Eiji Ichishima

PRODUCTION OF HUMAN COMPATIBLE HIGH MANNOSE-TYPE (MAN5GLCNAC2) SUGAR CHAINS IN SACCHAROMYCES CEREVISIAE

A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for α-1,2-mannosidase with the “HDEL” endoplasmic reticulum retention/retrieval tag was designed and expressed inSaccharomyces cerevisiae. An in vitroα-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 α-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 Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.

An alkaline proteinase of an alkalophilicBacillus sp.

An alkaline serine proteinase was purfied from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight was estimated to be 22,000 by a gel filtration method and 31,000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 8.2. The amino acid composition and CD spectrum were determined. The alkaline proteinase had a pH optimum at 10–11 for milk casein digestion. The specific activity of the alkaline proteinase was 0.35 katal/kg of protein at pH 10.0 for milk casein hydrolysis.The substrate specificity of the alkaline proteinase was studied by using the oxidized, insulin B-chain and angiotensin. An initial cleavage site was observed at Leu15-Tyr16, secondary site at Leu11-Val12, and additional sites at Gln4-His5, Tyr26-Thr27, and Asn3-Gln4 in the oxidized insulin B-chain at pH 10.0. In comparison with the subtilisins Carlsberg and Novo, the alkaline proteinase fromBacillus sp. showed a unique specificity toward the oxidized insulin B-chain. Hydrolysis of angiotensin at pH 10.0 with the alkaline proteinase was observed at Tyr4-Ile5. The proteinase has aKm of 0.1 mM andkcat of 3.3 s−1 with angiotensin as substrate.

Purification of an acidic α-d-mannosidase from Aspergillus saitoi and specific cleavage of 1,2-α-d-mannosidic linkage in yeast mannan

Abstract An acidic α- d -mannosidase (α- d -mannoside mannohydrolase, EC 3.2.1.24) has been isolated from culture filtrate of Aspergillus saitoi. The extracellular α-mannosidase was homogeneous in polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 51 000 and the isoelectric point pH 4.5. The purified enzyme has a pH optimum of 5.0, a Km of 0.45 mM with bakers yeast mannan and has no activity towards p- nitrophenyl -α- d -mannoside . The mode of action of the enzyme has been studied with bakers yeast mannan and sake yeast mannan. The enzyme cleaves specifically the 1.2-α-linked side chain, producing free mannose.

An α-mannosidase purified from Aspergillus saitoi is specific for α1,2 linkages

Abstract The substrate specificity of an α-mannosidase purified from Aspergillus saitoi was studied in detail. This enzyme hydrolyzes yeast mannan partially but does not act on p-nitrophenyl α-mannopyranoside. Survey of the action of the enzyme on various oligosaccharides liberated from glycoproteins indicated that the enzyme hydrolyzes Manα1→2Man linkage but not Manα1→3Man and Manα1→6 Man linkages at all. All Manα1→2 residues in intact bovine pancreatic ribonuclease B were removed completely by incubation with the α-mannosidase.

Molecular cloning and nucleotide sequence of the 1,2-α-d-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells

A full-length cDNA encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) from Aspergillus saitoi was cloned. Analysis of the 1718 bp nucleotide sequence of the cDNA revealed a single open reading frame with 1539 nucleotides of 1,2-alpha-D-mannosidase gene, msdS. The predicted amino-acid sequence of 1,2-alpha-D-mannosidase consists of 513 residues with a molecular mass of 55,767 and is 70%, 26% and 35% identity with those of Penicillium citrinum 1,2-alpha-D-mannosidase, yeast alpha-mannosidase, and mouse alpha-mannosidase. The cDNA of the msdS gene has been cloned and expressed in yeast cells. To identify the activity of expression product methyl-2-O-alpha-mannopyranosyl-alpha-mannopyranoside (Man alpha 1-->2Man-OMe) was used as a substrate at pH 5.0.

