Biomedical subjects

H Clausen

Publications and source records attributed to H Clausen.

At least 37 records · Page 2Linked to original sources

PubMed2000

Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.

Animals↗

PubMed1999

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions.

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.

Animals↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Cloning and expression of a proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I. A seventh member of the human beta4-galactosyltransferase gene family.

A seventh member of the human beta4-galactosyltransferase family, beta4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of beta4Gal-T7 depicts a type II transmembrane protein with sequence similarity to beta4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the beta4Gal-T family. The genomic organization of beta4Gal-T7 was different from previous beta4Gal-Ts. Expression of beta4Gal-T7 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity with beta-xylosides, and the linkage formed was Galbeta1-4Xyl. Thus, beta4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein beta1, 4-galactosyltransferase; EC 2.4.1.133), which attaches the first galactose in the proteoglycan linkage region GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Sequence analysis of beta4Gal-T7 from a fibroblast cell line of a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, previously shown to exhibit reduced galactosyltransferase I activity (Quentin, E., Gladen, A., Rodén, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342-1346), revealed two inherited allelic variants, beta4Gal-T7(186D) and beta4Gal-T7(206P), each with a single missense substitution in the putative catalytic domain of the enzyme. beta4Gal-T7(186D) exhibited a 4-fold elevated K(m) for the donor substrate, whereas essentially no activity was demonstrated with beta4Gal-T7(206P). Molecular cloning of beta4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.

Amino Acid Sequence↗

PubMed1999

Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy.

The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.

Amino Acid Sequence↗

PubMed1999

Incorporation of N-acetylgalactosamine into consecutive threonine residues in MUC2 tandem repeat by recombinant human N-acetyl-D-galactosamine transferase-T1, T2 and T3.

An oligopeptide containing three consecutive Thr residues mimicking the tandem repeat portion of MUC2 (PTTTPLK) was investigated for the acceptor specificity to UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase isozymes, UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T1, T2 and T3. The enzymatic reaction products were fractionated by the reversed-phase high performance liquid chromatography, then characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry and by a peptide sequencing analysis. A maximum of two, one or three N-acetyl-D-galactosamine residues was transferred by UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T1, T2 or T3, respectively. The preferential orders of N-acetyl-D-galactosamine incorporation were Thr-2, then Thr-4 for UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T1, Thr-2 for UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T2 and Thr4, Thr-3, then Thr-2 for UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T3.

Acetylgalactosamine↗

PubMed1999

Partial vapor-phase hydrolysis of peptide bonds: A method for mass spectrometric determination of O-glycosylated sites in glycopeptides.

In this study we present a method for determination of O-glycosylation sites in glycopeptides, based on partial vapor-phase acid hydrolysis in combination with mass spectrometric analysis. Pentafluoropropionic acid and hydrochloric acid were used for the hydrolysis of glycosylated peptides. The reaction conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified by mass spectrometric analysis of the hydrolytic cleavage products. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosylation sites. Examples are shown with mannosyl- and mucin-type glycopeptides. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities from the reagents, thus allowing analysis of the reaction products without further purification both by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

Amino Acid Sequence↗

PubMed1999

Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-Acetylgalactosamine:Peptide N-acetylgalactosaminyltransferases. site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions.

In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitro addition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcalpha or Galbeta1-3GalNAcalpha at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position -6 (Thr-10) or -10 (Ser-6) and is inhibited by disaccharide at position -11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.

Acetylgalactosamine↗

PubMed1999

Regulation of I-branched poly-N-acetyllactosamine synthesis. Concerted actions by I-extension enzyme, I-branching enzyme, and beta1,4-galactosyltransferase I.

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching beta1, 6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta1, 6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta1, 4-galactosyltransferases (beta4Gal-Ts), beta4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta4Gal-TI and i-extension beta1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta1-->4GlcNAcbeta1-->3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta4Gal-TI adds a galactose poorly to beta1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta1, 3-N-acetylglucosaminyltransferase and beta4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.

Animals↗

PubMed1999

Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression.

Intestinal metaplasia is a well-established premalignant condition of the stomach that is characterized by mucin carbohydrate modifications defined by histochemical methods. The purpose of the present study was to see whether the expression of mucin core proteins was modified in the different types of intestinal metaplasia and to evaluate the putative usefulness of mucins as "molecular markers" in this setting. We used a panel of monoclonal antibodies with well-defined specificities to MUC1, MUC2, MUC5AC, and MUC6 to characterize the expression pattern of mucins. In contrast to normal gastric mucosa, the complete form or type I intestinal metaplasia (n = 20) displayed little or no expression of MUC1, MUC5AC, or MUC6 in the metaplastic cells and strong expression of the intestinal mucin MUC2 in the goblet cells of all cases. The incomplete forms of intestinal metaplasia, type II (n = 25) and type III (n = 16), expressed MUC1 and MUC5AC in every case, both in goblet and in columnar cells. MUC6 was also expressed in 16 cases of type II intestinal metaplasia and in 11 cases of type III intestinal metaplasia. The intestinal mucin MUC2 was expressed in every case of incomplete intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the different types of intestinal metaplasia allows the identification of two patterns: one defined by decreased levels of expression of "gastric" mucins (MUC1, MUC5AC, and MUC6) and expression of MUC2 intestinal mucin, which corresponds to type I intestinal metaplasia, and the other defined by coexpression of "gastric mucins" (MUC1, MUC5AC, and MUC6) together with the MUC2 mucin, encompassing types II and III intestinal metaplasia. Our results challenge the classical sequential pathway of intestinal metaplasia (from type I to type III via a type II intermediate step).

