A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190-fold from calf liver crude microsomes by a four-step procedure involving (a) differential salt/ detergent extraction, (b) affinity chromatography on AH-Sepharose 4B with N-5-carboxypentyl-1-deoxymannojirimycin as ligand, (c) ConA-Sepharose and (d) DEAE-Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA-Sepharose, indicating the absence of highmannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2 + being most effective. It is inhibited by 1 -deoxymannojirimycin (dMM), N-methyl-dMM and N-5-carboxypentyl-dMM with Ki = 7 pM, 75 pM, and 140 pM, respectively.Man9 mannosidase cleaves three of the four a1 ,2-linked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrolyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl a-mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N-linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Gl~),(Man),(GlcNAc)~ intermediate, we assume that the enzyme is located in the endoplasmic reticulum.The structural diversity of N-linked oligosaccharides is the result of extensive modifications of a common (Gl~)~(Man),-(GlcNAc)2 precursor which is preassembled on a lipid carrier and, after transfer en bloc to the polypeptide chain, processed to high-mannose, hybrid and/or complex-type oligosaccharides. Processing begins with the stepwise removal of the three glucose residues by presumably two enzymes, glucosidase I and glucosidase 11. The resulting high-mannose intermediate is either preserved as such or acted upon by several u-mannosidases before other sugars, such as N-acetylglucosamine, galactose, sialic acid and fucose, are added [I].Up to now, four different mannosidase activities assumed to be involved in the mannose trimming reactions (endoplasmic reticulum mannosidase, Golgi mannosidase IA, IB and 11), have been identified and partially characterized on the basis of differences in their detergent extractability, response toward various inhibitors and some enzymatic properties [2-61. Here, we report on the isolation and purification to homogeneity of a processing mannosidase (Man9 mannosidase) from calf liver microsomes. Though similar in some respects, the calf liver enzyme differs from the abovementioned mannosidases especially in its substrate specificity. Thus, Man9 mannosidase hydrolyses three of the four a1,2-linked mannose residues from the natural (Man)9(GlcNAc)2 oligosaccharide, but does not cleave the remaining (Man)6-(GlcNAc)2 structure and is not active against aryl a-mannoCorrespondence to E. Bause, Institut fur Biochemie, Universitat Koln, Ziilpicher Stra...

Trimming glucosidase I has been purified about 400‐fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N‐glycoprotein as shown by its binding to concanavalin A—Sepharose and its susceptibility to endo‐β‐N‐acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non‐specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N‐linked high‐mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal α1–2‐linked glucose residue from natural Glc3‐Man9‐GlcNAc2, but is inactive towards Glc2‐Man9‐GlcNAc2 or nitrophenyl‐/methyl‐umbelliferyl‐α‐glucosides. The enzyme displays a pH optimum close to 6.4. does not require metal ions for activity and is strongly inhibited by 1‐deoxynojirimycin (Ki∼ 2.1 μM), N,N‐dimethyl‐1‐deoxynojirimycin (Ki∼ 0.5 μM) and N‐(5‐carboxypentyl)‐1‐deoxynojirimycin (Ki∼ 0.45 μM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross‐react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross‐reactivity.

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Man,-mannosidase, an a1 ,2-specific enzyme located in the endoplasmic reticulum and involved in N-linked-oligosaccharide processing, has been isolated from crude pig-liver microsomes and its substrate specificity studied using a variety of free and peptide-bound high-mannose oligosaccharide derivatives. The purified enzyme displays no activity towards synthetic a-mannosides, but removes three ctl,2-mannose residues from the natural Man,-(GlcNAc), substrate (M9). The a1 ,Zrnannosidic linkage remaining in the M6 intermediate is cleaved about 40-fold more slowly. Similar kinetics of hydrolysis were determined with Man,-(GlcNAc)2 N-glycosidically attached to the hexapeptide TyrAsn-Lys-Thr-Ser-Val (GP-M9), indicating that the specificity of the enzyme is not influenced by the peptide moiety of the substrate. The a1 ,2-mannose residue which is largely resislant to hydrolysis, was found to be attached in both the M6 and GP-M6 intermediate to the al,3-mannose of the peripheral al,3/al,6-branch of the glycan chain. Studies with glycopeptides varying in the size and branching pattern of the sugar chains, revealed that the relative rates at which the various al,2-mannosidic linkages were cleaved, differed depending on their structural complexity. This suggests that distinct sugar residues in the aglycon moiety may be functional in substrate recognition and binding. Reduction or removal of the terminal GlcNAc residue of the chitobiose unit in M, increased the hydrolytic susceptibility of the fourth (previously resistant) al,2-mannosidic linkage significantly. We conclude from this observation that, in addition to peripheral mannose residues, the intact chitobiose core represents a structural element affecting Man,-mannosidase specificity. A possible biological role of the enzyme during N-linked-oligosaccharide processing is discussed.The biosynthesis of N-linked oligosaccharides is a complex multi-step process involving the formation of a lipid-linked (GlcNAc),-Man9-Glc3 precursor, the transfer of the precursor from dolichyl diphosphate (Dol-PP) to the nascent polypeptide chain and the processing of the protein-bound glycan to the mature structure [l]. Remodelling of the oligosaccharide precursor begins with the removal of the terminal al,2-glucose by glucosidase I, followed by cleavage of the two innermost al,3-linked residues by glucosidase 11, with the resulting Mang-GlcNAc, becoming the target for several al,2-mannosidases. A key step in the conversion of high-mannose to complex oligosaccharides is the addition of a GlcNAc to Man5-(GlcNAc)2. After this transfer mannosidase I1 removes the peripheral al,3/al,6-mannosyl branch producing a Glc-N A C -M~~, -( G~C N A C )~ hybrid. GlcNAc, galactose, NeuAc and fucose residues are then added yielding complex diantennary, triantennary or tetraantennary structures [l].Little information is available on the biological significance of distinct glycan intermediates in this pathway or on the enLymes involved in their formation. This lack of information relates particularly to the ear...

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.

Glucosidase I was purified about 1900-fold from yeast microsomal preparations by DEAE-Sephacel chromatography, affinity chromatography on AH-Sepharose 4B-linked N-S-carboxypentyl-1-deoxynojirimycin and Con A-Sepharose chromatography. The enzyme is a glycoprotein with a subunit molecular mass of 95 kDa. Its reaction has a pH optimum close to 6.8 and does not require metal ions. Purified glucosidase I hydrolyses the distal al,Zlinked glucose residue from the Glq-Mang-GlcNAcz chain of its natural substrate, but is not active against Glc2-Mang-GlcNAcz and aryl-a-glucosides. Like glucosidase I from calf liver, the yeast enzyme is strongly inhibited by I-deoxynojirimycin (dNM), N-methyl-dNM and N-5-carboxypentyl-dNM with K, values of 16,0.3 and 3 PM, respectively. Trimming enzyme Glucosidase I Enzyme pur@cation (S. cerevisiae)

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.

An a ,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-1-deoxymannojirimycin (CP-dMM). On