Glycoproteins are proteins that carry N- and O-glycosidically-linked carbohydrate chains of complex structures and functions. N-glycan chains are assembled in the endoplasmic reticulum and the Golgi by a controlled sequence of glycosyltransferase and glycosidase processing reactions involving dolichol intermediates. The assembly of O-glycans occurs in the Golgi and does not involve dolichol. For most reactions, families of glycosyltransferases exist; the expression of the individual enzymes within a family is often subject to complex regulation. The biosynthesis of N- and O-glycan is controlled at the level of gene expression, mRNA, enzyme protein activity and localization, and through substrate and cofactor concentrations at the site of synthesis. This complex regulation results in many hundreds of structures, the range of which varies in different species, cell types, tissue types, states of development and differentiation. In diseased cells, the relative proportions of these structures are often characteristically different from normal, and may be useful for the assessment of the stage of the disease and for diagnosis. Knowledge of disease-specific glycoprotein structures and their functions may be used therapeutically, in immunotherapy, in blocking cell adhesion or interfering with other binding or biological processes. Recently, some of the mechanisms underlying glycoprotein alterations in disease have been elucidated. This opens the possibility of an active interference in the disease process. The functions of glycans in diseased cells will become more clear with the tools of molecular biology and transgenic animal models.

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-␤-D-GlcUA-(134)-␤-D-Glc-(13] n ) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn 2؉ or Mg 2؉ with maximal levels seen with 10 -20 mM Mn 2؉ . High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.

Galactosyltransferases are important enzymes for the extension of the glycan chains of glycoproteins and glycolipids, and play critical roles in cell surface functions and in the immune system. In this work, the acceptor specificity and several inhibitors of bovine beta1,4-Gal-transferase T1 (beta4GalT, EC 2.4.1.90) were studied. Series of analogs of N-acetylglucosamine (GlcNAc) and GlcNAc-carrying glycopeptides were synthesized as acceptor substrates. Modifications were made at the 3-, 4- and 6-positions of the sugar ring of the acceptor, in the nature of the glycosidic linkage, in the aglycone moiety and in the 2-acetamido group. The acceptor specificity studies showed that the 4-hydroxyl group of the sugar ring was essential for beta4GalT activity, but that the 3-hydroxyl could be replaced by an electronegative group. Compounds having the anomeric beta-configuration were more active than those having the alpha-configuration, and O-, S- and C-glycosyl compounds were all active as substrates. The aglycone was a major determinant for the rate of Gal-transfer. Derivatives containing a 2-naphthyl aglycone were inactive as substrates although quinolinyl groups supported activity. Several compounds having a bicyclic structure as the aglycone were found to bind to the enzyme and inhibited the transfer of Gal to control substrates. The best small hydrophobic GlcNAc-analog inhibitor was found to be 1-thio-N-butyrylGlcNbeta-(2-naphthyl) with a K(i) of 0.01 mM. These studies help to delineate beta4GalT-substrate interactions and will aid in the development of biologically applicable inhibitors of the enzyme.

Dolichyl-phospho-mannose synthase, or C1DPmannose:dolichyLphosphate mannosyltransferase (EC 2.4.1.831, was solubilized from rat liver microsomes with 2 .O% Nonidet P-40 and the enzyme was further purified by column chromatography on DEAE-cellulose in the presence of 0.1% Nonidet P-40. The purified enzyme preparation (880-fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P-40, Triton X-100, octyl b-glucoside, or deoxycholate. Detergent-free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. These results suggest that dolichyl-P-mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non-bilayer structural organization in isolation. Heat-inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl-P. The PtdEtn-reconstituted enzyme catalyzed the reversible transfer of mannose from GDP-mannose to dolichyl-P. The K,,, for GDP-mannose was found to be 0.69 pM and the apparent K,,, for dolichyl-P was 0.3 pM. GMP, GDP, and GTP inhibited mannosyltransfer 50% at concentrations of 16 pM, 1.3 pM and 3 pM respectively.GDPmannose : dolichyl-phosphate mannosyltransferase (EC 2.4.1.83) is involved in the formation of a key intermediate in glycoprotein biosynthesis. Although a large number of investigations have been carried out with this enzyme [I -51, the enzyme has never been substantially purified from mammalian tissues. Heifetz and Elbein [6] reported a 5-fold purification of the enzyme from porcine aorta, but the enzyme was unstable and was only partially characterized. Babczinski et al. [7] and Haselbeck and Tanner [8] have purified the enzyme 280-fold from yeast membranes, and Carlo and Villemez have studied a soluble form of the enzyme isolated from Acurztkamoeha costrlluni [9]. The yeast enzyme was shown to be active, both in nonionic detergents and when reconstituted into liposomes composed of commercial grade plant lecithin [8]. We would now like to report a greatly improved purification of the mannosyltransferase from rat liver microsomes. In contrast to the aorta and yeast enzymes, the partially purified liver enzyme was quite stable. Crude microsomal and solubilized preparations of the enzyme were active in the presence of nonionic detergents, but more highly Abbreviations. PtdCho, phosphatidylcholine; PtdELn, phosphatidylethanolaminc: Ptdlns, phosphatidylinositol; PtdSer, phosphatidylserine. These and other lipid abbreviations (see Table 2) follow IUB Recommendations, see Eur. J. Biochem. 7Y, 11 -21 (1977) and Eur. J. Biochem. 104, 322 (1980). E n q m e s . Dolichyl-phospho-mannose synthase, GDPmannose : dolichyl-phosphate mannosyltransferase (EC 2.4.1 3 3 ) ; mannosyltransferase 11, GDPmannose : glycolipid 1,3-~-~-mannosyltransferase (EC 2.4.1.132).~~ purified enzyme f...

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