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A yeast mutant capable of producing Man 5 GlcNAc 2 human compatible sugar chains on glycoproteins was constructed. An expression vector for ␣-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/ retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro ␣-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an ␣-1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man 5 GlcNAc 2 . This is the first report of a recombinant S. cerevisiae able to produce Man 5 GlcNAc 2 -oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.Saccharomyces cerevisiae is useful for the production of recombinant proteins of biological interest because of the established expression system, and it can be easily grown in large quantities. Moreover, yeast share the early steps of the mammalian Asn-linked glycosylation pathway. However, the mature Asn-linked oligosaccharides of yeast are mannan glycans and are highly antigenic against mammals. Thus, it would be necessary to eliminate the antigenicity of the sugar chains when recombinant therapeutic glycoproteins are produced in yeast.Several genes concerned with the biosynthesis of yeast sugar chains have been cloned, and the glycosylation pathway of yeast has been clarified. The OCH1 gene encodes an ␣-1,6-mannosyltransferase that initiates ␣-1,6-polymannose outer chain formation on the Asn-linked inner oligosaccharide Man 8 GlcNAc 2 in S. cerevisiae (1). MNN1 has been proposed as the structural gene for the ␣-1,3-mannosyltransferase that elongates the outer chain and the inner core oligosaccharide (2, 3). The ⌬och1 mnn1 double mutant accumulated a single oligosaccharide moiety, Man 8 GlcNAc 2 , a high mannose-type structure (1). This mutant may be useful to produce recombinant therapeutic glycoproteins without any antigenicity toward humans.On the other hand, some glycoproteins of therapeutic value require complex-type sugar chains for their efficacy. Erythropoietin (EPO), 1 a hematopoietic glycoprotein factor produced in the kidney, has three complex-type Asn-linked sugar chains and one mucin-type sugar chain. It is reported that the composition and structure of each sugar chain affected the biological activity, the efficiency of secretion, and had profound effects on the half-life of EPO in the blood circulation (4). It seems that the most active form of the EPO molecule requires tetraantennary Asn-linked sugar chains (5) with full sialylation, to prevent serum clearance by the action of the hepatic asialoglycoprotein binding protein (6, 7). When EPO was expressed in the ⌬och1 mnn1 mutant yeast, the recombinant EPO should have high mannose-type oligosaccharides, which are trapped by the...

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The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and glycine ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained. Though 30 mM diethyl pyrocarbonate (DEP) also inactivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was modified by DEP, 50% of the activity was still retained. The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC. However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivation of the sucrose-cleaving activity. In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group. Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE. A fluorescent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala.(ABSTRACT TRUNCATED AT 250 WORDS)