Involvement of cell wall β‐glucan in the action of HM‐1 killer toxin

HM‐1 killer toxin secreted from Hansenula mrakii inhibits the growth of Saccharomyces cerevisiae cells by interfering with β‐1,3‐glucan synthesis. We found that HM‐1 killer toxin killed intact cells but not protoplasts. In addition, cells lacking the functional KRE 6 allele (kre6Δ) became resistant to higher concentration of HM‐1 killer toxin. As reported by Roemer and Bussey [(1991) Proc. Natl. Acad. Sci. 88 11295–11299], cells lacking functional KPE6 had a reduced level of the cell wall β‐1,6‐glucan compared to that in cells harboring the normal KRE6. These results suggest that the cell wall ⨿‐glucan is involved in the action of HM‐1 killer toxin. Addition of HM‐1 killer toxin with several kinds of oligosaccharides revealed that either ⨿‐1,3‐ or β‐1,6‐glucan blocked the cytocidal action of HM‐1 killer toxin whereas α‐1,4‐glucan and chitin did not. Mannan also interfered with HM‐1 killer toxin action, but this inhibitory effect was much weaker than that observed with β‐1,3‐ or β‐1,6‐glucans. Thus, it appears that the cell wall β‐glucan interacts with HM‐1 killer toxin, and that this toxin‐β‐glucan commitment is required for the action of HM‐1 killer toxin.

Molecular cloning and nucleotide sequence of the genomic DNA for 1,2-α-d-mannosidase gene, msdC from Penicillium citrinum

A gene encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) was cloned from Penicillium citrinum genomic DNA using the polymerase chain reaction (PCR). The coding region of the gene, msdC, occupied 1737 bp and was separated into four exons by three introns. The predicted protein consisted of 511 amino acid residues with M(r) 56,569. Penicillium enzyme had a hydrophobic signal peptide at the N-terminal region as did mammalian membrane-bound alpha-mannosidases, but in this case a proteolytic cleavage occurred at Lys-35-Ser-36 to remove the signal sequence during cell growth. Parts of amino acid sequences were similar to those of mammalian Golgi alpha-mannosidase IA and IB, but the sequence around the aspartic acid residue which interacted with 1-deoxymannojirimycin (Yoshida et al. (1994) Biochem. J. 303, 97-103) was unique in Penicillium enzyme.

Action of crystalline acid carboxypeptidase from Penicillium janthinellum

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.

Aspzincin, a Family of Metalloendopeptidases with a New Zinc-binding Motif IDENTIFICATION OF NEW ZINC-BINDING SITES (His128, His132, and Asp164) AND THREE CATALYTICALLY CRUCIAL RESIDUES (Glu129, Asp143, and Tyr106) OF DEUTEROLYSIN FROMASPERGILLUS ORYZAE BY SITE-DIRECTED MUTAGENESIS

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His128 and His132 with Arg, of Glu129 with Gln or Asp, of Asp143 with Asn or Glu, of Asp164 with Asn, and of Tyr106 with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu129, Asp143, and Tyr106 are catalytically crucial residues in the enzyme. Glu129 may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn2+ metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the “HEXXH + D” motif and an aspartic acid as the third zinc ligand.

Purification, crystallisation and characterisation of carboxypeptidase from wheat bran

Abstract A carboxypeptidase was purified and crystallised from wheat bran. Disc gel electrophoresis at pH 4·0 and ultracentrifugal analysis revealed that the enzyme was essentially homogeneous. The sedimentation constant and isoelectric point were determined to be 6·3 S and 6·0, respectively. The molecular weight of the enzyme was estimated to be 118,000 by a gel filtration method. The enzyme liberated carboxyl terminal amino acid residues from a wide range of N -substituted dipeptides and tripeptides which contain l -proline. It had a pH optimum at pH 4·0 for Z -Glu-Tyr ( Z -benzyloxycarbonyl). The K m and k cat values for Z -Glu-Tyr at pH 4·0 and 30°C were 0·19 m m and 20 s −1 , respectively. The enzyme hydrolysed Z -Gly-Pro-Leu-Gly-Pro and bradykinin sequentially at pH 4·0 from their carboxyl terminal amino acid residues. The enzyme activity was completely inhibited by DFP.