Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.
Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.
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DOI：
10.1084/jem.168.5.1699
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发表时间：
1988-11-01
期刊：
影响因子：
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通讯作者：
Fearon DT
中科院分区：
文献类型：
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作者：
Klickstein LB;Bartow TJ;Miletic V;Rabson LD;Smith JA;Fearon DT
编码人CR 1的NH 2-末端区域的互补DNA克隆已被分离和测序。推导的人CR 1的F同种异型的完整氨基酸序列包含2，039个残基，包括41个残基的信号肽、1，930个残基的胞外结构域、25个氨基酸的跨膜结构域和43个氨基酸的胞质区。细胞外结构域仅由30个短共有重复序列（SCR）组成，这是C3/C4结合蛋白家族的特征。28个NH 2-末端SCR被组织为四个长同源重复序列（LHR），每个长同源重复序列具有七个SCR。新测序的LHR，LHR-A，与LHR-B在NH 2-末端的两个SCR中有61%的同一性，在COOH-末端的五个SCR中有99%以上的同一性。将8个cDNA克隆拼接以形成单个构建体piABCD，其含有巨细胞病毒启动子下游的整个CR 1编码序列。转染piABCD的COS细胞瞬时表达重组CR 1，其在SDS-PAGE上与红细胞CR 1的F同种异型共迁移，并介导与携带C4 b和C3 b的绵羊红细胞形成玫瑰花结。重组CR 1还具有裂解C3（ma）的因子I-辅因子活性。对COS细胞中表达的6个缺失突变体的分析表明，LHR-A的NH 2-末端两个SCR含有决定C4特异性的位点，LHR-B和-C的NH 2-末端两个SCR各含有决定C3特异性的位点。CR 1中这三个不同位点的存在可能使受体能够与经典和替代途径激活期间产生的C4 b/C3 b和C3 b/C3 b复合物多价相互作用。
Complementary DNA clones encoding the NH2-terminal region of human CR1 have been isolated and sequenced. The deduced complete amino acid sequence of the F allotype of human CR1 contains 2,039 residues, including a 41-residue signal peptide, an extracellular domain of 1,930 residues, a 25-amino acid transmembrane domain, and a 43-amino acid cytoplasmic region. The extracellular domain is composed exclusively of 30 short consensus repeats (SCRs), characteristic of the family of C3/C4-binding proteins. The 28 NH2-terminal SCRs are organized as four long homologous repeats (LHRs) of seven SCRs each. The newly sequenced LHR, LHR-A, is 61% identical to LHR-B in the NH2-terminal two SCRs and greater than 99% identical in the COOH-terminal five SCRs. Eight cDNA clones were spliced to form a single construct, piABCD, that contained the entire CR1 coding sequence downstream of a cytomegalovirus promoter. COS cells transfected with piABCD transiently expressed recombinant CR1 that comigrated with the F allotype of erythrocyte CR1 on SDS-PAGE and that mediated rosette formation with sheep erythrocytes bearing C4b and C3b. Recombinant CR1 also had factor I-cofactor activity for cleavage of C3(ma). Analyses of six deletion mutants expressed in COS cells indicated that the NH2-terminal two SCRs of LHR- A contained a site determining C4 specificity and the NH2-terminal two SCRs of LHR-B and -C each had a site determining C3 specificity. The presence of these three distinct sites in CR1 may enable the receptor to interact multivalently with C4b/C3b and C3b/C3b complexes generated during activation of the classical and alternative pathways.