Isolation of novel extracellualr matrix component and its function related with tissue fragility

1992 Fiscal Year Final Research Report Summary

• Project/Area Number: 03670527
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Dermatology
• Research Institution: Oita Medical University
• Principal Investigator: SHINKAI Hiroshi Oita Medical University Associate Professor, 医学部, 助教授 (90030957)
• Co-Investigator(Kenkyū-buntansha): KATAGIRI Kazumoto Oita Medical University Instructor, 医学部, 助手 (00204420) ; FUJIWARA Sakuhei Oita Medical University Assistant Professor, 医学部, 講師 (90181411)
• Project Period (FY): 1991 – 1992
• Keywords: Extracellular matrix / 45kD protein / Fibrosis

Research Abstract

To isolate novel component of extracellular matrix, new-born calf skin was extracted with 6 M urea or 4 M guanidine. The protein was separated from collagen by ion exchange chromatography and SDS-polyacrylamide gel electrophoresis. The proteins corresponded to Mr of 45 kD were cut from the gel and injected to rabbit. The antibodies did not shown cross reactivity against known extracellular matrix components though, reacted to 45 kD protein. Immunohistochemical findings showed that the protein coexisted around collagen fiber and in endothellial cells. The proteins were synthesized by cultured fibroblasts, though the synthesis was much lower compared to collagen. Three polypeptides were obtained from this protein after CNBr treatment. Following amino acid sequences were shown, DINGGAATLPQKLYQT;WLVNDTAVLP GKLYYVGVGFA ; SPDFAAST LAGLDDATKV from CB-peptides. N-terminal sequences from the protein was corresponded to large fragment of CB-peptides. Several oligonucleotides were synthesized from the amino acid sequence. To isolate the gene from cDNA library from fibroblasts and endothellial cells, antibodies and PCR method using synthesized DNA were used. No cross reactive colonies nor complementaly DNA were obtained from these libraries.