UDP-N-acetyl-D-galactosamine as a donor substrate for the glycosyltransferase encoded by the B gene at the human blood group ABO locus

The properties of the enzyme in the serum of blood group B individuals that catalyses the transfer of small amounts of N-acetyl-D-galactosamine to H-active precursor structures were compared with those of the blood group B gene-associated alpha-(1----3)-D-galactosyltransferase and with the blood group A gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases in the serum of blood group A1 and A2 individuals. The biosynthetic products formed by the enzyme in B serum were identical with the A-active structures synthesised by the A1 and A2 gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases but the enzyme differed from the A1 and A2 transferases in its apparent Km for UDP-N-acetyl-D-galactosamine, its heat susceptibility, its failure to bind to Sepharose 4B, and its adsorption to H-active sites on group O red cell ghosts under conditions which bind the B transferase but fail to adsorb the A1 and A2 transferases. The correlation between the levels of alpha-(1----3)-D-galactosyltransferase and alpha-(1----3)-N-acetyl-D-galactosaminyltransferase activities in all the group B serum samples tested, the maintenance of the same ratio of activities after successive cycles of binding to group O red cell ghosts, the retention of the ability to convert blood group O to A-active cells after treatment of the serum with Sepharose 4B, and the failure to detect any comparable activity in group O serum samples tested under the same conditions indicated that the enzyme in group B serum that utilises UDP-N-acetyl-D-galactosamine to make blood group A-active structures is the B gene-associated alpha-(1----3)-D-galactosyltransferase.     

An enzyme basis for blood type A intermediate status.

The blood type A is known to be subclassified as A1, A2, and A1-A2 intermediate (Aint), depending upon red cell agglutinability with anti-A1 and anti-H lectins. Approximately 80% of the blood group H-sites remained unglycosylated in type Aint erythrocyte membranes. Plasma from Aint individuals contains a special blood group GalNAc transferase (UDP-GalNAc:2''-fucosylgalactoside-alpha-3-N-acetylgalactosaminyl transferase), which is different from the enzyme in A1 plasma and the enzyme in A2 plasma. A1-enzyme has strong affinity to UDP-GalNAc and 2''-fucosyllactose, A2-enzyme has low affinity to both substrates, and Aint-enzyme has strong affinity to UDP-GalNAc and very low affinity to 2''-fucosyllactose, which is a soluble analog of the H-substances. The low degree of glycosylation of the blood group H-sites due to the low affinity of Aint-enzyme with the H-substances can account for the lower A activity and higher H activity in Aint red cells than in A1 red cells. The blood group A allele can be subdivided into three common alleles, A1, A2, and Aint, each controlling the formation of different types of blood group GalNAc transferases.     

Isolation of a porcine UDP-GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1

In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an alpha(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl-alpha(1,2)galactopyranoside; the enzyme did not use phenyl-beta-D-galactopyranoside (phenyl-beta-D-Gal) as an acceptor. Because the alpha(1,3)GalNAc transferase gene product requires an alpha(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the alpha(1,2)fucosyltransferase gene product is necessary for the functioning of the alpha(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. ABBREVIATIONS: CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fucalpha(1,2)Gal: fucosyl-alpha(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Galalpha(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl-beta-D-Gal: phenyl-beta-D-galactopyranoside; R: Galbeta1-4Glcbeta1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine     

Separation of blood group A-active oligosaccharides by high-pressure liquid affinity chromatography using a monoclonal antibody bound to concanavalin A silica

A column for high-pressure liquid affinity chromatography is prepared by binding a murine monoclonal anti-blood group A antibody of IgM isotype to concanavalin A-coated silica particles. The column specifically retards blood group A-active oligosaccharides with the nonreducing immunodominant trisaccharide sequence, GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1- ..., and separates three A-active oligosaccharides with different core structures. Retention of the oligosaccharides on the column diminishes with increasing temperatures, permitting thermal elution in the range 25-50 degrees C.     

Characterization and partial purification of beta-N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Urine from Sd(a+) individuals was found to contain a beta-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3'-sialyllactose and glycoproteins carrying the terminal NeuAc alpha-3Gal beta group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn2+ but not for detergents. The Km for UDP-GalNAc was 66 X 10(-6) M, using fetuin as an acceptor. Like beta-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [3H]GalNAc to 3'-sialyllactose, we propose that the enzyme attaches GalNAc in beta-anomeric configuration to O-4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a-) donor lacking beta-GalNAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the beta-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171-184) the urinary transferase is able to transfer beta-GalNAc to the NeuAc alpha-3Gal beta-3(NeuAc alpha-6)GalNAc chains bound to the native glycophorin.     

