Biomedical subjects

E Ichishima

Publications and source records attributed to E Ichishima.

At least 37 records · Page 2Linked to original sources

PubMed1996

HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.

Bacterial Outer Membrane Proteins↗

PubMed1995

Molecular cloning and nucleotide sequence of the 1,2-alpha-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.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and nucleotide sequence of the genomic DNA for 1,2-alpha-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.

Amino Acid Sequence↗

PubMed1995

Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis.

Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).

Amino Acid Sequence↗

PubMed1995

The structure of subtilisin ALP I from alkalophilic Bacillus sp. NKS-21.

The gene for an alkaline serine protease from alkalophilic Bacillus sp. NKS-21 (subtilisin ALP I) was cloned, and its nucleotide sequence was determined. The gene (aprQ) contained an open reading frame of 1125 bp, encoding a primary product of 374 amino acids. The mature protease, composed of 272 amino acids, was preceded by a putative signal sequence of 37 amino acids and a pro-sequence of 65 amino acids. The mature protease conserved the catalytic triad, Asp, His, and Ser, as subtilisin BPN' or other subtilisins, and the subtilisin ALP I might belong to the subtilisin super family. The primary structure of subtilisin ALP I was compared and discussed with those of 13 subtilisins, 5 subtilisins from alkalophilic Bacillus, and 8 from neutrophiles. Low homology was shown between subtilisin ALP I and subtilisins from alkalophiles or subtilisins from neutrophiles. Forty-five amino acid residues of the mature protein of subtilisin ALP I were entirely independent of other subtilisins. According to the homology of ALP I with other subtilisins, subtilisin ALP I might be in the middle point between alkaline subtilisins and neutral ones.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and nucleotide sequence of the 90k serine protease gene, hspK, from Bacillus subtilis (natto) No. 16.

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.

Amino Acid Sequence↗

PubMed1995

A new alkaline serine protease from alkalophilic Bacillus sp.: cloning, sequencing, and characterization of an intracellular protease.

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.

Amino Acid Sequence↗

PubMed1995

Molecular and enzymatic properties of an aspartic proteinase from Rhizopus hangchow.

An aspartic proteinase, rhizopuspepsin (EC 3.4.23.21), from Rhizopus hangchow was purified. The M(r) and isoelectric point were determined as ca 37,000 and 4.5, respectively. The first 19 amino acids in the N-terminal region were SGSGVVPMTDYEYDIEYYG. The contents of the alpha-helix, beta-structure and random coil were calculated to be ca 7.5, 88.9 and 2.7%, respectively. The enzyme can activate trypsinogen at pH 3.0. The activity was completely inactivated by pepstatin A. The specificity and mode of action of the enzyme were investigated with oxidized insulin B-chain at pH 3. The enzyme hydrolysed primarily two peptide bonds, the Leu15-Tyr16 bond and the Tyr16-Leu17 bond, while additional cleavage of the bonds, Ala14-Leu15 and Phe24-Phe25 was also noted.

Amino Acid Sequence↗

PubMed1995

Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS.

We purified a new extracellular serine proteinase (designated subtilisin Sendai) from the culture broth of alkalophilic Bacillus sp. G-825-6, and its properties were characterized. Its optimum pH was at 10.0, when succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-4-methylcoumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) was used as a substrate. The substrate specificity of subtilisin Sendai was determined with oxidized insulin B-chain and fluorogenic peptidyl-MCA substrates. The isoelectric point of subtilisin Sendai was over 11.0. The molecular mass of the enzyme was estimated as 28,000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The circular dichroism spectrum of the enzyme was measured, and we discuss the relationship between the secondary structure of the enzyme and alkaline stability at pH 12 in comparison with that of subtilisin NAT. The structural gene (aprS) was cloned and sequenced. The deduced amino acid sequence for the mature protein (269 amino acids) was preceded by a putative signal sequence of 27 residues and a putative pro-sequence of 86 amino acids. The homology of the primary structure for 13 subtilisins was compared. The catalytic triad (Asp32, His64, and Ser221 with the numbering of subtilisin BPN') and the amino acid sequences near these amino acid residues were well conserved. As a special feature, it was observed that there was an extensive number of negatively charged amino acids in the pro-region of subtilisin Sendai and alkaline subtilisins. This was different from those of subtilisin from neutrophiles.

