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Molecular Biological Study on the Macrophage Lectin

巨噬细胞凝集素的分子生物学研究

基本信息

批准号：
04454593
负责人：
KAWASAKI Toshisuke
金额：
$ 4.1万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

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.

在我们之前的研究中，我们发现大鼠腹腔巨噬细胞上的Gal/GalNAc特异性凝集素（巨噬细胞去唾液酸糖蛋白结合蛋白，M-ASGP-BP）在结构上与大鼠肝凝集素（RHL）相似，并且与RHL的主要成分RHL-1高度同源。在这项研究中，我们证明，转染的cDNA克隆，编码一个单一的多肽，M-ASGP-BP，是足够的表达的内吞受体无唾液酸类粘蛋白（ASOR）的COS-1细胞表面。ASOR的K摄取值为12.5 nM，与腹腔巨噬细胞的K摄取值（23 nM）相似，细胞表面结合的ASOR数量约为5 × 10 5/细胞，是腹腔巨噬细胞的数百倍。凝胶过滤研究表明，M-ASGP-BP是42 kDa的单链多肽的六聚体或八聚体。从其cDNA序列推断，M-ASGP-BP的氨基端在其胞质尾区含有Tyr 5-Glu 6-Asn 7-Phe 8序列。通过分别转染用谷胱甘肽定向诱变制备的野生型和突变型cDNA，测量COS-1细胞上表达的野生型和突变型M-ASGP-BP的内吞作用，研究了这种假定的内化信号在胞质尾区中的作用。在Tyr 5缺失或用丙氨酸替换时，ASOR的内化降低到野生型的大约四分之一。这些结果表明，重组M-ASGP-BP中的Tyr 5是其快速内化所必需的。从PCR扩增的DNA片段和基因组DNA克隆的序列中确定了M-ASGP-BP的基因结构。大鼠M-ASGP-BP基因由10个外显子和9个内含子组成，外显子-内含子结构与RHL基因高度同源。