Novel Quinic Acid Glycerates from Tussilago farfara Inhibit Polypeptide GalNAc-Transferase.

The discovery of a bioactive inhibitor tool for human polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), the initiating enzyme for mucin-type O-glycosylation, remains challenging. In the present study, we identified an array of quinic acid derivatives, including four new glycerates (1-4) from Tussilago farfara, a traditional Chinese medicinal plant, as active inhibitors of GalNAc-T2 using a combined screening approach with a cell-based T2-specific sensor and purified enzyme assay. These inhibitors dose-dependently inhibited human GalNAc-T2 but did not affect O-linked N-acetylglucosamine transferase (OGT), the other type of glycosyltransferase. Importantly, they are not cytotoxic and retain inhibitory activity in cells lacking elongated O-glycans, which are eliminated by the CRISPR/Cas9 gene editing tool. A structure-activity relationship study unveiled a novel quinic acid-caffeic acid conjugate pharmacophore that directs inhibition. Overall, these new natural product inhibitors could serve as a basis for developing an inhibitor tool for GalNAc-T2.

Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli.

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (ß-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (ß-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides.

Biochemical characterization and mutational analysis of silkworm Bombyx mori ß-1,4-N-acetylgalactosaminyltransferase and insight into the substrate specificity of ß-1,4-galactosyltransferase family enzymes.

Silkworm Bombyx mori is one of the insect hosts for recombinant protein production at academic and industrial levels. B. mori and other insect cells can produce mammalian proteins with proper posttranslational modifications, such as N-glycosylation, but the structures of N-glycans in B. mori are mainly high mannose- and paucimannose-type, while mammals also produce hybrid- and complex-type glycans. Recently, complex-type N-glycans whose structures are different from mammalian ones have been identified in some insect cell N-glycomes at very low levels compared with levels of high mannose- and paucimannose-type glycans. However, their functions and the enzymes involved in the biosynthesis of insect complex-type N-glycans are not clear, and complex-type N-glycans, except for N-acetylglucosamine-terminated glycans, are still not identified in the B. mori N-glycome. Here, we focused on the ß-1,4-galactosyltransferase family (also known as glycosyltransferase family 7, GT7) that contains mammalian ß-1,4-galactosyltransferase and insect ß-1,4-N-acetylgalactosaminyltransferase. A gene for a GT7 protein (BmGalNAcT) from B. mori was cloned, expressed in a soluble form using a silkworm expression system, and the gene product showed strict ß-1,4-N-acetylgalactosaminyltransferase activity but not ß-1,4-galactosyltransferase activity. A mutation in Ile298 or Ile310, which are predicted to be located in the active site, reduced its glycosyltransferase activity, suggesting that these residues and the corresponding residues are responsible for substrate specificity of GT7. These results suggested that BmGalNAcT may be involved in the complex-type N-glycans, and moreover, bioinformatics analysis revealed that B. mori might have an extra gene for a GT7 enzyme with different specificity in addition to the known insect GT7 glycosyltransferases.

Molecular cloning, expression, and characterization of UDP N-acetyl-α-d-galactosamine: Polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum.

Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, but effective treatments are lacking. Cryptosporidium employs mucin-like glycoproteins with O-glycans to attach to and infect host intestinal epithelial cells. The Tn antigen (GalNAcα1-Ser/Thr) is an O-glycan essential for these processes, as Tn-specific lectins and a Tn-specific monoclonal antibody block attachment to and infection of host cells in vitro. The enzymes in Cryptosporidium catalyzing their synthesis, however, have not been studied. Previously, we identified four genes encoding putative UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, expression, purification, and characterization of one of the four enzymes Cryptosporidium parvum (Cp)-ppGalNAc-T4. This enzyme contains the characteristic domains and motifs conserved in ppGalNAc-Ts and is expressed at multiple time points during in vitro infection. Recombinant soluble Cp-ppGalNAc-T4 was enzymatically active against an unmodified EA2 peptide suggesting that it may function as an "initiating" ppGalNAc-T. Cp-ppGalNAc-T4 also exhibited a strong preference for UDP-GalNAc over other nucleotide sugar donors and was active against unmodified and O-glycosylated versions of the C. parvum gp40-derived peptide, with a preference for the former, suggesting it may play a role in modifying this glycoprotein in vivo. Given the importance of mucin-type O-glycosylation in Cryptosporidium spp., the enzymes that catalyze their synthesis may serve as potential therapeutic targets.

Functional control of polypeptide GalNAc-transferase 3 through an acetylation site in the C-terminal lectin domain.