Enzymatic synthesis of blood group A and B trisaccharide analogues

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.     

A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover

The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF-binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.     

一株人抗人A—血型物质单克隆抗体特异性的研究

本文对一株人抗人A-血型物质单克隆抗体,用定量免疫沉淀法以及ELISA研究其与多种单糖、双糖及寡糖的反应性,从而确定了其结合部位的结构特异性。实验发现其结合部位互补于含有双分子岩藻糖残基的A-t糖:这一研究进一步强调了含有双分子岩藻糖残基的A血型抗原决定簇的重要性。     

Purification of Forssman and human blood group A glycolipids by affinity chromatography on immobilized Helix pomatia lectin

A method for the affinity purification of intact glycolipids having nonreducing terminal alpha 1-3 linked N-acetylgalatosamine residues has been developed. This technique relies on the retention of the carbohydrate-binding specificity of immobilized Helix pomatia lectin in aqueous solutions of tetrahydrofuran. Both Forssman glycolipid and a mouse blood group A-active hexaosylceramide were bound by columns of the lectin equilibrated in a solvent containing 95% tetrahydrofuran and 5% water. After application of a step gradient of increasing water content up to 50%, the specifically bound glycolipids were eluted in solvent containing N-acetylgalactosamine. The Forssman and A-active glycolipids were similarly purified in a single chromatographic step from total lipid extracts of sheep and human type A erythrocyte stroma, respectively. Nonspecifically bound lipids and glycolipids were eluted from this column by simply increasing the water content of the eluting buffer. The extension of this method to other carbohydrate-binding proteins including lectins and monoclonal antibodies may provide a rapid purification of glycolipids based on their carbohydrate structures.     

Further characterization of type 2 and type 3 chain blood group A glycosphingolipids from human erythrocyte membranes

Blood group A glycosphingolipids with slow chromatographic mobilities have been separated systematically with an improved chromatographic procedure, and their structures have been analyzed by application of a panel of monoclonal antibodies defining A determinants carried by type 1, type 2, type 3, and type 4 carbohydrate chains as well as by 1H NMR spectroscopy and methylation analysis. Of several A-active fractions, previously termed Aa, Ab, Ac, and Ad, in decreasing order of thin-layer chromatographic mobility, the third fraction (Ac) was characterized as containing one type 3 chain A component and one type 2 chain A component without branching, which have been termed type 3 chain Ab and nor-Ac, respectively. (Formula: see text). The major component present in the fourth A-active fraction (Ad) was isolated and characterized as a branched type 2 chain glycolipid formerly termed Ac. The major component in the fifth A-active fraction (Ae) was identified as a branched type 2 chain A previously termed Ad. The structures of Ac (n = 1) and Ad (n = 2) are (Formula: see text).     

Separation and partial sequence analysis of blood group A-active oligosaccharides by affinity chromatography using monoclonal antibodies

An affinity column containing an anti-blood group A monoclonal antibody coupled to Sepharose beads specifically retards oligosaccharides with the nonreducing trisaccharide sequence GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-R. Three A-active oligosaccharides, A-tetra, A-penta, and A-hepta, elute as retarded peaks, well-separated from unbound sugars. A-hepta, which contains a difucosylated type 1 (Leb) core structure, elutes much later than A-tetra or A-penta and can be completely separated from the latter oligosaccharides by affinity chromatography. The order of elution of the oligosaccharides agrees with their previously determined specific molar activities as inhibitors of quantitative immune precipitation [H.-T. Chen, and E. A. Kabat, (1985) J. Biol. Chem. 260, 13208-13217]. Treatment of A-hepta with Charonia lampas alpha-galactosaminidase abolishes its binding by the anti-A affinity column and converts it to a Leb-active oligosaccharide (lacto-N-difucohexaose I) that is specifically retarded on a second affinity column containing an anti-Leb monoclonal antibody.     

