature at which hybridization and / or washing is performed , and b ) the ionic strength and polarity ( e . g ., formamide ) of the hybridization and washing solutions , as well as other parameters . hybridization requires that the two nucleic acids contain substantially complementary sequences ; depending on the stringency of hybridization , however , mismatches may be tolerated . typically , hybridization of two sequences at high stringency ( such as , for example , in an aqueous solution of 0 . 5 × ssc , at 65 ° c .) requires that the sequences exhibit some high degree of complementarity over their entire sequence . conditions of intermediate stringency ( such as , for example , an aqueous solution of 2 × ssc at 65 ° c .) and low stringency ( such as , for example , an aqueous solution of 2 × ssc at 55 ° c . ), require correspondingly less overall complementarily between the hybridizing sequences . ( 1 × ssc is 0 . 15 m nacl , 0 . 015 m na citrate .) 5 . an “ isolated ” nucleic acid or polypeptide as used herein refers to a component that is removed from its original environment ( for example , its natural environment if it is naturally occurring ). an isolated nucleic acid or polypeptide contains less than about 50 %, preferably less than about 75 %, and most preferably less than about 90 %, of the cellular components with which it was originally associated . 6 . a “ probe ” refers to a nucleic acid that forms a hybrid structure with a sequence in a target region due to complementarily of at least one sequence in the probe with a sequence in the target region . 7 . a nucleic acid that is “ derived from ” a designated sequence refers to a nucleic acid sequence that corresponds to a region of the designated sequence . this encompasses sequences that are homologous or complementary to the sequence , as well as “ sequence - conservative variants ” and “ function - conservative variants ”. sequence - conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position . function - conservative variants of β4gal - t2 are those in which a given amino acid residue in the polypeptide has been changed without altering the overall conformation and enzymatic activity ( including substrate specificity ) of the native polypeptide ; these changes include , but are not limited to , replacement of an amino acid with one having similar physico - chemical properties ( such as , for example , acidic , basic , hydrophobic , and the like ). 8 . a “ donor substrate ” is a molecule recognized by , e . g ., a galactosyltransferase and that contributes a galactosyl moiety for the transferase reaction . for β4gal - t2 , a donor substrate is udp - galactose . an “ acceptor substrate ” is a molecule , preferably a saccharide or oligosaccharide , that is recognized by , e . g ., a galatosyltransferase and that is the target for the modification catalyzed by the transferase , i . e ., receives the galatosyl moiety . for β4gal - t2 , acceptor substrates include without limitation oligosaccharides , glycoproteins , o - linked glcnac - glycopeptides , and glycosphingolipids containing the sequences glcnacβ1 - 3gal , glcnacβ1 - 6gal , glcnacβ1 - 6galnac , glcnacβ1 - 3galnac , glcnacβ1 - 2man , glcnacβ1 - 4man , glcnacβ1 - 6man , glcnacβ1 - 3man , glcβ1 - ceramide . the present invention provides the isolated dna molecules , including genomic dna and cdna , encoding the udp - galactose : β - n - acetylglucosamine β - 1 , 4 - galactosyltransferase ( β4gal - t2 ). β4gal - t2 was identified by analysis of est database sequence information , and cloned based on est and 5 ′ race cdna clones . the cloning strategy may be briefly summarized as follows : 1 ) synthesis of oligonucleotides derived from est sequence information , designated eber102 and eber 104 ; 2 ) successive 5 ′- rapid amplification of cdna ends ( 5 ′ race ) using commercial marathon - ready cdna ; 3 ) cloning and sequencing of 5 ′ race cdna ; 4 ) identification of a novel cdna sequence corresponding to βgal - t2 ; 5 ) construction of expression constructs by reverse - transcription - polymerase chain reaction ( rt - pcr ) using colo205 human cell line mrna ; 6 ) expression of the cdna encoding β4gal - t2 in sf9 ( spodoptera frugiperda ) cells . more specifically , the isolation of a representative dna molecule encoding a novel second member of the mammalian udp - galactose : β - n - acetylglucosamine β - 1 , 4 - galactosyltransferase family involved the following procedures described below . novel human dna sequences with apparent homology to the human β4gal - t1 gene ( masri et al ., 1988 ) were identified by sequence similarity searches of the dbest database at the national center for biotechnology information , usa , using the blastn and tblastn algorithms . composites for identified novel genes were compiled and analysed for sequence similarity to human β4gal - t1 . est cdna clones with the longest inserts ( fig1 ) were obtained from genome systems inc , usa . two partly overlapping ests with predicted sequence similarity to β4gal - t1 were identified ( fig1 ). sequencing of the inserts revealed an open reading frame which potentially encoded a sequence similar to β4gal - t1 , but the 5 ′ sequence was shorter and without an initiation codon . further 5 ′ sequence was obtained by 5 ′ race using human fetal brain marathon - ready cdna ( clontech ) in combination with anti - sense primers