O-GalNAc glycans are important structures in cellular homeostasis. Their biosynthesis is initiated by members of the polypeptide GalNAc-transferase (ppGalNAc-T) enzyme family. Mutations in ppGalNAc-T3 isoform cause diseases (congenital disorders of glycosylation) in humans. The K626 residue located in the C-terminal ß-trefoil fold of ppGalNAc-T3 was predicted to be a site with high likelihood of acetylation by CBP/p300 acetyltransferase. We used a site-directed mutagenesis approach to evaluate the role of this acetylation site in biological properties of the enzyme. Two K626 mutants of ppGalNAc-T3 (T3K626Q and T3K626A) had GalNAc-T activities lower than that of wild-type enzyme. Direct and competitive interaction assays revealed that GalNAc recognition by the lectin domain was altered in the mutants. The presence of GlcNAc glycosides affected the interaction of the three enzymes with mucin-derived peptides. In GalNAc-T activity assays, the presence of GlcNAc glycosides significantly inhibited activity of the mutant (T3K626Q) that mimicked acetylation. Our findings, taken together, reveal the crucial role of the K626 residue in the C-terminal ß-trefoil fold in biological properties of human ppGalNAc-T3. We propose that acetylated residues on ppGalNAc-T3 function as control points for enzyme activity, and high level of GlcNAc glycosides promote a synergistic regulatory mechanism, leading to a metabolically disordered state.

Identification of a chondroitin synthase from an unexpected source, the green sulfur bacterium Chlorobium phaeobacteroides.

Glycosaminoglycans (GAGs) are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria Pasteurella multocida Type F and Escherichia coli K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, Chlorobium phaeobacteroides DSM 266, which was very similar (~62% identical) to the open reading frames of the known bifunctional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant E. coli-derived Chlorobium enzyme preparations were found to possess bona fide chondroitin synthase activity in vitro. This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in C. phaeobacteroides is unexpected for several reasons including (a) a free-living nonpathogenic organism should not "need" an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5-3 billion years ago and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.

An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2.

Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif, which is present in the R-type lectin group. The acetylation site K521 is part of the QKW motif of ß-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and αGalNAc-glycosylated mucin peptides indicated that the degree of interaction of lectin domain with αGalNAc depends on the peptide sequence of mucin. Studies of the inhibitory effect of various carbohydrates on the interactions of ppGalNAc-T2 with MUC1αGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for αGalNAc. K521Q mutation resulted in an enzyme activity lower than that of the wild-type ppGalNAc-T2, similar to the acetylation of ppGalNAc-T2. We conclude that an acetylation site in the QKW motif of the lectin domain modulates carbohydrate recognition specificity and catalytic activity of ppGalNAc-T2 for partially preglycosylated acceptors and a certain naked peptide. Posttranslational modifications of ppGalNAc-Ts, such as acetylation, may play key roles in modulating the functions of the R-type lectin domains in cellular homeostasis.

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.

[Prokaryotic expression and antibody preparation of human GALNT3-sol protein].

AIM: In order to detect the expression of GALNT3 in various tumor tissues, the prokaryotic expression vector of human GALNT3-sol (a truncation of GALNT3 being deleted of the hydrophobic trans-membrane domain) was constructed, and then the recombinant GALNT3-sol protein was expressed and purified from E.coli, followed by the preparation of polyclonal antibody against GALNT3-sol and characterization of its properties. METHODS: The human cDNA of GALNT3-sol (1 755 bp)was amplified from MKN45 cell line and cloned into expression vector pET5b/GALNT3-sol, then transformed into E.coli BL21(DE3), in which the GALNT3-sol protein was induced by IPTG and then purified by Electrophoresis.Mice were immunized with the purified protein and the anti-serum was collected at different time intervals.Properties of the anti-serum were further detected by ELISA and Western blot. RESULTS: The prokaryotic expression vector of pET15b/GALNT3-sol was constructed successfully.Human GALNT3-sol protein was expressed in E.coli after IPTG induction.The titer of the obtained anti-serum reached 1:25 600, and its specificity was proved by Western blot. CONCLUSION: Human GALNT3-sol protein can be successfully expressed in E.coli, and the specific anti-human GALNT3-sol antibody can be obtained by immunization of mice, which makes it possible to further investigate the role of GALNT3 in the progression of various tumors.

Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates.

A large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) catalyzes the first step of mucin-type protein O-glycosylation by transferring GalNAc to serine and threonine residues of acceptor polypeptides. The acceptor peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly characterized and poorly understood, despite the fact that mutations in two individual isoforms are deleterious to man and the fly. In this work a series of oriented random peptide substrate libraries, based on the GAGAXXXTXXXAGAGK sequence motif (where X = randomized positions), have been used to obtain the first comprehensive determination of the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 isoforms. ppGalNAc T-glycosylated random peptides were isolated by lectin affinity chromatography, and transferase amino acid preferences were determined by Edman amino acid sequencing. The results reveal common and unique position-sensitive features for both transferases, consistent with previous reports of the preferences of ppGalNAc T1 and T2. The random peptide substrates also reveal additional specific features that have never been described before that are consistent with the x-ray crystal structures of the two transferases and furthermore are reflected in a data base analysis of in vivo O-glycosylation sites. By using the transferase-specific preferences, optimum and selective acceptor peptide substrates have been generated for each transferase. This approach represents a relatively complete, facile, and reproducible method for obtaining ppGalNAc T peptide substrate specificity. Such information will be invaluable for identifying isoform-specific peptide acceptors, creating isoform-specific substrates, and predicting O-glycosylation sites.

