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A sequence highly homologous to β1,4-N-acetylgalactosaminyltransferase III (β4GalNAc-T3) was found in a database of human expressed sequence tags. The full-length open reading frame of the gene, β4GalNAc-T4 (GenBank accession number AB089939), was cloned using the 5′ rapid amplification of cDNA ends method. It encodes a typical type II transmembrane protein of 1039 amino acids having 42.6% identity with β4GalNAc-T3. The recombinant enzyme transferred N-acetylgalactosamine to N-acetylglucosamine-β-benzyl with a β1,4-linkage to form N,N′-diacetyllactosediamine as did β4GalNAc-T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to β4GalNAc-T3 in vitro, however, the tissue distributions of the two enzymes were quite different. These results indicated that the two enzymes have similar roles in different tissues. [Copyright &y& Elsevier]

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to the 120 kDa APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analysed the various glycans on the 120 kDa APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of the 4 possible N-glycosylation sites are occupied with fucosylated paucimannose {Man2–3(Fuc1–2)GlcNAc2-peptide} type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal “stalk” region of the protein. It is likely that 5–6 of the 13 sites are occupied, probably with simple {GalNAc-peptide} type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc rich, is the most likely binding site for Cry1Ac. [Copyright &y& Elsevier]

O128 is one of the most common atypical enteropathogenic Escherichia coli isolated from diarrhea patients worldwide. The primary structure of E. coli O128 repeat units has previously been determined as →3)-β-D-GalNAc-(1→4)-α-D-Gal-(1→3)-β-D-GalNAc-(1→6)-[α-L-Fuc-(1→2)]-β-D-Gal-(1→ pentasaccharide. Here we report the complete sequencing of E. coli O128 antigen biosynthesis gene cluster and its flanking regions. Comparative sequence analysis revealed the expected O128 antigen process genes, GDP-fucose biosynthesis genes and four potential glycosyltransferase genes responsible for the assembly of E. coli O128 antigen repeats. WbsJ was shown to encode an α-1,2-fucosyltransferase by enzymatic assays and nuclear magnetic resonance spectroscopy analysis. [Copyright &y& Elsevier]

The carbohydrate-binding site of galectin 1, a vertebrate β-galactoside-binding lectin, has a pronounced specificity for the βGal(1→3)- and βGal(1→4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6′-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3′-OSO3-LacNAc, 3′-OSO3-Galβ(1→3)GlcNAcβ1-OBzl and 3-OSO3-Galβ1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2′-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions. [ABSTRACT FROM AUTHOR]

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (∼25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a ‘bottle-brush’ model. [ABSTRACT FROM AUTHOR]