eber102 and eber104 . the 5 ′ race products were cloned and multiple clones were sequenced . the entire sequence was confirmed by sequencing genomic p1 clones . the composite sequence contained an open reading frame of 1116 bp ( fig2 ), with an overall sequence identity of approximately 63 % to β4gal - t1 . the predicted open reading frame has one potential initiation codon in agreement with kozak &# 39 ; s rule ( kozak , 1992 ). the predicted coding sequence depicts a type ii transmembrane glycoprotein with a 11 amino acid residue n - terminal cytoplasmic domain , a transmembrane segment of 21 residues , and a stem region and catalytic domain of 340 residues , with three potential n - linked glycosylation sites ( fig2 ). multiple alignment analysis ( clustalw ) of human β4gal - t1 ( accession # m22921 ), human β4gal - t2 , and human β4gal - t3 ( accession # y12510 ) presented in fig3 demonstrated sequence significant similarities especially in the central and c - terminal region and conservation of cysteine residues . the n - terminal regions show no sequence similarity . a 3 ′ untranslated region without polyadenylation signals was included in the oligo - dt primed est cdna clones sequenced . the 3 ′ ests ( stsg4681 ) were linked to chromosome 1 between d1s2861 and d1s211 microsatellite markers at 73 - 75 cm ( ncbi ). an expression construct designed to encode amino acid residues 31 - 372 of β4gal - t2 was prepared by rt - pcr with mrna from colo205 cell line , using the primer pair eber100for and eber114 ( fig2 ). expression of a soluble construct of β4gal - t2 in sf9 cells ( pharmingen ) resulted in marked increase in galactosyltransferase activity using the βglcnac - benzyl acceptor substrate compared to uninfected cells or cells infected with control constructs for polypeptide galnac - transferases or histo - blood group a and o genes ( bennett et al ., 1996 ; gentzsch and tanner , 1996 ) ( table i ). table i substrate specificity of β4gal - transferases β4gal - t2 a ( nmol / min / ml ) substrate concentration 1 mm 3 mm 9 mm d - glcnac 1 . 4 3 . 2 4 . 8 bz - β - d - glcnac 6 . 8 3 . 6 1 . 5 bz - α - d - glcnac 0 . 4 1 . 1 1 . 7 o - nph - α - d - glcnac 0 . 4 0 . 8 1 . 5 p - nph - β - d - glcnac 3 . 0 2 . 3 0 . 9 p - nph - 1 - thio - β - d - glcnac 1 . 2 1 . 6 0 . 2 4 - me - lumb - β - d - glcnac 0 . 8 0 . 6 0 . 4 β - d - glcnac -( 1 - 3 )- β - d - gal - 1 - ome 5 . 8 7 . 7 nd b β - d - glcnac -( 1 - 6 )- α - d - man - 1 - ome 8 . 5 11 . 3 nd bz - 2 -( 2 - β - d - glcnac )- α - d - glcnac 9 . 9 2 . 6 1 . 3 4 - me - lumb - β - d - galnac nd 0 . 0 nd o - nph - β - d - galnac nd 0 . 0 nd bz -*- d - galnac nd 0 . 0 nd 4 - me - lumb - β - d - gal nd 0 . 0 nd o - nph - β - d - gal nd 0 . 0 nd analysis of the substrate specificity of the soluble β4gal - t2 activity showed that only βglcnac - benzyl and not αglcnac - benzyl or agalnac - benzyl was an acceptor substrate . free glucose was not an acceptor , but in the presence of increasing concentrations of α - lactalbumin incorporation rates similar to bovine milk β4gal - transferase was observed ( fig4 panel a ). differences in the concentration of a - lactalbumin to achieve maximum activity with glc were observed with 0 . 4 mg / ml required for β4gal - t2 and only 0 . 1 mg / ml for the bovine milk enzyme . the activities of both β4gal - t2 and the bovine milk enzyme with glcnac were inhibited by a - lactalbumin , but β4gal - t1 ( bovine milk transferase preparation ) was overall more sensitive to inhibition ( fig4 panel b ). the apparent km for benzyl - βglcnac was 0 . 16 mm , and the km for udp - gal using benzyl - βglcnac was 0 . 011 mm . the bovine milk β4 - galactosyltransferase showed higher km for udp - gal in agreement with previous studies ( fujita - yamaguchi and yoshida , 1981 ; paquet and moscarello , 1984 ; furukawa et al ., 1990 ; nakazawa et al ., 1991 ; malissard et al ., 1996 ), and the measured km for glcnac was similar to that determined in some studies ( powell and brew , 1974 ; moscarello et al ., 1985 ), but 5 - 10 fold higher than compared to other studies ( fujita - yamaguchi and yoshida , 1981 ; paquet and moscarello , 1984 ; nakazawa et al ., 1991 ; malissard et al ., 1996 ). as shown in fig5 β4gal - t2 was inhibited at high concentrations of both benzyl - βglcnac and free n - acetylglucosamine to higher degree than bovine milk β4gal - transferase and β4gal - t3 ( shur , 1982 ). β4gal - t2 showed strict donor substrate specificity for udp - gal and did not utilise udp - galnac or udp - glcnac with the acceptor substrates tested . β4gal - t2 utilised the lc 3 cer glycosphingolipid substrates , and the product formed with this substrate was confirmed by 1 h - nmr to be nlc 3 cer similar to what was found for the activity of β4gal - t3 ( almeida et al ., 1997 ). β4gal - t2 exhibited the overall best activities with the glycoprotein acceptors ovalbumin , asialo - agalacto - fetuin , and asialo - agalacto - transferrin ( table ii ). table ii substrate specificity of β4 - galactosyltransferases with glycopeptide and glycoproten acceptors β4gal - t2 β4gal - t3 bovine milk β4gal - t acceptor substrate a nmol / min / ml nmol / min / ml nmol / min / μg β - d - glcnac - 1 - bz 3 . 5 3 . 9 3 . 4 β - d - glcnac - 1 - 1 . 3 0 . 9 ( fapgsypal ) *- d - galnac - 1 - 0 . 0 0 . 0 0 . 0 ( fapsnypal ) hen egg albumin 2 . 0 1 . 0 0 . 7 asialo - agalacto - 2 . 8 0 . 