Microassay for oligosaccharyltransferase: separation of reaction components by partitioning in detergent solution followed by ultrafiltration.

Oligosaccharyltransferase catalyzes the transfer of oligosaccharide from a lipid dolichol to asparagine acceptor sites on nascent polypeptides. We have developed an assay for this enzyme which is based on the specific distribution of the substrate and product of the reaction in detergent solution. GlcNAc-[3H]GlcNAc-PP-Dol was synthesized for use as a carbohydrate donor. Benzoyl-Asn-Leu-Thr-amide, a commercially available peptide, was used as the oligosaccharyltransferase glycan acceptor substrate. In the presence of Triton X-100, GlcNAc-[3H]GlcNAc-PP-Dol partitions into detergent micelles while glycosylated acceptor peptide partitions into the intermicellar aqueous compartment. Separation of GlcNAc-[3H]GlcNAc-PP-Dol and glycopeptide was achieved by ultrafiltration. With this method oligosaccharyltransferase activity in microsomal preparations could be measured with as little as 1 microgram of protein.

New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.

[Synthesis of moraprenylpyrophosphate disaccharides--precursors of the biosynthesis of Salmonella O-antigenic polysaccharides of C2 and C3 serotypes]. / Sintez moraprenilpirofosfatdisakharidov--predshestvennikov biosinteza O-antigennykh polisakharidov bakterii sal'monella serogrupp C2 i C3.

Disaccharides Man(alpha 1-3)Gal and Man(beta 1-3)Gal and their acetates have been synthesized, from which alpha-glycosyl phosphates were obtained by the MacDonald reaction. These compounds were converted into corresponding moraprenylpyrophasphate disaccharides by treating with moraprenylphosphoimidazolidate.

Transmembrane assembly of N-linked glycoproteins. Studies on the topology of saccharide synthesis.

The mechanism of synthesis of dolichol-linked saccharides has been examined using sealed hen oviduct microsomes. N,N'-Diacetylchitobiosylpyrophosphoryldolichol (chitobiosyl-lipid) was shown to be inaccessible to a soluble galactosyltransferase during its synthesis from UDP-GlcNac. In contrast, exogenous chitobiosyl-lipid added to microsomes is accessible to galactosyltransferase and does not appear to be unidirectionally translocated to the lumen. These results imply that assembly of chitobiosyl-lipid does not occur on the cytoplasmic face of the rough endoplasmic reticulum followed by a rapid unidirectional translocation to the lumen. Two lines of evidence rule out sugar nucleotide uptake and utilization within the lumen as a mechanism for chitobiosyl-lipid synthesis. GDP-mannose, which is involved in the elongation of chitobiosyl-lipid to form oligosaccharide-lipid, also does not permeate microsomal vesicles. This observation regarding GDP-mannose raises two important questions. 1) What is the topology of the oligosaccharide in sealed microsomes? and 2) how is the chitobiosyl-lipid elongated by addition of mannosyl units to form an oligosaccharide-lipid? During the synthesis of oligosaccharide-lipid in freeze-thawed microsomes, approximately 40% of the oligosaccharide is released from the lipid by hydrolysis and remains entrapped within the sealed microsomes. These data suggest that oligosaccharide-lipid is transiently present at the luminal face of the rough endoplasmic reticulum during its synthesis. Most of the enzymes involved in the synthesis of oligosaccharide-lipid can be inactivated by external proteolysis of microsomal vesicles. These data are discussed in terms of a model in which synthesis of chitobiosyl-lipid and its elongation to oligosaccharide-lipid are coupled processes that occur in a transmembrane multienzyme complex.