1994 Fiscal Year Final Research Report Summary
Molecular Biological Study on the Macrophage Lectin
| Project/Area Number |
04454593
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KAWASAKI Toshisuke Dept.Biol.Chem., Fac.Pharm.Sci., Kyoto.U.Professor, 薬学部, 教授 (50025706)
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| Co-Investigator(Kenkyū-buntansha) |
KOZUTSUMI Y Dept.Biol.Dhem., Fac.Pharm.Sci., Kyoto.U.Associate Professor, 薬学部, 助教授 (70205425)
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| Project Period (FY) |
1992 – 1994
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| Keywords | macrophage / lectin / endocytosis / internalization signal / phosphotyrosine / cDNA / recombinant lectin / galactose |
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| Research Abstract |
In our previous study, we found that the Gal/GalNAc-specific lectin on rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP) is structurally similar to rat hepatic lectin (RHL) and is highly homologous with the major component of RHL,RHL-1. In this study, we demonstrated that transfection with a cDNA clone which encodes a single polypeptide, M-ASGP-BP,was sufficient for the expression of an endocytotic receptor for asialoorosomucoid (ASOR) on the COS-1 cell surface. The Kuptake value for ASOR was 12.5 nM,which is similar to that of peritoneal macrophages (23 nM) , and the number of ASOR bound on the cell surface was about 5 x 10 5/cell, this being hundreds of times larger than that for peritoneal macrophages. Gel filtration study indicated that M-ASGP-BP is a hexamer or octamer of a single polypeptide chain of 42 kDa. The aminoterminus of M-ASGP-BP deduced from its cDNA sequence contained the sequence, Tyr5-Glu6-Asn7-Phe8, in its cytoplasmic tail. The role of this putative internalization signal in the cytoplasmic tail was studied by measuring the endocytic of the wild type and mutant M-ASGP-BPs expressed on COS-1 cells through transfection wiht the wild type and mutant cDNAs prepared by oligonucleotide-directed mutagenesis, respectively. On the deletion of Tyr5 of replacement of it with alanine, the internalization of ASOR decreased to approximately on fourth that in the case of wild type. These results suggest that the Tyr5 in the recombinant M-ASGP-BP is necessary for its rapid internalization. The gene organization of M-ASGP-BP was determined from the sequences of PCR amplified DNA fragments and genomic DNA clones. The rat M-ASGP-BP gene consists of ten exons separated by nine introns, and the overall exon-intron organization is haighly homologous with that of RHL-gene.
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