Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q.

Abstract

SummaryThis work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gClq-R, was first isolated from Raji cells and was found to bind to the globular "heads" of Clq molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NHzterminal amino acid sequence of the first 24 residues of the Clq-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to Clq may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity This recombinant protein was also able to inhibit the complementmediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gd filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the Clq-binding protein is expressed at high levels in Raji and Dandi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 call line. However, it is not expressed in the K562 cell line. Comparison of gClq-R NHz-terminal sequence with that of the receptor for the collagen-like domain ofClq (cClq-R) showed no similarity. Furthermore, antibodies to gClq-R or an 18-amino acid residue-long NHz-terminal synthetic gClq-R peptide did not cross-react with antibodies to cClq-R. Anti-gClq-R immunoblotted a 33-kD Raft cell membrane protein, whereas anti cClq-R recognized a molecule of ,v60 kD. The NH2-terminal sequence of gClg-R appears to be displayed extracdlularly since anti-gClg-R peptide reacted with surface molecules on lymphocytes, polymorphonudear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses. In addition, all or part of the gClq binding domain may reside within the 24 amino acid stretch of the NHz-terminal sequence of gClq-R since the 18 amino acid residue long-synthetic peptide corresponding to this region inhibited serum Clq hemolytic activity The data presented in this report suggest that there are at least two types of Clq-R which appear to be expressed on the same type of ceils and these receptors individually or in concert may contribute to the diversity of Clq-mediated responses.

Arginase II Contributes to the Ca 2+ /CaMKII/eNOS Axis by Regulating Ca 2+ Concentration Between the Cytosol and Mitochondria in a p32‐Dependent Manner

“…p32 has both mitochondrial and non‐mitochondrial localization, although p32 predominantly targets to mitochondria with an N‐terminal mitochondria‐targeting sequence 25. In this study, we have fractionated the cell lysate into mitochondria and cytosol and found that p32 was abundantly presented in the mitochondria.…”

“…Although p32 can be existed in multicellular compartments, it is predominantly targeted to mitochondria because of the mitochondrial targeting sequence contained in the 73N‐terminal amino acids 25. Functional studies have shown that p32 is required for the induction of mitochondria‐dependent cell death26, 27 and for the metabolic shift between oxidative phosphorylation and aerobic glycolysis 28.…”

Platelet Mediated Complement Activation

“…In contrast, the amino terminal, globular domain of C1q binds the 33kDa gC1qR/p33 (gC1qR) (Ghebrehiwet, Lim, Peerschke, Willis, and Reid 1994). gC1qR is a ubiquitously expressed cellular protein (Ghebrehiwet and Peerschke 1998;Ghebrehiwet, Lim, Kumar, Feng, and Peerschke 2001).…”

C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms

“…Polyclonal and monoclonal antibodies to human gC1qR were prepared as described by Ghebrehiwet et al, [8,26]. Rabbit polyclonal antibodies to C1q were raised against purified human C1q.…”

“…Three C1q-bps, which interact with the collagen tail of C1q, of molecular masses 126, 57 and 68 kDa, have been described on the surface of neutrophils [4][5][6]. Another C1q-bp, called gC1qR, is known to interact with globular head regions of C1q [7,8]. gC1qR is a highly acidic protein and a Abbreviations used : cC1q, collagen-like tails of C1q ; gC1q, globular heads of C1q ; C1q-bp, C1q-binding protein ; FMLP, N-formylmethioninyl-leucylphenylalanine ; PLC, phospholipase C ; PT, pertussis toxin.…”