7 0 . 8 fetuin asialo - fetuin 0 . 2 0 . 0 0 . 1 the activities of the b4gal - transferases were analysed relative to benzyl - β - glcnac , and β4gal - t2 showed 2 - 3 fold higher activity than other β4gal - transferases tested . β4gal - t2 also showed the best activity with a synthetic o - linked βglcnac - glycopeptide ( table ii ), suggesting that this enzyme will show higher sensitivity in labeling o - linked βglcnac - glycoproteins as well . northern analysis with mrna from 16 human adult organs showed a single transcript of both genes of approximately 2 . 2 kb ( fig6 ). β4gal - t2 was expressed weakly in several adult organs with highest expression in prostate , testis , ovary , intestine , and muscle . the present invention also provides isolated genomic dna molecules encoding β4gal - t2 . a human p1 library ( dupont merck pharmaceutical company human foreskin fibroblast p1 library ) was screened using primer pairs eber100 and eber102 . three clones ; dpmc - hff # 10638 : 515 : g9 , dpmc - hff # 10639 : 516 : g4 , and dpmc - hff # 10640 : 924 : a11 , were obtained from genome systems . southern blot analysis with various oligonucleotides covering the 3 ′ and 5 ′ coding sequence of the existing full length β4gal - t2 cdna indicated that the entire coding sequence was included in the p1 clone . a comparative southern blot analysis between cloned p1 dna and total human genomic dna using a full length cdna as probe gave similar patterns , validating the use of cloned p1 dna as a model . the coding region of β4gal - t2 were found in six exons ( fig7 ). human and mouse β4gal - t1 is encoded in six exons ( hollis et al ., 1989 ; mengle - gaw et al ., 1991 ). comparison of the intron / exon boundaries of β4gal - t1 , - t2 , and - t3 , revealed that the five introns in the coding regions of the three genes are placed identically . fig8 and 9 depict a pcr strategy and primer sequences for amplification of all coding exons in β4gal - t2 using genomic dna . in practicing the present invention , many conventional techniques in molecular biology , microbiology , recombinant dna , and immunology , are used . such techniques are well known and are explained fully in , for example , sambrook et al ., 1989 , molecular cloning : a laboratory manual , second edition , cold spring harbor laboratory press , cold spring harbor , n . y . ; dna cloning : a practical approach , volumes i and ii , 1985 ( d . n . glover ed . ); oligonucleotide synthesis , 1984 , ( m . l . gait ed . ); nucleic acid hybridization , 1985 , ( hames and higgins ); transcription and translation , 1984 ( hames and higgins eds . ); animal cell culture , 1986 ( r . i . freshney ed . ); immobilized cells and enzymes , 1986 ( irl press ); perbal , 1984 , a practical guide to molecular cloning ; the series , methods in enzymology ( academic press , inc . ); gene transfer vectors for mammalian cells , 1987 ( j . h . miller and m . p . calos eds ., cold spring harbor laboratory ); methods in enzymology vol . 154 and vol . 155 ( wu and grossman , and wu , eds ., respectively ); immunochemical methods in cell and molecular biology , 1987 ( mayer and waler , eds ; academic press , london ); scopes , 1987 , protein purification : principles and practice , second edition ( springer - verlag , n . y .) and handbook of experimental immunology , 1986 , volumes i - iv ( weir and blackwell eds .). the invention encompasses isolated nucleic acid fragments comprising all or part of the nucleic acid sequence disclosed herein as seq id no : 1 . the fragments are at least about 8 nucleotides in length , preferably at least about 12 nucleotides in length , and most preferably at least about 15 - 20 nucleotides in length . the invention further encompasses isolated nucleic acids comprising sequences that are hybridizable under stringency conditions of 2 × ssc , 55 ° c ., to seq id no : 1 ; preferably , the nucleic acids are hybridizable at 2 × ssc , 65 ° c . ; and most preferably , are hybridizable at 0 . 5 × ssc , 65 ° c . the nucleic acids may be isolated directly from cells . alternatively , the polymerase chain reaction ( pcr ) method can be used to produce the nucleic acids of the invention , using either chemically synthesized strands or genomic material as templates . primers used for pcr can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites , if desirable , to facilitate incorporation into a given vector for recombinant expression . the nucleic acids of the present invention may be flanked by natural human regulatory sequences , or may be associated with heterologous sequences , including promoters , enhancers , response elements , signal sequences , polyadenylation sequences , introns , 5 ′- and 3 ′- noncoding regions , and the like . the nucleic acids may also be modified by many means known in the art . non - limiting examples of such modifications include methylation , “ caps ”, substitution of one or more of the naturally occurring nucleotides with an analog , internucleotide modifications such as , for example , those with uncharged linkages ( e . g ., methyl phosphonates , phosphotriesters , phosphoroamidates , carbamates , etc .) and with charged linkages ( e . g ., phosphorothioates , phosphorodithioates , etc .). nucleic acids may contain one or more additional covalently linked moieties , such as , for example , proteins ( e . g ., nucleases , toxins , antibodies , signal peptides , poly - l - lysine , etc . ), intercalators ( e . g ., acridine , psoralen , etc . ), chelators ( e . g ., metals , radioactive metals , iron , oxidative metals , etc . ), and alkylators . the nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage . furthermore , the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal , either directly or indirectly . exemplary labels include radioisotopes , fluorescent molecules , biotin , and the like . according to the present invention , useful probes comprise a probe sequence at least eight nucleotides in length that consists of all or part of the sequence from among the sequences designated seq id no : 1 or sequence - conservative or function - conservative variants thereof , or a complement thereof , and that has been labelled as described above . the invention also provides nucleic acid vectors comprising the disclosed sequence or derivatives or fragments thereof . a large number of vectors , including plasmid and fungal vectors , have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts , and may be used for gene therapy as well as for simple cloning or protein expression . recombinant cloning vectors will often include one or more replication systems for cloning or expression , one or more markers for selection in the host , e . g . antibiotic resistance , and one or more expression cassettes . the inserted coding sequences may be synthesized by standard methods , isolated from natural sources , or prepared as hybrids , etc . ligation of the coding sequences to transcriptional regulatory elements and / or to other amino acid coding sequences may be achieved by known methods . suitable host cells may be transformed / transfected / infected as appropriate by any suitable method including electroporation , cacl 2 mediated dna uptake , fungal infection , microinjection , microprojectile , or other established methods . appropriate host cells included bacteria , archebacteria , fungi , especially yeast , and plant and animal cells , especially mammalian cells . of particular interest are saccharomyces cerevisiae , schizosaccharomyces pombi , sf9 cells , c129 cells , 293 cells , neurospora , and cho cells , cos cells , hela cells , and immortalized mammalian myeloid and lymphoid cell lines . preferred replication systems include m13 , cole1 , sv40 , baculovirus , lambda , adenovirus , and the like . a large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts . examples of these regions , methods of isolation , manner of manipulation , etc . are known in the art . under appropriate expression conditions , host cells can be used as a source of recombinantly produced β4gal - t2 derived peptides and polypeptides . advantageously , vectors may also include a transcription regulatory element ( i . e ., a promoter ) operably linked to the β4gal - t2 - coding portion . the promoter may optionally contain operator portions and / or ribosome binding sites . non - limiting examples of bacterial promoters compatible with e . coli include : â - lactamase ( penicillinase ) promoter ; lactose promoter ; tryptophan ( trp ) promoter ; arabinose bad operon promoter ; lambda - derived p1 promoter and n gene ribosome binding site ; and the hybrid tac promoter derived from sequences of the trp and lac uv5 promoters . non - limiting examples of yeast promoters include 3 - phosphoglycerate kinase promoter , glyceraldehyde - 3 phosphate dehydrogenase ( gapdh ) promoter , galactokinase ( gali ) promoter , galactoepimerase promoter , and alcohol dehydrogenase ( adh ) promoter . suitable promoters for mammalian cells include without limitation viral promoters such as that from simian virus 40 ( sv40 ), rous sarcoma virus ( rsv ), adenovirus ( adv ), and bovine papilloma virus ( bpv ). mammalian cells may also require terminator sequences and poly a addition sequences and enhancer sequences which increase expression may also be included ; sequences which cause amplification of the gene may also be desirable . furthermore , sequences that facilitate secretion of the recombinant product from cells , including , but not limited to , bacteria , yeast , and animal cells , such as secretory signal sequences and / or prohormone pro region sequences , may also be included . these sequences are known in the art . nucleic acids encoding wild - type or variant polypeptides may also be introduced into cells by recombination events . for example , such a sequence can be introduced into a cell , and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene . other recombination - based methods such as nonhomologous recombinations or deletion of endogenous genes by homologous recombination may also be used . the nucleic acids of the present invention find use , for example , as probes for the detection of β4gal - t2 in other species and as templates for the recombinant production of peptides or polypeptides . these and other embodiments of the present invention are described in more detail below . the present invention encompasses isolated peptides and polypeptides encoded by the disclosed genomic sequence . peptides are preferably at least five residues in length . nucleic acids comprising protein - coding sequences can be used to direct the recombinant expression of polypeptides in intact cells or in cell - free translation systems . the known genetic code , tailored if desired for more efficient expression in a given host organism , can be used to synthesize oligonucleotides encoding the desired amino acid sequences . the phosphoramidite solid support method of matteucci et al ., 1981 , j . am . chem . soc . 