Complete purification and characterization of alpha-3-N-acetylgalactosaminyltransferase encoded by the human blood group A gene

Human alpha-3-N-acetylgalactosaminyltransferase has been purified 27,000,000-fold from A1 plasma by (NH4)2SO4 fractionation and affinity chromatography on Sepharose 4B, anti-human group O plasma antibodies-Sepharose 4B, and Blue Dextran-Sephadex G-25. A modified procedure in the Sepharose 4B step was developed by batch adsorption and desorption experiments. Cibacron Blue F3G-A, the chromophore of Blue Dextran, was found to bind to the enzyme. UDP is an effective inhibitor of this binding. The pure transferase has an apparent molecular weight of 35,000 as judged by SDS-PAGE in the presence of a reducing agent. The specific activity is 16 pmol/min.ng enzyme, which is comparable to that (30 pmol/min.ng enzyme) of alpha-3-N-acetylgalactosaminyltransferase from porcine submaxillary glands [Schwyzer and Hill (1977) J. Biol. Chem. 252, 2338-2355]. The apparent Km values for UDP-GalNAc, 2'-fucosyllactose, and lacto-N-fucopentaose I are 13, 270, and 350 microM, respectively. The reaction velocity was found to fall off again at high concentrations of oligosaccharide acceptor substrates. The apparent Ki values for UDP and UDP-galactose are 8.6 and 6.2 microM, respectively. The pure enzyme also catalyzes the transfer of galactose in alpha-linkage to 2'-fucosyllactose though the transfer rate of galactose is much lower than that of N-acetylgalactosamine.     

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66

The membrane-bound UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66, has been purified 48,100-fold, mainly by affinity chromatography in aqueous Triton X-100 on apomucin (deglycosylated bovine submaxillary mucin) coupled to Sepharose. The purified preparation behaved homogeneously on gel filtration on Sephadex G-150 in aqueous Triton X-100 and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 55,000. The enzyme requires Mn2+, and only UDP-GalNAc served as a sugar donor. Apomucin, A1 protein, kappa-casein, apofetuin, and apoantifreeze glycoproteins served as acceptors, but the rate and amount of the transfer varied considerably from one acceptor to another. The transfer reaction terminated at the level of glycosylation of from only a few to at most about 40% of the serine plus threonine residues from which mucin-type oligosaccharides had been removed. This indicates that the transferase requires a certain conformation surrounding the acceptor site, but suggests also that a special mechanism may be functioning in vivo for frequent glycosylation of the abundant serine plus threonine residues of mucins. Lacto-N-fucopentaose I, ceramide di- and trihexosides, and globoside were not acceptors.     

Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli

Large amounts of globotriose (Galalpha-4Galbeta-4Glc) are shown to be produced by the high cell density culture of an Escherichia coli strain over-expressing the Neisseria meningitidis lgtC gene for alpha-1,4-Gal transferase. The strain which was devoid of both alpha and beta galactosidase activity was fed with glycerol as the energy and carbon source and with lactose as precursor for globotriose synthesis. After complete exhaustion of lactose, globotriose could serve as an alternative acceptor for LgtC and the formation of a series of polygalactosylated compounds was observed. The system was extended to the synthesis of globotetraose (GalNAcbeta-3Galalpha-4Galbeta-4Glc) by overexpressing two additional genes: lgtD from Haemophilus influenzae Rd which encodes a beta-1,3-GalNAc transferase and wbpP from Pseudomonas aeruginosa which encodes a UDP-GalNAc C4 epimerase. Globotetraose could also be produced from exogenous globotriose which was shown to be actively taken up by the cells.     

Immunolocalization of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and blood group A substance in the trans-tubular network of the Golgi apparatus and mucus of intestinal goblet cells

The subcellular distribution of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and its product was studied in human intestinal goblet cells by immunoelectron microscopy. The O-glycosylation step yielding blood group A-active glycoconjugates occurred in the trans region of the Golgi apparatus as indicated by the presence of immunolabel for both antigens. In the Golgi apparatus, immunoreactive alpha 1,3N-acetylgalactosaminyltransferase was detectable in trans cisternae and in the trans-tubular network which was found to be continuous with the cisternal stack and exhibited acid phosphatase activity. This demonstrates that in intestinal goblet cells (i) the trans-tubular network does not constitute a compartment distinct from trans cisternae, and (ii) structures corresponding to GERL are structurally and functionally part of the Golgi apparatus. In addition to immunolabel for transferase at the inner surface of the cisternal membranes, luminally located immunolabel indicating the presence of free, not membrane-associated transferase became first detectable in the trans-tubular network and early forming mucus droplets contained therein. Further, the content of mature mucus droplets as well as the extracellular mucus layer were labeled. Absence of immunolabel in blood group 0 subjects lacking the blood group A gene specified transferase and the apparent non-reactivity of the antibodies with carbohydrate epitopes indicates that free alpha 1,3N-acetylgalactosaminyltransferase is present in mucus droplets and becomes secreted by intestinal goblet cells.     

