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Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.

ABSTRACT: 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.

SUBMITTER: Klickstein LB

PROVIDER: S-EPMC2189104 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

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Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.

Klickstein L B LB Bartow T J TJ Miletic V V Rabson L D LD Smith J A JA Fearon D T DT

The Journal of experimental medicine 19881101 5

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 prote ...[more]

PMID: 2972794

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Project description:In order to study the mechanisms of COVID-19 damage following the complement activation phase occurring during the innate immune response to SARS-CoV-2, CR1 (the regulating complement activation factor, CD35, the C3b/C4b receptor), C4d deposits on Erythrocytes (E), and the products of complement activation C3b/C3bi, were assessed in 52 COVID-19 patients undergoing O2 therapy or assisted ventilation in ICU units in Rheims France. An acquired decrease of CR1 density on E from COVID-19 patients was observed (Mean = 418, SD = 162, N = 52) versus healthy individuals (Mean = 592, SD = 287, N = 400), Student's t-test p < 10-6, particularly among fatal cases, and in parallel with several parameters of clinical severity. Large deposits of C4d on E in patients were well above values observed in normal individuals, mostly without concomitant C3 deposits, in more than 80% of the patients. This finding is reminiscent of the increased C4d deposits on E previously observed to correlate with sub endothelial pericapillary deposits in organ transplant rejection, and with clinical SLE flares. Conversely, significant C3 deposits on E were only observed among ¼ of the patients. The decrease of CR1/E density, deposits of C4 fragments on E and previously reported detection of virus spikes or C3 on E among COVID-19 patients, suggest that the handling and clearance of immune complex or complement fragment coated cell debris may play an important role in the pathophysiology of SARS-CoV-2. Measurement of C4d deposits on E might represent a surrogate marker for assessing inflammation and complement activation occurring in organ capillaries and CR1/E decrease might represent a cumulative index of complement activation in COVID-19 patients. Taken together, these original findings highlight the participation of complement regulatory proteins and indicate that E are important in immune pathophysiology of COVID-19 patients. Besides a potential role for monitoring the course of disease, these observations suggest that novel therapies such as the use of CR1, or CR1-like molecules, in order to down regulate complement activation and inflammation, should be considered.

Project description:The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

Dataset Information

Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.

Publications

Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.

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