Amino Acid Sequence↗

PubMed1995

Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant.

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.

Amino Acid Sequence↗

PubMed1994

Chemical modification of Penicillium 1,2-alpha-D-mannosidase by water-soluble carbodi-imide: identification of a catalytically important aspartic acid residue.

1,2-alpha-D-Mannosidase from Penicillium citrinum was inactivated by chemical modification with 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide (EDC). Most of the activity was lost after modification in the absence of a nucleophile, glycine ethyl ester. 1-Deoxymannojirimycin (dMM), a competitive inhibitor of the enzyme, showed partial protection against the inactivation. After the modification by EDC without the presence of a nucleophile, proteolytic digests of the enzyme were analysed by reversed-phase h.p.l.c. and a unique peptide was shown to decrease when dMM was present during the modification. The peptide was absent from the digests of unmodified enzyme. The amino acid sequence of the peptide (A; Ile-Gly-Pro) was identical in part with that of the adjacent peptide (B; Ile-Gly-Pro-Asp-Ser-Trp-Gly-Trp-Asp-Pro-Lys). When cholecystokinin tetrapeptide (Trp-Met-Asp-Phe-NH2) was modified by EDC alone, the modified peptide could be separated from unmodified peptide by reversed-phase h.p.i.c., and Edman degradation was stopped before the modified aspartic acid residue. This suggested that, in the enzyme, peptide A was derived from peptide B by the modification. Consequently, Asp-4 in peptide B was assumed to be masked by dMM during the modification, and to be involved in the interaction of the enzyme with its substrate.

1-Deoxynojirimycin↗

PubMed1994

Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, and 18 kDa metalloendopeptidase gene from Penicillium citrinum.

A full-length cDNA encoding the penicillolysin, an 18 kDa metalloendopeptidase from Penicillium citrinum, was cloned. Analysis of the 1284 base pair nucleotide sequence of the cDNA revealed a single open reading frame coding for 351 amino acid residues. The coding region of penicillolysin gene, plnC, occupies 1053 base pairs of the cDNA. The sequence consists of a putative 19-residue signal sequence, a 155-residue propeptide segment, and the 177-residues of penicillolysin with a molecular weight of 18,529. The deduced primary structure of penicillolysin is unique and the enzyme is a member of a new metalloendopeptidase family. Two histidine residues, His-128 and His-132, and glutamic acid residue, Glu-65 in penicillolysin were assumed to correspond to zinc ligands in the homologous thermolysin.

Amino Acid Sequence↗

PubMed1994

Involvement of cell wall beta-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 beta-1,3-glucan synthesis. We found that HM-1 killer toxin killed intact cells but not protoplasts. In addition, cells lacking the functional KRE6 allele (kre6 delta) 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 KRE6 had a reduced level of the cell wall beta-1,6-glucan compared to that in cells harboring the normal KRE6. These results suggest that the cell wall beta-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 beta-1,3- or beta-1,6-glucan blocked the cytocidal action of HM-1 killer toxin whereas alpha-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 beta-1,3- or beta-1,6-glucans. Thus, it appears that the cell wall beta-glucan interacts with HM-1 killer toxin, and that this toxin-beta-glucan commitment is required for the action of HM-1 killer toxin.

Cell Wall↗

PubMed1994

Primary structure of aspergillopepsin I deduced from nucleotide sequence of the gene and aspartic acid-76 is an essential active site of the enzyme for trypsinogen activation.