103 : 3185 , the method of yoo et al ., 1989 , j . biol . chem . 764 : 17078 , or other well known methods can be used for such synthesis . the resulting oligonucleotides can be inserted into an appropriate vector and expressed in a compatible host organism . the polypeptides of the present invention , including function - conservative variants of the disclosed sequence , may be isolated from native or from heterologous organisms or cells ( including , but not limited to , bacteria , fungi , insect , plant , and mammalian cells ) into which a protein - coding sequence has been introduced and expressed . furthermore , the polypeptides may be part of recombinant fusion proteins . methods for polypeptide purification are well - known in the art , including , without limitation , preparative disc - gel elctrophoresis , isoelectric focusing , hplc , reversed - phase hplc , gel filtration , ion exchange and partition chromatography , and countercurrent distribution . for some purposes , it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification , such as , but not limited to , a polyhistidine sequence . the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid - phase matrix . alternatively , antibodies produced against a protein or against peptides derived therefrom can be used as purification reagents . other purification methods are possible . the present invention also encompasses derivatives and homologues of polypeptides . for some purposes , nucleic acid sequences encoding the peptides may be altered by substitutions , additions , or deletions that provide for functionally equivalent molecules , i . e ., function - conservative variants . for example , one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties , such as , for example , positively charged amino acids ( arginine , lysine , and histidine ); negatively charged amino acids ( aspartate and glutamate ); polar neutral amino acids ; and non - polar amino acids . the isolated polypeptides may be modified by , for example , phosphorylation , sulfation , acylation , or other protein modifications . they may also be modified with a label capable of providing a detectable signal , either directly or indirectly , including , but not limited to , radioisotopes and fluorescent compounds . the present invention encompasses antibodies that specifically recognize immunogenic components derived from β4gal - t2 . such antibodies can be used as reagents for detection and purification of β4gal - t2 . β4gal - t2 specific antibodies according to the present invention include polyclonal and monoclonal antibodies . the antibodies may be elicited in an animal host by immunization with β4gal - t2 components or may be formed by in vitro immunization of immune cells . the immunogenic components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems . the antibodies may also be produced in recombinant systems programmed with appropriate antibody - encoding dna . alternatively , the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains . the antibodies include hybrid antibodies ( i . e ., containing two sets of heavy chain / light chain combinations , each of which recognizes a different antigen ), chimeric antibodies ( i . e ., in which either the heavy chains , light chains , or both , are fusion proteins ), and univalent antibodies ( i . e ., comprised of a heavy chain / light chain complex bound to the constant region of a second heavy chain ). also included are fab fragments , including fab ′ and f ( ab ) 2 fragments of antibodies . methods for the production of all of the above types of antibodies and derivatives are well - known in the art . for example , techniques for producing and processing polyclonal antisera are disclosed in mayer and walker , 1987 , immunochemical methods in cell and molecular biology , ( academic press , london ). the antibodies of this invention can be purified by standard methods , including but not limited to preparative disc - gel elctrophoresis , isoelectric focusing , hplc , reversed - phase hplc , gel filtration , ion exchange and partition chromatography , and countercurrent distribution . purification methods for antibodies are disclosed , e . g ., in the art of antibody purification , 1989 , amicon division , w . r . grace & amp ; co . general protein purification methods are described in protein purification : principles and practice , r . k . scopes , ed ., 1987 , springer - verlag , new york , n . y . anti - β4gal - t2 antibodies , whether unlabeled or labeled by standard methods , can be used as the basis for immunoassays . the particular label used will depend upon the type of immunoassay used . examples of labels that can be used include , but are not limited to , radiolabels such as 32 p , 125 i , 3 h and 14 c ; fluorescent labels such as fluorescein and its derivatives , rhodamine and its derivatives , dansyl and umbelliferone ; chemiluminescers such as luciferia and 2 , 3 - dihydrophthal - azinediones ; and enzymes such as horseradish peroxidase , alkaline phosphatase , lysozyme and glucose - 6 - phosphate dehydrogenase . the antibodies can be tagged with such labels by known methods . for example , coupling agents such as aldehydes , carbodiimides , dimaleimide , imidates , succinimides , bisdiazotized benzadine and the like may be used to tag the antibodies with fluorescent , chemiluminescent or enzyme labels . the general methods involved are well known in the art and are described in , e . g ., chan ( ed . ), 1987 , immunoassay : a practical guide , academic press , inc ., orlando , fla . the following examples are intended to further illustrate the invention without limiting its scope . a : identification of cdna homologous to b4gal - t1 by analysis of est database sequence information . database searches were performed with the coding sequence of the human β4gal - t1 sequence ( masri et al ., 1988 ) using the blastn and tblastn algorithms against the dbest database at the national center for biotechnology information , usa . the blastn algorithm was used to identify ests representing the query gene ( identities of ≦ 95 %), whereas tblastn was used to identify non - identical , but similar est sequences . ests with 50 - 90 % nucleotide sequence identity were regarded as different from the query sequence . the results of tblastn searches were evaluated by visual inspection after elimination of ests regarded as identical to the query sequence (& lt ; 95 % nucleotide sequence identity ). ests with several apparent short sequence motifs and cysteine residues arranged with similar spacing were selected for further sequence analysis . initially , the identified ests ( 5 ′ sequence ) were used in blastn searches of the dbest database to search for overlapping ests ( 95 - 100 % identity in at least 30 bp ) ( fig1 ). if new ests were identified , the procedure was repeated and sequences merged . in addition , all identified ests were analysed in the unigene database in order to confirm that they were from the same gene transcript , and to select cdna clones with the longest inserts as well as identify additional ests with a non - overlapping 5 ′ sequence . composites of all the sequence information for each set of ests were compiled and analysed for sequence similarity to human β4gal - t1 . two partly overlapping ests were identified ( fig1 ). sequencing of the inserts revealed an open reading frame which potentially encoded a sequence similar to β4gal - t1 , but the 5 ′ sequence was shorter and without an initiation codon . further 5 ′ sequence was obtained by 5 ′ race using human fetal brain marathon - ready cdna ( clontech ) in combination with anti - sense primers eber102 ( 5 ′- gaaactgagccttactcaggc ) and eber104 ( 5 ′- tccacatcgctgaagatgaagc ) for 35 cycles at 95 ° c ., 45 sec ; 55 ° c ., 15 sec ; 68 ° c ., 3 min , using the expand kit enzyme ( boehringer mannheim ). the race products were cloned into the bamhi site of pt7t3u19 and multiple clones were sequenced . the entire sequence was confirmed by sequencing genomic p1 clones . an expression construct designed to encode amino acid residues 31 - 372 of β4gal - t2 was prepared by rt - pcr with mrna from colo205 cell line , using the primer pair eber100for ( 5 ′- tactttgacgtctacgcccag ) and eber114 ( 5 ′- gaaaacagagcccagctcag ) with bamh1 restriction sites ( fig2 ). the pcr product were cloned into the bamhi site of pacgp67 ( pharmingen ), and the construct sequenced to verify correct insertion and sequence . the plasmid pacgp67 - β4gal - t2 - sol was co - transfected with baculo - goldä dna ( pharmingen ) as described previously ( bennett et al ., 1996 ). recombinant baculo - virus were obtained after two successive amplifications in sf9 cells grown in serum - containing medium , and titres of virus were estimated by titration in 24 - well plates with monitoring of enzyme activities . controls included pacgp67 - β4gal - t3 - sol ( almeida et al ., 1997 ) and pacgp67 - galnac - t3 - sol ( bennett et al ., 1996 ). standard assays were performed in 50 ml total reaction mixtures containing 25 mm tris ( ph 7 . 5 ), 10 mm mncl 2 , 0 . 25 % triton x - 100 , 100 mm udp -[ 14 c ]- gal ( 2 , 300 cpm / nmol ) ( amersham ), and varying concentration of acceptor substrates ( sigma ) ( see table i for structures ). the soluble constructs were assayed with 5 - 20 ml of culture supernatant from infected cells , whereas the full length construct was assayed with 1 % triton x - 100 homogenates of washed cells . bovine milk β1 , 4gal - transferase ( sigma ) was used as control . assays used for determination of km of acceptor substrates were modified to include 200 mm udp -[ 14 c ]- gal , and assays for donor substrate km were performed with 2 mm ( for β4gal - t3 and bovine milk gal - t ) or 0 . 25 mm βglcnac - benzyl . reaction products were quantified by dowex - 1 chromatography . assays with hen egg ovalbumin ( sigma ), asialo - fetuin ( sigma ), and asialo - agalacto - fetuin ( sigma , treated with bgalactosidase ) were performed with the standard reaction mixture modified to contain 200 mm udp - gal , 54 mm nacl , and 0 . 