Susumu Tsunasawa

Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.

When stimulated with antigen, B cells are influenced by T cells to proliferate and differentiate into antibody-forming cells. Since it was reported1,2 that soluble factors could replace certain functions of helper T cells in the antibody response, several different kinds of lymphokines and monokines have been reported in B-cell growth and differentiation3,4. Among these, human B-cell differentiation factor (BCDF or BSF-2) has been shown to induce the final maturation of B cells into immunoglobulin-secreting cells5–8. BSF-2 was purified to homogeneity9 and its partial NH2-terminal amino-acid sequence was determined10. These studies indicated that BSF-2 is functionally and structurally unlike other known proteins. Here, we report the molecular cloning, structural analysis and functional expression of the cDNA encoding human BSF-2. The primary sequence of BSF-2 deduced from the cDNA reveals that BSF-2 is a novel interleukin consisting of 184 amino acids.

Molecular structure of human lymphocyte receptor for immunoglobulin E

We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.

Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity

Two antifungal peptides (Pn-AMP1 and Pn-AMP2) have been purified to homogeneity from seeds of Pharbitis nil. The amino acid sequences of Pn-AMP1 (41 amino acid0 residues) and Pn-AMP2 (40 amino acid residues) were identical except that Pn-AMP1 has an additional serine residue at the carboxyl-terminus. The molecular masses of Pn-AMP1 and Pn-AMP2 were confirmed as 4299.7 and 4213.2 Da, respectively. Both the Pn-AMPs were highly basic (pI 12.02) and had characteristics of cysteine/glycine rich chitin-binding domain. Pn-AMPs exhibited potent antifungal activity against both chitin-containing and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/ml for Pn-AMP1 and from 0.6 to 75 micrograms/ml for Pn-AMP2. The Pn-AMPs penetrated very rapidly into fungal hyphae and localized at septum and hyphal tips of fungi, which caused burst of hyphal tips. Burst of hyphae resulted in disruption of the fungal membrane and leakage of the cytoplasmic materials. To our knowledge, Pn-AMPs are the first hevein-like proteins that show similar fungicidal effects as thionins do.

Purification and characterization of a novel solvent-tolerant lipase from Fusarium heterosporum

Abstract A microorganism producing a solvent-tolerant lipase was identified as Fusarium (F.) heterosporum. The lipase was purified from the culture filtrate to homogeneity as judged by disc-PAGE and SDS-PAGE. The purification included SP-Sephadex chromatography, gel filtration and isoelectric focusing, and the recovery yield was 38%. The lipase was a monomeric protein with a molecular weight of 31 kDa estimated by SDS-PAGE, and a pI of 7.0. The optimum pH at 40°C and optimum temperature at pH 5.6 were 5.5–6.0 and 45–50°C, respectively, when olive oil was used as the substrate. The lipase was stable over a pH range of 4–10 at 30°C for 4 h, and up to 40°C at pH 5.6 for 30 min. Furthermore, the enzyme was not inactivated even after incubation at 30°C in 50% solvent such as dimethylsulfoxide (DMSO), hexane, benzene and ether for 20 h. The activity did not decrease in a reaction with stirring in a mixture containing 50% DMSO or dimethylformamide. The lipase preferably reacted on middle-chain fatty acid triglycerides (6≤C≤12), and cleaved only 1,3-ester bonds of triolein. The enzyme had an N-terminal sequence of Ala-Val-Thr-Val-Thr-Thr-Gln-Asp-Leu-Ser, which has not previously been found in any other protein. We compared the properties of lipases from F. heterosporum and another strain F. oxysporum.

[14] Amino-terminal acetylation of proteins: An overview

Publisher Summary This chapter focuses on amino-terminal acetylation of proteins. N α -acetylation is considered one of the typical modification of proteins in living organisms. Experiments with ovalbumin, α -crystallin, and histone have shown that N α -acetylation is a cotranslational event. In the biosynthesis of ovalbumin, a secretory protein, the amino-terminal methionine is removed when the nascent peptide chain has extended about 20 residues, while the N α -acetylation of the new amino-terminal glycine takes place after the peptide chain has been elongated up to about 40 residues. Thus, for both secretory and nonsecretory proteins, N α -acetylation occurs at a stage when the amino-terminal portion of the growing chain has protruded from the ribosome. N α -acetylation is essentially an enzyme-catalyzed reaction in which the protein accepts the acetyl group from acetyl-CoA. The enzyme N α –acetyltransferase is found in various cells and tissues such as rabbit reticulocytes, rat liver, calf lens, rat pituitary, and hen oviduct. Based on the mode of action in catalysis, the transferases are classified in two major groups. One group includes the enzyme that catalyzes N α -acetylation of the nascent peptide chain growing on ribosomes. The enzymes in this group are probably ribosome bound or membrane bound. The other group of enzymes includes those that are associated with the processing of bioactive peptides and mature proteins.

Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor.

The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S‐carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide‐containing peptides. The inhibitor consists of 112 amino acids with an M r of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human γ‐trace, human salivary acidic protein and chicken egg‐white cystatin.

DL-2-haloacid dehalogenase from Pseudomonas sp. 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme- substrate ester intermediate

dl-2-Haloacid dehalogenase fromPseudomonas sp. 113 (dl-DEX 113) catalyzes the hydrolytic dehalogenation of d- andl-2-haloalkanoic acids, producing the correspondingl- and d-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (l-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of dl-DEX 113. When a single turnover reaction of dl-DEX 113 was carried out with a large excess of the enzyme in H2 18O with a 10 times smaller amount of the substrate, either d- or l-2-chloropropionate, the major product was found to be18O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H2 18O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained 18O. These results indicate that the H2 18O of the solvent directly attacks the α-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.

Reaction Mechanism of Fluoroacetate Dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxellasp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) fromXanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborský, J., Koča, J., and Janssen, D. B. (1997)Biochemistry 36, 9571–9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H2 18O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99–Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the α-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 → Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.

A simple and highly successful C‐terminal sequence analysis of proteins by mass spectrometry

A simple and efficient method for C‐terminal sequencing of proteins has long been pursued because it would provide substantial information for identifying the covalent structure, including post‐translational modifications. However, there are still significant impediments to both direct sequencing from C termini of proteins and specific isolation of C‐terminal peptides from proteins. We describe here a highly successful, de novo C‐terminal sequencing method of proteins by employing succinimidyloxycarbonylmethyl tris (2,4,6‐trimethoxyphenyl) phosphonium bromide and mass spectrometry.

Terminal proteomics: N‐ and C‐terminal analyses for high‐fidelity identification of proteins using MS

In proteomics, MS plays an essential role in identifying and quantifying proteins. To characterize mature target proteins from living cells, candidate proteins are often analyzed with PMF and MS/MS ion search methods in combination with computational search routines based on bioinformatics. In contrast to shotgun proteomics, which is widely used to identify proteins, proteomics based on the analysis of N‐ and C‐terminal amino acid sequences (terminal proteomics) should render higher fidelity results because of the high information content of terminal sequence and potentially high throughput of the method not requiring very high sequence coverage to be achieved by extensive sequencing. In line with this expectation, we review recent advances in methods for N‐ and C‐terminal amino acid sequencing of proteins. This review focuses mainly on the methods of N‐ and C‐terminal analyses based on MALDI‐TOF MS for its easy accessibility, with several complementary approaches using LC/MS/MS. We also describe problems associated with MS and possible remedies, including chemical and enzymatic procedures to enhance the fidelity of these methods.