Mucin-type O-glycosylation is initiated by UDP-Nacetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear.In this report we characterized the specificity and kinetic properties of three purified recombinant GalNActransferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNActransferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.To date three human UDP-N-Acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (1-3) (GalNAc-transferases) 1 have been identified and characterized (1-4). Although the three GalNAc-transferases show similarities in primary structure with regard to predicted domain structures, sequence motifs, and conserved cysteine residues, the overall amino acid sequence similarity of only 45% suggests that the members of the GalNAc-transferase family have undergone significant changes during evolution. The genes encoding these enzymes are located on different chromosomes and have distinct structures, although some intron positions are conserved, suggesting an evolutionary relationship. 2 The genes are differentially expressed in organs as revealed by Northern analysis (1-3); in particular GalNAc-T3 exhibited a restricted expression pattern. One question addressed here is whether these three GalNAc-transferases are isoenzymes with redundant or unique functions.Hennet et al. (5) recently addressed this question by analyzing mice rendered deficient in a close homologue of GalNAc-T1 by gene targeting. No obvious phenotypic differences were observed and preliminary characterization of the residual GalNAc-transferase activity with a few substrates did not reveal differences in enzyme activities. There was a reduction in GalNAc-transferase activity in ES cells in which the gene was inactivated. It is difficult to assess the full significance of these findings because the enzyme deleted in these studies is not well characterized with respect to substrate specificity and tissue expression pattern. Disruption of Dol-P-Man:polypeptide mannosyltransferases which initiate O-gly...

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:␤-N-acetylglucosamine ␤-1,3-galactosyltransferase, designated ␤3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to ␤3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:␤-N-acetylglucosamine ␤-1,3-galactosyltransferase (␤3Gal-T2) with similar kinetic properties as ␤3Gal-T1. Another gene represented a UDP-galactose:␤-N-acetyl-galactosamine ␤-1,3-galactosyltransferase (␤3Gal-T4) involved in G M1 /G D1 ganglioside synthesis, and this gene was highly similar to a recently reported rat G D1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. ␤3Gal-T1 mRNA was expressed in brain, ␤3Gal-T2 was expressed in brain and heart, and ␤3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. ␤3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous ␤3-galactosyltransferase genes.

In order to investigate the expression of MUC5AC mucin in normal gastric mucosa and gastric carcinomas, we produced 3 monoclonal antibodies (MAbs) using a MUC5AC synthetic peptide. The immunohistochemical study was performed using one of these MAbs (CLH2) which reacted with the different designs of peptides based on the MUC5AC tandem repeat and with native and deglycosylated mucin extracted from gastric tissues. CLH2 immunoreactivity was restricted to foveolar and mucopeptic neck cells in normal gastric mucosa. Mucins are glycoproteins with a high degree of O-glycosylation which are major components of the mucous viscous gel covering the surfaces of epithelial tissues (for review, see Lesuffleur et al., 1994). Several human genes encoding distinct mucin proteins (designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7 and MUC8) have been identified by molecular cloning (for review, see Gendler and Spicer, 1995). Mucins have a common domain structure which includes one or more regions consisting of tandem repeats. These repeated regions are rich in serine and threonine residues which are potentially O-glycosylated (Gendler and Spicer, 1995).Among the mucins identified to date, MUC5AC (previously called MUC5A and MUC5C) was cloned from tracheobronchial (Aubert et al., 1991;Crepin et al., 1990) and stomach (Guyonnet Duperat et al., 1995) cDNA libraries. Northern blotting, in situ hybridization and immunohistochemical studies showed that MUC5AC is expressed in normal gastric mucosa (Audie et al., 1993;Carrato et al., 1994;DeBolos et al., 1995; Ho et al., 1995a,b;Sotozono et al., 1996). Moreover, these studies revealed that gastric epithelium has a unique organ and cell-type expression pattern which is characterized by high levels of MUC5AC expression in the surface foveolar cells and absence of MUC5AC expression in deep glands of the gastric body and pyloric glands of the antrum (Audie et al., 1993;Carrato et al., 1994;DeBolos et al., 1995; Ho et al., 1995a,b;Sotozono et al., 1996).Neoplastic transformation in the stomach has been reported to be associated with a decrease in MUC5AC expression Ho et al., 1995b). However, little is known about the expression pattern of MUC5AC in the different morphologic sub-types of gastric carcinoma, or about the putative relationship between MUC5AC expression and the pathologic features of the cases.In the present report, we describe the development of monoclonal antibody (MAb) CLH2 against a synthetic peptide based on the MUC5AC tandem repeat. The immunohistochemical properties of MAb CLH2 were characterized in several gastrointestinal and non-gastrointestinal normal tissues. MAb CLH2 was used to detect the expression of MUC5AC in 66 gastric carcinomas, which included a representative number of the different morphologic sub-types (intestinal, diffuse and atypical). The results we obtained indicate that the down-regulation of MUC5AC is dependent upon tumor type and stage. MATERIAL AND METHODS Synthetic peptidesThe synthetic peptides used in this study (Table I) were HPLC-pur...

Mucins are high molecular-weight glycoproteins involved in the protection and lubrication of respiratory, gastrointestinal, and reproductive tracts. Hypersecretory diseases such as cystic fibrosis (CF), chronic bronchitis, and asthma result in dysregulated levels of mucin production stemming from increased abundance of mucin-secreting cell types in the surface airway epithelium and submucosal glands. The isolation of at least nine mucin genes has prompted studies to characterize the cellular expression patterns of these mucins in normal and diseased tissues. In the present study, in situ hybridization and immunocytochemical methods were used to determine the cellular distribution of MUC5B and MUC7 expression in CF and non-CF human bronchus. Our findings indicate that MUC5B and MUC7 have expression patterns in human bronchial airways that are limited exclusively to submucosal glands. Specifically, MUC5B expression was confined to all mucous tubules, whereas MUC7 expression was seen in a subset of lysozyme expressing serous tubules of submucosal glands. Interestingly, heterogeneity of MUC7 expression between glands of the same bronchus ranged from 0 to 93% of serous tubules, suggesting that functional diversity may exist between glands within the same bronchial sample. No remarkable differences were observed in the expression patterns of MUC5B or MUC7 between CF (n ϭ 7) and non-CF (n ϭ 10) bronchial samples. In conclusion, MUC5B and MUC7 expressions define different cellular compartments within submucosal glands of human bronchus and lend insight into the heterogeneity of mucin production in the lung.