5 mg ovalbumin . the transfer of gal was evaluated after precipitation by filtration through whatman gf / c glass fiber filters . c : stable expression of full coding sequence of βgal - t2 in cho cells . a cdna sequence encoding the full coding sequence of the putative β4gal - t2 gene was derived by rt - pcr using primers eber 120 ( 5 ′- agcggatccatgagcagactgctgggg - 3 ′) and eber 114 with bamhi restriction sites introduced . the pcr product was designed to yield a β4gal - t2 protein with a hydrophobic transmembrane retention signal in order to have the enzyme expressed and positioned in the appropriate golgi compartment of the transfected cell . the pcr product was inserted into the bamhi site of a mammalian expression vector pcdna3 ( invitrogen ), and the construct , pcdna3 - β4gal - t2 - mem , was transfected into cho and stable transfectants were selected . d : stable expression of the soluble form of bgal - t2 in cho cells . cdna pacgp67 - b4gal - t2 - sol containing the coding sequence of the putative soluble b4gal - t2 enzyme was cloned into the bamhi site of a modified mammalian expression vector pcdna3 ( invitrogen ). pcdna3 had been modified by insertion of an interferon signal peptide sequence into the kpni / bamhi site of ensuring secretion of the expressed product when cloned into the vector . the pcdna3γinf - β4gal - t2 - sol construct was transfected into cho and stable transfectants were selected . human multiple tissue northern blots were obtained from clontech . the soluble expression construct of β4gal - t2 was used as probe . the probe was random primed labelled using αp 32 dctp ( amersham ) and an oligo labelling kit ( pharmacia ). the blots were probed 18 hours at 42 ° c . as previously described ( bennett et al ., 1996 ), and washed 2 × 10 min at rt with 2 × ssc , 1 % na4p202 ; 2 × 20 min at 65 ° c . with 0 . 2 × ssc , 1 % sds , 1 % na 4 p 2 o 2 ; and once 10 min with 0 . 2 × ssc at rt . a human foreskin genomic p1 library ( dupont merck pharmaceutical company human foreskin fibroblast p1 library ) was screened using primer pair eber100 ( 5 ′- tgaaggaggatgccgcctatgac )/ eber102 ( 5 ′- gaaactgagccttactcaggc ). p1 clones were obtained from genome systems inc , and dna from p1 phages prepared as recommended by genome systems inc . the entire coding sequence of each gene was sequenced in full using automated sequencing ( abi377 , perkin elmer ) with dye terminator chemistry . intron / exon boundaries were determined by comparison with the cdna sequences optimising for the gt / ag rule ( breathnach and chambon , 1981 ). primer pairs as described in fig8 and 9 have been used for pcr amplification of individual coding sequence of the 6 exons . each pcr product was subcloned and the sequence of 10 clones containing the appropriate insert was determined assuring that both alleles of each individual are characterized . from the foregoing it will be evident that , although specific embodiments of the invention have been described herein for purposes of illustration , various modifications may be made without deviating from the spirit and scope of the invention . 1 . almeida , r ., amado , m ., david , l ., levery , s . b ., holmes , e . h ., merkx , g ., van kessel , a . g ., hassan , h ., bennett , e . p ., and clausen , h . ( 1997 ) a family of human b4 - galactosyltransferases : cloning and expression of two novel udp - galactose : b - n - acetylglucosamine b1 , 4 - galactosyltransferases , b4gal - t2 and b4gal - t3 . j . biol . chem ., 272 , 31979 - 31992 . 2 . asano , m ., furukawa , k ., kido , m ., matsumoto , s ., umesaki , y ., kochibe , n ., and iwakura , y . ( 1997 ) growth retardation and early death of b - 1 , 4 - galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells . embo j ., 16 , 1850 - 1857 . 3 . axford , j . s ., alavi , a ., bond , a ., and hay , f . c . ( 1994 ) differential b lymphocyte galactosyltransferase activity in the mrl mouse model of rheumatoid arthritis . autoimmunity ., 17 , 157 - 163 . 4 . bennett , e . p ., hassan , h ., and clausen , h . ( 1996 ) cdna cloning and expression of a novel human udp - n - acetyl - alpha - d - galactosamine . polypeptide n - acetylgalactosaminyltransferase , galnac - t3 . j . biol . chem ., 271 , 17006 - 17012 . 5 . breathnach , r . and chambon , p . ( 1981 ) organization and expression of eucaryotic split genes coding for proteins . ann rev biochem ., 50 , 349 - 383 . 6 . brew , k ., vanaman , t . c ., and hill , r . l . ( 1968 ) the role of alpha - lactalbumin and the a protein in lactose synthetase : a unique mechanism for the control of a biological reaction . proc natl acad sci usa ., 59 , 491 - 497 . 7 . d &# 39 ; agostaro , g ., bendiak , b ., and tropak , m . ( 1989 ) cloning of cdna encoding the membrane - bound form of bovine beta 1 , 4 - galactosyltransferase . eur j biochem ., 183 , 211 - 217 . 8 . fujita - yamaguchi , y . and yoshida , a . ( 1981 ) purification and characterization of human serum galactosyltransferase ( lactose synthetase a protein ). j . biol . chem ., 256 , 2701 - 2706 . 9 . furukawa , k ., matsuta , k ., takeuchi , f ., kosuge , e ., miyamoto , t ., and kobata , a . ( 1990 ) kinetic study of a galactosyltransferase in the b cells of patients with rheumatoid arthritis . int immunol ., 2 , 105 - 112 . 10 . gentzsch , m . and tanner , w . ( 1996 ) the pmt gene family : protein o - glycosylation in saccharomyces cerevisiae is vital . embo j ., 15 , 5752 - 5759 . 11 . hollis , g . f ., douglas , j . g ., shaper , n . l ., shaper , j . h ., stafford - hollis , j . m ., evans , r . j ., and kirsch , i . r . ( 1989 ) genomic structure of murine beta - 1 , 4 - galactosyltransferase . biochem biophys res comm ., 162 , 1069 - 1075 . 12 . keusch , j ., lydyard , p . m ., isenberg , d . a ., and delves , p . j . ( 1995 ) beta 1 , 4 - galactosyltransferase activity in b cells detected using a simple elisa - based assay . glycobiology ., 5 , 365 - 700 . 