A血型单克隆抗体的抗原结合部位结构多样性研究

应用杂交瘤技术,以Ａ型红细胞,Ａ１血型物质ＭＳＭ（Ａ１）和Ａ－ＲＢＣ＋ＭＳＭ（Ａ１）为免疫原,制备了一组抗人Ａ血型单克隆抗体：Ａ１２１８,Ｂ５７,ＤＥ９２３－Ｇ８,Ｄ２８６－Ｅ１２经Ｔａｋａｔｓｙ微量血细胞凝集试验证明：这组单抗仅能凝集Ａ１,Ａ２及ＡＢ型红细胞,不能凝集Ｂ,Ｏ型红细胞．采用ＥＬＩＳＡ定量抑制试验法,精确测定了它们抗原结合部位的结构,互补于Ａ活性寡糖。Ａ１２１８互补于具有双岩藻糖结构的Ａ活性五糖（Ａ－Ｐｅｎｔａ）;Ｂ５７,ＤＥ９２３－Ｇ８互补于具有单岩藻糖结构的Ａ活性六糖（Ａ－Ｈｅｘａ）;而Ｄ２８６－Ｅ１２则互补于具有单岩藻糖的Ａ活性四糖（Ａ－Ｔｅｔｒａ）．结果表明：血凝特异性相同的抗Ａ单抗,其抗原结合部位的结构可呈现多样性。即Ａ活性寡糖的糖基组成数目和含有岩藻糖数目均可不相同,各种抑制剂对不同单抗的抑制作用强弱也不相同。     

Purification and characterization of UDP-N-acetylgalactosamine: globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase, a synthase of human blood group P antigen, from canine spleen

A UDP-N-acetylgalactosamine:globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase which catalyzes the conversion of human blood group Pk antigen into P antigen has been purified over 18,000-fold in 4% yield from a Triton X-100 extract of canine spleen microsomes by affinity chromatography on UDP-hexanolamine-Sepharose and globotriaosylceramide acid-Sepharose. The purified enzyme migrates as two major bands with apparent molecular weights of 64,000 and 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Mn2+ was required for activity, and the pH optimum was 6.9. Km values for UDP-GalNAc and globotriaosylceramide were 14 and 2.5 microM, respectively. Studies on substrate specificities indicate that the preferred substrates have the general structure Gal alpha 1-4Gal-OR in which the nature of the R moiety has relatively little effect on activity. An antibody against the purified enzyme eliminated the activity of the enzyme, but did not neutralize the alpha-3-N-acetylgalactosaminyltransferase involved in the biosynthesis of Forssman glycolipid.     

Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.     

Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from porcine submaxillary glands was purified to electrophoretic homogeneity. IgG prepared from antisera against the pure enzyme immunoprecipitated the transferase in Triton X-100 extracts of submaxillary glands. The submaxillary transferase is a membrane-bound enzyme in contrast to the pure bovine colostrum enzyme, which is soluble in the absence of detergents. Both transferases have similar properties but also differ significantly. Examination of the acceptor substrate specificity of the submaxillary gland transferase showed that it specifically transferred N-acetylgalactosamine from UDP-GalNAc to the hydroxyl group of threonine and was devoid of transferase activity toward serine-containing peptides. These results imply that more than one transferase is involved in forming the GalNAc-threonine and the GalNAc-serine linkages found in O-linked oligosaccharides in glycoproteins. The amino acid sequence adjacent to glycosylated threonine residues may influence the rate of glycosylation by the pure transferase. For example, the second threonine residue in the sequence, Thr-Thr, appears to be glycosylated about twice as fast as the first and more rapidly than single, isolated threonine residues. However, no unique consensus sequence for glycosylation of threonine residues is evident, and any accessible threonine residue appears to be a potential acceptor substrate.