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

TitleIsolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular 'heads' of C1q
Authors
Issue Date1994
PublisherRockefeller University Press. The Journal's web site is located at http://www.jem.org
Citation
Journal Of Experimental Medicine, 1994, v. 179 n. 6, p. 1809-1821 How to Cite?
AbstractThis work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular 'heads' of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH 2-terminal amino acid sequence of the first 24 residues of the C1q- 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 NH 2 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 C1q 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 complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH 2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue- long NH 2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of ~60 kD. The NH 2- terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses. In addition, all or part of the gC1q binding domain may reside within the 24 amino acid stretch of the NH 2-terminal sequence of gC1q-R since the 18 amino acid residue long- synthetic peptide corresponding to this region inhibited serum C1q hemolytic activity. The data presented in this report suggest that there are at least two types of C1q-R which appear to be expressed on the same type of cells and these receptors individually or in concert may contribute to the diversity of C1q-mediated responses.
Persistent Identifierhttp://hdl.handle.net/10722/53488
ISSN
2015 Impact Factor: 11.24
2015 SCImago Journal Rankings: 10.762
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DC FieldValueLanguage
dc.contributor.authorGhebrehiwet, Ben_HK
dc.contributor.authorLim, BLen_HK
dc.contributor.authorPeerschke, EIBen_HK
dc.contributor.authorWillis, ACen_HK
dc.contributor.authorReid, KBMen_HK
dc.date.accessioned2009-04-03T07:21:16Z-
dc.date.available2009-04-03T07:21:16Z-
dc.date.issued1994en_HK
dc.identifier.citationJournal Of Experimental Medicine, 1994, v. 179 n. 6, p. 1809-1821en_HK
dc.identifier.issn0022-1007en_HK
dc.identifier.urihttp://hdl.handle.net/10722/53488-
dc.description.abstractThis work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular 'heads' of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH 2-terminal amino acid sequence of the first 24 residues of the C1q- 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 NH 2 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 C1q 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 complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH 2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue- long NH 2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of ~60 kD. The NH 2- terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses. In addition, all or part of the gC1q binding domain may reside within the 24 amino acid stretch of the NH 2-terminal sequence of gC1q-R since the 18 amino acid residue long- synthetic peptide corresponding to this region inhibited serum C1q hemolytic activity. The data presented in this report suggest that there are at least two types of C1q-R which appear to be expressed on the same type of cells and these receptors individually or in concert may contribute to the diversity of C1q-mediated responses.en_HK
dc.languageengen_HK
dc.publisherRockefeller University Press. The Journal's web site is located at http://www.jem.orgen_HK
dc.relation.ispartofJournal of Experimental Medicineen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThe Journal of Experimental Medicine. Copyright © Rockefeller University Press.en_HK
dc.subject.meshAntigens, CD44en_HK
dc.subject.meshCarrier Proteinsen_HK
dc.subject.meshChromatography, Affinityen_HK
dc.subject.meshComplement C1q - metabolismen_HK
dc.subject.meshReceptors, Complement - biosynthesis - isolation & purification - metabolismen_HK
dc.titleIsolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular 'heads' of C1qen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0022-1007&volume=179&issue=6&spage=1809&epage=1821&date=1994&atitle=Isolation,+cDNA+cloning,+and+overexpression+of+a+33-kD+cell+surface+glycoprotein+that+binds+to+the+globular+heads+of+C1qen_HK
dc.identifier.emailLim, BL: bllim@hkucc.hku.hken_HK
dc.identifier.authorityLim, BL=rp00744en_HK
dc.description.naturepublished_or_final_versionen_HK
dc.identifier.doi10.1084/jem.179.6.1809en_HK
dc.identifier.pmid8195709-
dc.identifier.pmcidPMC2191527en_HK
dc.identifier.scopuseid_2-s2.0-0028290256en_HK
dc.identifier.hkuros9687-
dc.identifier.volume179en_HK
dc.identifier.issue6en_HK
dc.identifier.spage1809en_HK
dc.identifier.epage1821en_HK
dc.identifier.isiWOS:A1994NN51000007-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridGhebrehiwet, B=7005039582en_HK
dc.identifier.scopusauthoridLim, BL=7201983917en_HK
dc.identifier.scopusauthoridPeerschke, EIB=7005991940en_HK
dc.identifier.scopusauthoridWillis, AC=7202176800en_HK
dc.identifier.scopusauthoridReid, KBM=7202780648en_HK

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