Makoto Yaguchi

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and capillary liquid chromatography-electrospray mass spectrometry (cLC-ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC-ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man8GlcNAc2) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC-PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.

A xylanase gene from Bacillus subtilis: nucleotide sequence and comparison with B. pumilus gene

A gene coding for xylanase (endo-1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) from Bacillus subtilis PAP115 has been isolated and its complete nucleotide sequence determined. Starting from an ATG initiator codon, an open reading frame coding for 213 amino acids was found. The N terminus of the processed enzyme as expressed in Escherichia coli was located by amino acid sequence analysis. The amino acid analysis and apparent molecular weight (22,000) of the expressed enzyme were consistent with the translated nucleotide sequence. A proposed 28-residue signal sequence of the enzyme shows features comparable with other Bacillus signal sequences, namely a negatively charged region close to methionine followed by a long hydrophobic string. The coding sequence is preceded by a possible ribosome binding site and, further upstream, by potential transcription initiation signals. When the xylanase amino acid sequence was compared to a xylanase from B. pumilus, strong evidence for homology was found, with over 50% identities in the processed enzymes.

The Glu residue in the conserved ASN-Glu-Pro sequence of two highly divergent endo-β-1,4-glucanases is essential for enzymatic activity

We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.

The 5S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.

Complete amino acid sequence of goose erythrocyte H5 histone and the homology between H1 and H5 histones

Summary Goose H5 histone has 193 amino acid residues and a molecular weight of 20,903. Residues 1–28 and 101–193 are probably extended while residues 29–100 are in a globular conformation. An alignment of availalbe sequences from other lysine-rich histones indicates H5 species variability is limited mostly to the N-terminal domain whereas variability between H1 and H5 proteins is within both N and C terminal domains. The constancy in size of the central domain may be important for the function of all lysine-rich histones.

Primary structure of protein S18 from the small Escherichia coli ribosomal subunit

Protein S18 is a very basic protein of the E. coli 30 S ribosomal subunit [l] . It has been implicated in aminoacyl-tRNA binding [2] , and it appears to be situated at the decoding site of mRNA because the bromoacetylated AUG analog was found to be irreversibly bound to S18 [3,4]. It has a sulfhydryl group which reacts readily with N-ethylmaleimide and its uptake paralleled ethylmaleimide-induced loss of incorporating activity [5] . The tryptic peptides of protein S18 have been isolated and their ammo acid compositions reported [6,7]. The N-terminal residue was found to be ‘blocked’ [7-91. The structural gene for the protein S18 maps at minute 84 [7,10,1 l] which is outside of the major cluster for ribosomal protein genes around minute 64 [ 121. An altered protein S18 was isolated from a temperaturesensitive mutant of E. coli and the alteration in the mutant protein consists of a replacement of arginine by cysteine [7]. This report summarizes the determination of the complete amino acid sequence of protein S18. More details will be published elsewhere. Protein S18 consists of 74 amino acids and the blocked N-terminal residue was found to be N-acetyl-alanine. The molecular weight of S18 is 8951.

Characterization of protein glycoforms by capillary-zone electrophoresis-nanoelectrospray mass spectrometry

Abstract The investigation of N- and O-linked glycoproteins using capillary-zone electrophoresis interfaced with nanoelectrospray mass spectrometry is described. The combination of high-resolution separation with high-sensitivity mass spectrometric detection provides analysis of glycoprotein digests at sample loadings of high femtomoles to low picomoles. Stepped-orifice voltage scanning is used to identify glycopeptides in complex proteolytic digests. Further structural information is obtained using capillary zone electrophoresis (CZE)–MS–MS to elucidate the composition of both N- and O-linked glycopeptide oligosaccharides. Collisional activation in the orifice/skimmer region is used to generate first-generation fragment ions which undergo subsequent dissociation in the r.f.-only collision cell of the triple quadrupole mass spectrometer. These experiments provided informative peptide backbone fragment ions usually not available from fragment ion spectra of multiply protonated glycopeptide ions. These methods were applied to the characterization of α-amylase inhibitor 1, a lectin from Lotus tetragonolobus , two N-linked glycoproteins, and to κ-casein, a glycoprotein comprising O-linked sialylated glycans.

A fungal cellulase shows sequence homology with the active site of hen egg-white lysozyme

The N-terminal amino acid sequence of an endo-beta-1,4-glucanase from the cellulase complex of the white-rot fungus Schizophyllum commune has been determined. The sequence from Glu-33 to Tyr-51 was homologous with the active site sequences of various hen egg-white type lysozymes, including lysozyme catalytic residues (Glu-35, Asp-52) and substrate binding residue Asn-44. The homology offers evidence for a lysozyme-type mechanism in enzymic hydrolysis of cellulose.

Genetic position and amino acid replacements of several mutations in ribosomal protein S5 from Escherichia coli.

SummaryThe relative genetic position of the following four mutations of ribosomal protein S5 has been determined: spc-13, a mutation to spectinomycin resistance; striN421 and strid1023, mutations suppressing dependence on streptomycin and sup0–1, a mutation suppressing partially the temperature-sensitive phenotype of an alanyl-tRNA synthetase mutation. The transduction experiments performed indicate that the spc-13 site is located in the S5 cistron proximal to the strA locus, that sup0–1 maps proximal to the aroE gene and that the striN421 and strid1023 loci are located between these two mutational sites.Proteinchemical analysis of the amino acid replacement in protein S5 of strain N421 (carrying the striN421 allele) has shown that an arginine residue is replaced by leucine which results in the appearance of a trypsin intensitive bond between the tryptic peptides T2 and T16. The same alteration has been previously found by Itoh and Wittmann (1973) in the S5 protein of strain d1023.Determination of the alteration of ribosomal protein S5 of strain 0–1 (sup0–1 allele) revealed that the C-terminal tryptic peptide is altered. It differs from that of the wild-type protein by the lack of five amino acids and the appearance of a C-terminal glycine residue instead of a lysine residue. This change can be explained by the deletion of eleven nucleotides in the S5 cistron of strain 0–1.The recent determination of the primary structure of ribosomal protein S5 (Wittmann-Liebold and Greuer, 1975) allows the ordering of the S5 alterations employed: The order is spc-13-strid1023 (striN421)-sup0–1 with the spc-13 amino acid replacement being located at the NH2-terminal portion of the S5 sequence and the alteration of strain 0–1 at the COOH-terminal end. The proteinchemical results are therefore in full agreement with the genetic data and unambiguously allow the conclusion that the S5 cistron is transcribed counterclock-wise on the Escherichia coli chromosome.

The primary structure of protein S10 from the small ribosomal subunit of Escherichia coli

As revealed by immuno electron microscopy studies [ 1,2] protein SlO is located on the ‘head’ of the 30 S subunit of the Escherichia coli ribosome. It can be crosslinked to proteins Sl and S3 by bifunctional reagents [3] suggesting the proteins are neighbours. Neutron scattering studies indicate [4] protein SlO is also in close probity to protein S9. Interaction between protein SlO and proteins 53 and S9 (as well as St4) has been observed during the in vitro assembly process. SlO is one of the proteins which is incorporated into the intermediate reconstitution particles at a rather late stage. Single component omission experiments indicated that protein SlO is involved in tRNA-binding to the ribosomes (reviewed in [5]). Here, we report the complete primary structure of protein SlO and present its secondary structure based on 4 prediction progr~mes. Furthermore, a comparison of the primary structure of protein SlO has been made with that of other ribosomal proteins.