No abstract

The catalytical role of the hydroxy amino acid in the "marker sequence" Asn-Xaa-Thr(Ser) for the N-glycosylation step of glycoprotein formation was investigated by using a series of hexapeptides derived from Tyr-Asn-Gly-Xaa-Ser-Val by substituting threonine, serine, cysteine, valine and O-methylthreonine respectively for Xaa. The results, which were obtained with calf liver microsomal fractions as enzyme source and dolichyl diphosphate di-N-acetyl [14C] chitobiose as glycosyl donor showed that the threonine-, serine- and cysteine-containing derivatives could be glycosylated, although at very different rates, whereas the valine and O-methylthreonine analogues did not work as glycosyl acceptors. Replacement of threonine by serine resulted in a 4-fold decrease in Vmax, and about a 10-fold increase in Km for glycosyl transfer. Replacement of serine by cysteine again decreased acceptor activity 2-3-fold. The various results, taken together, indicate an absolute requirement for a hydrogen-bond-donor function in the side chain of the hydroxy amino acid of the "marker sequence" and furthermore, point to a considerable influence of the structure of this amino acid on binding as well as on the glycosyl transfer itself. In order to explain the observed differences in the glycosyl-transfer rates, a model is proposed with a hydrogen-bond interaction between the amide of asparagine as the hydrogen-bond donor and the oxygen of the hydroxy group of the hydroxy amino acid as the hydrogen-bond acceptor. The participation of the hydroxy group in the catalytic mechanism of glycosyl transfer in the kind of proton-relay system is discussed.

Many secretory and membrane proteins are glycoproteins carrying asparagine-linked (N-linked) oligosaccharides. There are two types of N-linked glycans, referred to as high-mannose and complex type, respectively. Biosynthesis of N-linked glycans of the complex type proceeds via a high-mannose intermediate. After the initial transfer of a high-mannose oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2 from a lipid carrier to the nascent polypeptide chain, trimming reactions take place. Trimming glucosidases remove the glucose residues quantitatively and mannosidases IA/B and II can remove all but three mannose residues. After trimming, terminal sugars such as N-acetylglucosamine, galactose, sialic acid and fucose may be added and result in the conversion to a glycan of the complex type. Because suitable inhibitors were lacking, it was difficult to assess the importance of the trimming reactions for proper intracellular traffic, modification reactions other than the addition of terminal sugars, or as regulatory steps in glycoprotein processing. Here we describe the action of 1-deoxymannojirimycin (1,5-dideoxy-1,5-imino-D-mannitol, dMM; Fig. 1) on the biosynthesis of IgM and IgD. dMM is the mannose analogue of 1-deoxynojirimycin (dNM; Fig. 1), itself a glucosidase inhibitor. We present evidence that dMM is a mannosidase inhibitor. In vivo dMM inhibits the equivalent of the mannosidase IA/B activities and blocks conversion of high-mannose to complex oligosaccharides. It is the first such inhibitor to be reported. Interference with the biosynthetic pathway of N-linked glycans could prove to be a powerful way to manipulate carbohydrate structure in vivo.

Trimming glucosidase 1 and I1 have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and I1 and the nonspecific cc-glucosidase(s) were inhibited by 1 -deoxynojirimycin with median inhibitory concentration of 3 pM, 20 pM, 12 pM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of I-deoxynojirimycin and formed the basis for the design of the affinity ligand.Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel eletrophoresis of the purified enzyme revealed a subunit molecullar mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was % 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 -'C in the presence of detergent (t,,, z 20 days) and showed high specificity for the hydrolysis of the terminal ( E 1,2)-linked glucose residue in the natural substrate Glc,-Man,-(GlcNAc),.During N-glycoprotein formation oligosaccharides of the composition Glc,-Man,-(GlcNAc), are preassembled on a lipid carrier (dolichyl diphosphate, Doil-PP) and subsequently transferred 'en bloc' onto target asprragine residues of the nascent polypeptide chain. The protein-bound oligosaccharides are then modified by a sequence of reactions, generating N-glycans with either high mannoge orland complex type structures. The trimming sequence is initiated by the stepwise removal ofthe three glucose units, which is catalysed by at least two specific glucosidases (glucosidase I and 11). Cleavage of the glucose residues is followed by an einzymatic hydrolysis of several ( a 1,2)-linked mannose residues, resulting in high mannosc structures, which may be preserved as such or further modified to complex type oligosaccharides, the latter process requiring the concerted action of presumably other rx-mannosidases and various glycosyltransferhses (for review, see [I]).Since glucose residues are generally not found in mature N-glycoproteins, it is suggested that their transient occurrence may have an important function in the regulation of oligosaccharide trimming and processing. First evidence that the presence of the Glc, unit may give a signal for the transfer of the lipid-linked precursor oligosaccharide to protein, came from studies ofTurco et al. [2], who demonstrated that, at least under conditions in vitvo, the Glc,-containing structure is transferred more rapidly to protein acceptors than the intermediate devoid of glucose. A similar regulatory function was recently put forward by Spiro et al. [3], who discussed that the extent of N-glycosylation might depend...

The effects of 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), inhibitors of oligosaccharide trimming glucosidase I and mannosidase I, respectively, on the biosynthesis of vesicular stomatitis virus G protein, influenza virus hemagglutinin, and human class I histocompatibility antigens were investigated. Although the oligosaccharides of these membrane glycoproteins were greatly altered, neither dNM nor dMM interfered with their surface expression, as determined by a variety of assays, including accessibility to proteases and antibodies; neither did these drugs inhibit production of infectious virus particles.