Cloning and expression of a brain-specific putative UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase gene.

We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GalNAc-transferases (designated pt-GalNAc-T) showed that they contained structural features characteristic of the GalNAc-transferase family. It was also found that human pt-GalNAc-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmental disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GalNAc-T in brain in more detail, we first examined that of human pt-GalNAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GalNAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GalNAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GalNAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GalNAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.

Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5'-diphosphate-alpha-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferases T1 and T2: kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats.

A large family of uridine 5'-diphosphate (UDP)-alpha-N-acetylgalactosamine (GalNAc):polypeptide N-acetylgalactosaminyl transferases (ppGalNAc Ts) initiates mucin-type O-glycan biosynthesis at serine and threonine. The peptide substrate specificities of individual family members are not well characterized or understood, leaving an inability to rationally predict or comprehend sites of O-glycosylation. Recently, a kinetic modeling approach demonstrated neighboring residue glycosylation as a major factor modulating the O-glycosylation of the porcine submaxillary gland mucin 81 residue tandem repeat by ppGalNAc T1 and T2 [Gerken et al. (2002) J. Biol. Chem. 277, 49850-49862]. To confirm the general applicability of this model and its parameters, the ppGalNAc T1 and T2 glycosylation kinetics of the 80+ residue tandem repeat from the canine submaxillary gland mucin was obtained and characterized. To reproduce the glycosylation patterns of both mucins (comprising 50+ serine/threonine residues), specific effects of neighboring peptide sequence, in addition to the previously described effects of neighboring residue glycosylation, were required of the model. Differences in specificity of the two transferases were defined by their sensitivities to neighboring proline and nonglycosylated hydroxyamino acid residues, from which a ppGalNAc T2 motif was identified. Importantly, the model can approximate the previously reported ppGalNAc T2 glycosylation kinetics of the IgA1 hinge domain peptide [Iwasaki, et al. (2003) J. Biol. Chem. 278, 5613-5621], further validating both the approach and the ppGalNAc T2 positional weighting parameters. The characterization of ppGalNAc transferase specificity by this approach may prove useful for the search for isoform-specific substrates, the creation of isoform-specific inhibitors, and the prediction of mucin-type O-glycosylation sites.

Donor substrate regeneration for efficient synthesis of globotetraose and isoglobotetraose.

Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4Glc) and isoglobotetraose (GalNAcbeta1-->3Galalpha1-->3Galbeta1-->4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant beta-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans.

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal(beta)4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc(beta)4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase (Ce(beta)4GalNAcT) that synthesizes the LDN structure. Ce(beta)4GalNAcT is a member of the beta4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce(beta)4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDP-Gal, as a donor toward a variety of acceptor substrates containing terminal beta-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and (1)H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce(beta)4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic beta4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.

Natural and recombinant A and B gene encoded glycosyltransferases.

The human blood group A and B synthesizing enzymes are glycosyltransferases that catalyse the transfer of a monosaccharide residue from UDP-GalNAc and UDP-Gal donors, respectively, to alphaFuc1,2-Gal terminated blood group H acceptors. Extensive investigations of their substrate specificity and physical properties have been carried out since their initial discovery. These studies demonstrated a rigid specificity for the acceptor structure, crossover in donor specificity and immunological similarity along with chromatographic differences. Cloning of the enzymes has shown that they are highly homologous, differing in only four of their 354 amino acids. Changing the residues Arg176-->Gly, Gly235-->Ser, Leu266-->Met and Gly 268-->Ala converts the enzyme specificity from blood group A to blood group B glycosyltransferase. Structure function investigations have been carried out by systematic interchange and modification of these four critical amino acids. These studies have shown that donor specificity is attributed to the last two amino acids. Mutants have also been produced with greatly enhanced turnover rates as well as hybrid A/B enzymes that catalyse both reactions efficiently.

A novel human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates.

