Fumio Sakiyama

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.

Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility.

We isolated cDNA clones encoding five S-RNases (S1-,S3- , S5-, S6-, S7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51–66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S1- and S4-RNases (90.0%) and P. pyrifolia S3- and S5-RNases (95.5%). The positions of amino acid substitutions between S1- and S4-RNases were spread over the entire region, but in the pair of S3- and S5-RNases, amino acid substitutions were found in the 21–90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S3 and S5 pollen and to trigger the self-incompatible reaction.

Identification of regions in which positive selection may operate in S-RNase of Rosaceae: Implication for S-allele-specific recognition sites in S-RNase

A stylar S‐RNase is associated with gametophytic self‐incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S‐RNase is responsible for S‐allele‐specific recognition in the self‐incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (dS ) and non‐synonymous (dN ) substitutions in rosaceous S‐RNases detected four regions with an excess of dN over dS in which positive selection may operate (PS regions). The topology of the secondary structure of the S‐RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S‐RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S‐RNases the PS regions also form two sites and are candidates for the recognition sites for S‐allele‐specific discrimination.

[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.

Characterization of structural unit of phospholamban by amino acid sequencing and electrophoretic analysis

The partial amino acid sequence of phospholamban from canine cardiac sarcoplasmic reticulum was determined by sequence analysis of the peptides obtained from the protein cleaved by cyanogen bromide and with TPCK-trypsin. The sequence determined initiated with N alpha-acetylated methionine followed by 44 amino acid residues intervening two unidentified residues. This polypeptide would represent a structural unit (protomer) of phospholamban. Analysis of temperature-dependent conversion of phospholamban from 26 kDa to lower molecular weight form (6 kDa) suggested that phospholamban holoprotein is composed of five identical protomers.

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.

The imidazole 15N signals of histidine 64 (His64), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with 15N at ring-Nδ1 and Nϵ2, 13Cat ring-Cϵ1, 13C and 15N at all carbon and nitrogen, or 15N at the amide nitrogen and the labeled glycine with 13C at the carbonyl carbon. Using the pH dependence of ring-15N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (KT) of His64 is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His64 as both a general acid (a) and base (b): its ϵ2-nitrogen as (a) releases one proton into the bulk, whereas itsδ1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the δ1-nitrogen of His64. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Purification, Bacteriolytic Activity, and Specificity of .BETA.-Lytic Protease from Lysobacter sp. IB-9374.

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.

Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli

SummaryThe DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5′-phosphoryl and 3′-hydroxyl groups in DNA. It is essential for DNA replication and repair of damaged DNA strands. We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions. The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase. The ligase consists of 671 amino acid residues with a molecular weight of 73,690.