13 . kobata , a . ( 1992 ) structures and functions of the sugar chains of glycoproteins . eur j biochem ., 209 , 483 - 501 . 14 . kozak , m . ( 1992 ) regulation of translation in eukaryotic systems . ann rev cell biol ., 8 , 197 - 225 . 15 . lu , q ., hasty , p ., and shur , b . d . ( 1997 ) targeted mutation in beta1 , 4 - galactosyltransferase leads to pituitary insufficiency and neonatal lethality . develop biol ., 181 , 257 - 267 . 16 . malissard , m ., borsig , l ., di marco , s ., grutter , m . g ., kragl , u ., wandrey , c ., and berger , e . g . ( 1996 ) recombinant soluble beta - 1 , 4 - galactosyltransferases expressed in saccharomyces cerevisiae . purification , characterization and comparison with human enzyme . eur j biochem ., 239 , 340 - 348 . 17 . masri , k . a ., appert , h . e ., and fukuda , m . n . ( 1988 ) identification of the full - length coding sequence for human galactosyltransferase ( beta - n - acetylglucosaminide : beta 1 , 4 - galactosyltransferase ). biochem biophys res comm ., 157 , 657 - 663 . 18 . mengle - gaw , l ., mccoy - haman , m . f ., and tiemeier , d . c . ( 1991 ) genomic structure and expression of human beta - 1 , 4 - galactosyltransferase . biochem biophys res comm ., 176 , 1269 - 1276 . 19 . moscarello , m . a ., mitranic , m . m ., and vella , g . ( 1985 ) stimulation of bovine milk galactosyltransferase activity by bovine colostrum n - acetylglucosaminyltransferase i . biochim biophys acta ., 831 , 192 - 200 . 20 . nakazawa , k ., ando , t ., kimura , t ., and narimatsu , h . ( 1988 ) cloning and sequencing of a full - length cdna of mouse n - acetylglucosamine ( beta 1 - 4 ) galactosyltransferase . j biochem ., 104 , 165 - 168 . 21 . nakazawa , k ., furukawa , k ., kobata , a ., and narimatsu , h . ( 1991 ) characterization of a murine beta 1 - 4 galactosyltransferase expressed in cos - 1 cells . eur j biochem ., 196 , 363 - 368 . 22 . narimatsu , h ., sinha , s ., brew , k ., okayama , h ., and qasba , p . k . ( 1986 ) cloning and sequencing of cdna of bovine n - acetylglucosamine ( beta 1 - 4 ) galactosyltransferase . proc natl acad sci usa ., 83 , 4720 - 4724 . 23 . paquet , m . r . and moscarello , m . a . ( 1984 ) a kinetic comparison of partially purified rat liver golgi and rat serum galactosyltransferases . biochem j ., 218 , 745 - 751 . 24 . powell , j . t . and brew , k . ( 1974 ) the preparation and characterization of two forms of bovine galactosyl transferase . eur j biochem ., 48 , 217 - 228 . 25 . shaper , j . h ., joziasse , d . h ., meurer , j . a ., chou , t . - d . d ., schnaar , r . a ., and shaper , n . l . ( 1995 ) the chicken genome contains two functional non - allelic b1 , 4 - galactosyltransferase genes . glycoconjugate j ., 12 , 477 26 . shaper , n . l ., shaper , j . h ., meuth , j . l ., fox , j . l ., chang , h ., kirsch , i . r ., hollis , and gf . ( 1986 ) bovine galactosyltransferase : identification of a clone by direct immunological screening of a cdna expression library . proc natl acad sci usa ., 83 , 1573 - 1577 . 27 . shaper , n . l ., hollis , g . f ., douglas , j . g ., kirsch , i . r ., and shaper , j . h . ( 1988 ) characterization of the full length cdna for murine beta - 1 , 4 - galactosyltransferase . novel features at the 5 ′- end predict two translational start sites at two in - frame augs . j . biol . chem ., 263 , 10420 - 10428 . 28 . shaper , n . l ., meurer , j . a ., joziasse , d . h ., chou , t . d ., smith , e . j ., schnaar , r . a ., and shaper , j . h . ( 1997 ) the chicken genome contains two functional nonallelic b1 , 4 - galactosyltransferase genes : chromosomal assignment to syntenic regions tracks fate of the two gene lineages in the human genome . j . biol . chem ., 272 , 31389 - 31399 . 29 . sheares , b . t . and carlson , d . m . ( 1984 ) two distinct udp - galactose : 2 - acetamido - 2 - deoxy - d - glucose 4 beta - galactosyltransferases in porcine trachea . j . biol . chem ., 259 , 8045 - 8047 . 30 . shur , b . d . ( 1982 ) evidence that galactosyltransferase is a surface receptor for poly ( n )- acetyllactosamine glycoconjugates on embryonal carcinoma cells . j . biol . chem ., 257 , 6871 - 6878 . 31 . wilson , i . b ., platt , f . m ., isenberg , d . a ., and rademacher , t . w . ( 1993 ) aberrant control of galactosyltransferase in peripheral b lymphocytes and epstein - barr virus transformed b lymphoblasts from patients with rheumatoid arthritis [ see comments ]. j rheumatol ., 20 , 1282 - 1287 . met ser arg leu leu gly gly thr leu glu arg val cys lys ala val cys pro asp ser pro pro gly leu val gly arg leu leu ile glu phe thr ser pro met pro leu glu arg val gln arg glu asn pro gly val leu met gly gly arg tyr thr pro pro asp cys thr pro ala gln thr gly val tyr val ile asn gln his gly glu asp thr phe asn arg ala arg asn leu tyr arg cys gly asp gln pro arg his phe ala ile ala val ser gly leu ser lys ala gln phe leu arg ile asn gly phe pro pro leu phe thr asn ile thr val asp ile gly arg pro pro ser trp glu phe thr ser pro met pro leu glu arg val gln arg glu asn pro gly val leu met gly gly arg tyr thr pro pro asp cys thr pro ala gln thr val ala val ile ile pro phe arg his arg glu his his leu arg tyr gly val tyr val ile asn gln his gly glu asp thr phe asn ala ala 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