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase gene family, designated GalNAc-T7, was cloned and expressed. GalNAc-T7 exhibited different properties compared to other characterized members of this gene family, in showing apparent exclusive specificity for partially GalNAc-glycosylated acceptor substrates. GalNAc-T7 showed no activity with a large panel of non-glycosylated peptides, but was selectively activated by partial GalNAc glycosylation of peptide substrates derived from the tandem repeats of human MUC2 and rat submaxillary gland mucin. The function of GalNAc-T7 is suggested to be as a follow-up enzyme in the initiation step of O-glycosylation.

Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line.

We recently discovered a novel alpha-N-acetylgalactosaminyltransferase in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) and also in mouse mast cytoma cells (Lidholt et al., Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an alpha-GalNAc residue to the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, derived from proteoglycans. In this study, we characterized this enzyme using a preparation obtained from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl-Sepharose chromatography. Structural characterization by1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, as a substrate demonstrated that the enzyme was a UDP-GalNAc:GlcAbeta1-R alpha1,4-N -acetylgalactosaminyltransferase. This is the first identification of an alpha1,4-N-acetylgalactosaminyltransferase. Using N -acetylchondrosine GlcAbeta1-3GalNAc as an alternative substrate, the enzyme required divalent cations for the transferase reaction, with maximal activity at 20 mM Mn2+and exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM 2-( N -morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N -acetylchondrosine, the linkage tetrasaccharide-serine, and UDP-GalNAc were 1060 microM, 188 microM, and 27 microM, respectively. This suggested that the linkage tetrasaccharide-serine was a good acceptor substrate for the enzyme. In addition, the enzyme utilized glucuronylneolactotetraosylceramide GlcAbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcbeta1-1Cer but not sulfoglucuronylneolactotetraosylceramide GlcA(3-O -sulfate)beta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cbeta1-1Cer as acceptor substrates. The possibility of involvement of this enzyme in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.

Purification and characterization of UDP-GlcNAc:Galbeta1-4GlcNAcbeta1-3*Galbeta1-4Glc(NAc)-R(GlcNAc to *Gal) beta1,6N-acetylglucosaminyltransferase from hog small intestine.

A beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni2+-chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 Glc-PA. The apparent molecular weight of the purified enzyme was 76,000 under nonreducing conditions. The enzyme has a pH optimum at 7.0 and has no requirement for any divalent metal ions. The Km values for pyridylaminated lacto-N-neotetraose and UDP-GlcNAc were 0.96 and 2. 59 mM, respectively. For its activity, this enzyme was shown to have an absolute requirement of at least a complete LacNAc (LacNAc = Galbeta1-4GlcNAc) residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal LacNAc units. The data strongly suggest that this enzyme could be involved in generating branches to central positions of preformed as well as growing polylactosamine chains, but not in synthesizing the distal branches to growing polylactosamine chains.

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase from swine trachea epithelium.

UDP-GalNac: polypeptide N-acetylgalactosaminyltransferase from swine trachea epithelium was purified to homogeneity by procedures which included affinity chromatography on Sepharose 4B columns containing bound deglycosylated Cowper's gland mucin. The enzyme, purified 12,000-fold from microsomes with a yield of 40%, showed only a single band on dodecyl sulfate polyacrylamide gel electrophoresis. The homogenous enzyme has an apparent molecular mass of 70,000 Da, as determined by gel electrophoresis or gel filtration. The transferase has a broad pH optimum between 6.7-7.8 with maximal activity at pH 7.2, and required Mn2+ for activity with maximal activity at 5-7.5 mM. Higher concentrations of Mn2+, inhibited the enzyme. The purified transferase was specific for UDPGalNAc and glycosylated both threonine and serine residues in tryptic peptides prepared from deglycosylated Cowper's gland and swine and human trachea mucins. The apparent Km of the transferase for UDPGalNAc was 6.3 microM, and the Km values for deglycosylated Cowper's gland and human and swine trachea mucins were 0.83, 1.12 and 0.94 mg/ml, respectively. The Vmax of the purified enzyme was 2.1 micromol/min/mg with deglycosylated Cowper's gland mucin, as the glycosyl acceptor. However, the activities with peptides prepared from deglycosylated mucins by limited acid hydrolysis were 20-fold greater than the intact glycoprotein under identical conditions. The deglycosylated mucins and larger peptides aggregated with time of storage and precipitated from solution. Aggregation was accompanied by a corresponding loss of enzymatic activity even after dispersion of the aggregate by sonication. The deglycosylated mucins which were prepared by chemical treatment and periodate oxidation still contained about 20% of the N-acetylgalactosamine present in the intact mucin. When this residual amino sugar was removed by periodate oxidation the completely deglycosylated mucins became very poor substrates for the purified transferase. Data obtained in the current study indicate that the accessibility of serine and threonine in the polypeptide chains of mucin glycoproteins significantly influences the rate of glycosylation of these amino acids. The best substrates and affinity ligand for the enzyme were fragments of incompletely deglycosylated mucin polypeptide chains.