Keiichi Murayama

Highly thermostable neutral protease from Bacillus stearothermophilus

Abstract Bacillus stearothermophilus MK232, which produced a highly thermostable neutral protease, was isolated from a natural environment. By several steps of mutagenesis, a hyper-producing mutant strain, YG185, was obtained. The enzyme productivity was twice as much as that of the original strain. This extracellular neutral protease was purified and crystallized. The molecular weight of the enzyme was 34,000 by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.5 and 70°C, respectively, and the enzyme was stable at pH 5–10 and below 70°C. The thermostability and specific activity of the new protease are around 10% and 40% higher than those of thermolysin (the neutral protease from Bacillus thermoproteolyticus), respectively. The enzyme was inactivated by EDTA, but not by phenylmethylsulfonyl fluoride. These results indicate that the enzyme is a highly thermostable neutral-(metallo)protease.

F1α: A Novel Mucin Antigen Associated with Gastric Carcinogenesis

In a previous study, we obtained a novel monoclonal antibody (F1α-75) directed against a synthetic mucin antigen termed F1α, and demonstrated that this antigen was expressed in a high percentage (80.2%; 89/111) of gastric carcinomas. In the present study, we compared the expression of F1α with that of sialyl-Tn antigen, a mucin antigen similar to F1α, in 54 human early gastric carcinomas, intestinal metaplasia and adenomatous polyps of the stomach to determine how differences in the expression of these antigens correlated with gastric carcinogenesis. The rate of expression of F1α in early gastric carcinoma tissues, 81.5%, was higher than that of sialyl-Tn antigen, 57.4%. No correlation was found between the rate of expression and histological type, depth of cancerous invasion or lymph node metastasis. Sialyl-Tn antigen was extensively expressed in 77.5% of specimens of complete intestinal metaplasia and in 78.6% of those of incomplete intestinal metaplasia; however, the expression of F1α in those specimens was rare and sporadic, with rates of only 25.0 and 27.7%, respectively. In adenomatous polyps, the rate of expression of F1α is 6.25% and that of sialyl-Tn antigen is 43.8%. Our findings indicate that F1α is a more specific antigen for gastric cancer than sialyl-Tn antigen, and that F1α is an antigen associated with carcinogenesis of the stomach.

Fusion protein of interleukin-6 and interleukin-6 receptor without a polypeptide linker

FP6, a novel recombinant fusion protein of interleukin-6 (IL-6) and IL-6 receptor (IL-6R), was prepared in the methylotrophic yeast Pichia pastoris. This protein was a potent activator of a cell surface transducing glycoprotein, gp130 and is a potential therapeutical reagent in the hemopoietic field. A linker is generally thought to be required for two fused molecules to retain their proper structures although it should preferably be removed to reduce possible antigenicity. It was found that the C-terminal residue of IL-6R could be directly linked to the N-terminal residue of IL-6 without decreasing the ability of IL-6 to bind gp130 and send the IL-6 signal. It was also found that the peptide bond between Lys-37 and Asp-38 of IL-6 was prone to proteolytic cleavage and that the immunoglobulin (Ig)-like region of IL-6R underwent extensive and heterogeneous glycosylation when expressed in P. pastoris. Based on these findings, we designed FP6 without the Ig-like region, in which the C-terminal residue of Ala-333 of IL-6R was directly linked to Asp-38 of IL-6 by a peptide bond. Purified FP6 had both an in vitro effect on hemopoietic progenitors to generate various colonies and an in vivo effect on megakaryocyte progenitors to increase platelet counts. Four purified FP6s were obtained, which had the same molecular mass and different isoelectric points without any detectable modification in the course of purification. The difference in isoelectric points was shown to be due to microheterogeneity of the carbohydrate chains. Each FP6 had the same specific activity in the cell growth assay with or without endoglycosidase digestion. Homogeneous FP6 with respect to isoelectric point as well as molecular mass merits more detailed characterization and evaluation for possible clinical application.

Study of a Synthesized New Carbohydrate Antigen in Gastro-Intestinal Cancers

In this study, a novel monoclonal antibody (Flα-75) directed to a carbohydrate antigen (Flα), which was chemically synthesized, was raised and the expression of Flα was evaluated in gastric and colon cancers. The core structure of Flα is a mucin-type carbohydrate chain which has never been reported before (Galβl→4GlcNAcβl→6GalNAcαl→Cer). As a matter of fact, Flα was found in human cancerous tissues of stomach and colon, but not in normal tissues thereof. The positive rate of Flα was 79.0% in 81 gastric cancers and 38.4% in 73 colon cancers. Among histologic types of gastric cancer, the positive rate of Flα was highest for poorly differentiated adenocarcinoma and low for well differentiated tubular adenocarcinoma. The results suggested that Flα may be a novel type of tumor marker which has a high specificity for gastric cancers and that additional cancer-associated antigens can possibly be found by using this approach.