The coding region of the aspergillopepsin I (EC 3.4.23.18) gene occupies 1340 base pairs of the genomic DNA and is separated into four exons by three introns. The predicted amino-acid sequence of aspergillopepsin I consists of 325 residues and is 32% and 27% homologous with those of human pepsin and calf chymosin. The cDNA of the gene prepared from mRNA has been cloned and expressed in yeast cells. To identify the residue of the substrate binding pocket in determining the specificity of aspergillopepsin I towards basic substrates, this residue was replaced with a serine residue by site-directed mutagenesis. The mutation is a single amino-acid change, Asp-76 converted to Ser-D76S, in the enzyme. The striking feature of this is that only the trypsinogen activating activity was destroyed. We therefore concluded that Asp-76 is the binding site towards basic substrates.

Amino Acid Sequence↗

PubMed1994

A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization, and mode of action.

A 1,2-alpha-D-mannosidase was purified to homogeneity from the culture supernatant of Bacillus sp. M-90, which was isolated from soil by enrichment culture on baker's yeast mannan. The purified enzyme had M(r) 380,000 Da, and was comprised of two apparently identical 190,000 Da subunits. It had a neutral optimum pH (7.0) and an isoelectric point of 3.6. The enzyme was highly specific for alpha 1,2-linked D-mannose oligosaccharides. An N-linked high-mannose type oligosaccharide, Man9GlcNAc2, was a good substrate, yielding Man5GlcNAc2, and the alpha 1,2-linked side chains of Saccharomyces cerevisiae mannan were also specifically hydrolyzed by the enzyme. p-Nitrophenyl alpha-D-mannopyranoside and 1,2-alpha-D-mannobiitol were not hydrolyzed at all. Calcium ion, 1-deoxyman-nojirimycin, and swainsonine had no effect on the enzyme, but the activity was completely inhibited by EDTA. The mode of action on alpha 1,2-linked mannotetraose indicated that the enzyme is an exo-1,2-alpha-D-mannanase.

Amino Acids↗

PubMed1994

Functional changes of dextran-modified alkaline proteinase from alkalophilic Bacillus sp.

A serine alkaline proteinase (EC 3.4.21.62) from Bacillus sp. (ALPase I) was modified with the 2,4-dialdehyde derivative of clinical dextran (dialdehyde dextran). The modified preparation was purified using an ion-exchange column and gel filtration. The modified enzyme contained 75% carbohydrate by weight. The isoelectric point (pI) of ALPase I was converted from 8.2 to approximately 5.0 by this modification. The specific activity of the dextran-modified ALPase I was 56% of that of the native enzyme when milk casein was used as a substrate. It also had some superior characteristics: the thermostability of the modified enzyme at pH 10.0 was about 10-15 degrees C higher than that of control. In organic solvents such as n-hexane, benzene, and toluene, the hydrolysis reaction of the modified ALPase I for the fluorogenic substrate, succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-methylcoumaryl-7-am ide (Suc-Ala-Ala-Pro-Phe-MCA), was several times higher than that of the native. This modification greatly improved the stability of ALPase I against nonionic and anionic surfactants. After exposure to lauryl benzene sulfonate and sodium lauryl sulfonate the modified enzyme retained over 95 and 90% of its activity, respectively, but the native enzyme lost its activity. We conclude that modification of serine proteinases with dialdehyde-dextran might be a useful method for improving enzyme character for enzyme technology.

Amino Acid Sequence↗

PubMed1994

Inactivation of chemotactic peptides by aminopeptidase Ey from hen's (Gallus gallus domesticus) egg yolk.

Aminopeptidase Ey, purified from hen's (Gallus gallus domesticus) egg yolk, was studied for its specificity against N-blocked peptides. Only N-formylmethionyl peptides were hydrolyzed by the enzyme in the tested peptides. N-Formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) lost its chemotactic activity toward human neutrophil after incubation with aminopeptidase Ey.

Amino Acid Sequence↗

PubMed1994

Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis.

A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S. cerevisiae genomic library. This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame. The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites. The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively. Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein. Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S. cerevisiae genome, and the transcriptional level of HKR1 is extremely low. Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene. Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin. Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.

Amino Acid Sequence↗