Antibodies, Monoclonal↗

PubMed1999

Control of O-glycan branch formation. Molecular cloning of human cDNA encoding a novel beta1,6-N-acetylglucosaminyltransferase forming core 2 and core 4.

A novel human UDP-GlcNAc:Gal/GlcNAcbeta1-3GalNAcalpha beta1, 6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-alpha-D-glucosamine:acceptor beta1, 6-N-acetylglucosaminyltransferase (beta1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a beta1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in beta1,6GlcNAc-transferases capable of forming core 2 O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

Amino Acid Sequence↗

PubMed1999

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family.

Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc-transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.

Animals↗

PubMed1999

An alpha2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas.

The MUC1 mucin is expressed on the luminal surface of most simple epithelial cells but in carcinomas, especially those of the breast and ovary, MUC1 is upregulated and aberrantly glycosylated. MUC1 contains a large amount of O-linked glycans which, in the mucin expressed by normal mammary epithelial cells, consist mainly of core 2 based structures carrying polylactosamine chains. However, the mucin expressed by breast carcinomas has shorter side-chains, often consisting of sialylated core 1 (Galbeta1-3GalNAc). in situ hybridization of primary breast tissue showed that a sialyltransferase (ST3Gal I), responsible for adding sialic acid to core 1 thereby terminating chain extension, is elevated in primary breast carcinomas when compared to normal or benign tissue. Furthermore, the level of mRNA expression encoding ST3Gal I is correlated to the intensity of staining seen with the antibody SM3, which specifically recognises underglycosylated, tumour associated MUC1. Thus, the aberrant glycosylation of MUC1 seen in breast carcinomas appears to be due, at least in part, to the elevation of ST3Gal I.

Breast Neoplasms↗

PubMed1999

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

Animals↗

PubMed1998

Synthesis of poly-N-acetyllactosamine in core 2 branched O-glycans. The requirement of novel beta-1,4-galactosyltransferase IV and beta-1,3-n-acetylglucosaminyltransferase.

Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Lex. Poly-N-acetyllactosamines in mucin-type O-glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 beta-1,6-N-acetylglucosaminyltransferase. Using a beta-1, 4-galactosyltransferase (beta4Gal-TI) present in milk and the recently cloned beta-1,3-N-acetylglucosaminyltransferase, the formation of poly-N-acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAcbeta1-->6(Galbeta1-->3)GalNAcalpha-->R. Since the majority of synthesized oligosaccharides contained N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the beta4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant beta4Gal-Ts, beta4Gal-TIV was found to be most efficient in the addition of a single galactose residue to GlcNAcbeta1-->6(Galbeta1-->3)GalNAcalpha-->R. Moreover, beta4Gal-TIV, together with beta-1,3-N-acetylglucosaminyltransferase, was capable of synthesizing poly-N-acetyllactosamine in core 2 branched oligosaccharides. On the other hand, beta4Gal-TI was found to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans. In contrast to beta4Gal-TI, the efficiency of beta4Gal-TIV decreased dramatically as the acceptors contained more N-acetyllactosamine repeats, consistent with the fact that core 2 branched O-glycans contain fewer and shorter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that beta4Gal-TIV is responsible for poly-N-acetyllactosamine synthesis in core 2 branched O-glycans.

Animals↗

PubMed1998

Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat.

A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.

Amino Acid Sequence↗

PubMed1998

Cloning of a novel member of the UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glycosphingolipid biosynthesis.

A novel putative member of the human UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, designated beta4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of beta4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other beta1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of beta4Gal-T4 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal beta-GlcNAc; however, in contrast to beta4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalacto-transferrin, or ovalbumin. The catalytic activity of beta4Gal-T4 with monosaccharide acceptor substrates, N-acetylglucosamine as well as glucose, was markedly activated in the presence of alpha-lactalbumin. The genomic organization of the coding region of beta4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in beta4Gal-T1, -T2, and -T3. beta4Gal-T4 represents a new member of the beta4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.

Amino Acid Sequence↗

PubMed1998

Structural analysis of peptide substrates for mucin-type O-glycosylation.

The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.

Amino Acid Sequence↗