Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

Wong, Winnie W.; Jack, R M; Smith, John A.; Kennedy, Christine A.; Fearon, Douglas T.

doi:10.1016/0022-1759(85)90362-x

Cited by 31 publications

(18 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…They inhibit complement activation via different mechanisms in the complement activation cascade. [6][7][8] For example, MCP binds to complement activation products C3b and C4b, accelerating factor I-mediated inactivation of C3b and C4b [9][10][11] ; DAF inhibits the formation of C3 and C5 convertases; and CD59 inhibits formation of the membrane attack complex. Theoretically, concomitant expression of multiple hCRPs might inactivate complement more effectively than expression of a single hCRP.…”

Section: Introductionmentioning

confidence: 99%

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Yang¹,

Deng²,

Jiang³

et al. 2012

Exp Clin Transplant

View full text Add to dashboard Cite

Objectives: To analyze the protective effects against complement-mediated cytolysis of the MCP, DAF, and CD59 human complement regulatory proteins, alone and in combination, on NIH 3T3 mouse fibroblast cells. Materials and Methods:We constructed 3 doubleand 3 single-human complement regulatory protein plasmids (pIRES-hMCP-hDAF, pIRES-hMCP-hCD59, pIRES-hDAF-hCD59, pIRES-A-hMCP, pIRES-B-hDAF, and pIRES-B-hCD59). The plasmids were transfected into NIH 3T3 cells, and stable transfectants were obtained by treatment with 200 kg/m3 G418 for 2 weeks. Normal human serum (50%) as a source of complement was added to the culture medium of stable transfectants. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the protective ability of different human complement regulatory protein plasmids on complement-dependent cytolysis. Results: The viability of double-human complement regulatory protein stable transfectants was significantly higher than that of single-human complement regulatory protein stable transfectants (P < .05). Among the double-transfectants, cells expressing pIRES-hMCP-hDAF and pIRES-hMCPhCD59 survived better than cells expressing pIREShDAF-hCD59 (91.75% ± 3.30% and 84.88% ± 2.36% vs 66.19% ± 6.52%; P < .05). Among the singletransfectants, cells expressing pIRES-A-hMCP or pIRES-B-hDAF survived better than cells expressing pIRES-B-hCD59 or pIRES empty vector (53.76% ± 3.84% and 56.32% ± 2.83% vs 43.28% ± 0.96% and 40.27% ± 1.11%; P < .05). Conclusions: These results suggest that the MCP+DAF and MCP+CD59 combinations could be more effective than DAF+CD59 in protecting the NIH 3T3 cells from injury caused by complementdependent cytolysis, whereas MCP or DAF alone is stronger than CD59 alone in inhibiting membrane attack complex formation.

show abstract

Section: Introductionmentioning

confidence: 99%

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Yang¹,

Deng²,

Jiang³

et al. 2012

Exp Clin Transplant

View full text Add to dashboard Cite

show abstract

“…Neutrophil oxidative burst is initiated by interaction of cell-surface complement (CR) and immunoglobulin G (Fc R) receptors with immune complexes (ICs) [13]. The major CR1 (CD35) ligands are C3b, C4b [14], and C3bi, with lower affinity [15], whereas C3bi is the main CR3 (CD11b/CD18) ligand [16]. Both CR1 and CR3 are implicated in the phagocytosis of complementopsonized particles [17].…”

Section: Introductionmentioning

confidence: 99%

Activation of Complement Alternative Pathway in Rheumatoid Arthritis: Implications in Peripheral Neutrophils Functions

Paoliello-Paschoalato¹

2011

TOAUTOJ

View full text Add to dashboard Cite

Evaluation of the respiratory burst induced by receptors Fc R and CR was carried out in peripheral blood neutrophils (PBN) in rheumatoid arthritis (RA) patients with active and inactive disease. Simultaneously, cooperation between these receptors and their expression, PBN chemotaxis, and complement system systemic activity were also investigated. Neutrophils were stimulated with IC-IgG opsonized with normal human serum (NHS) or not, or with IC-IgG opsonized with RA human serum (RAHS). ROS production was increased in neutrophils of patients with active or inactive RA stimulated of IC-IgG opsonized with NHS compared to the response of the cells mediated by ICIgG. However, there was poor Fc R/CR cooperation in these RA neutrophils, as indicated by decreased ROS production upon stimulation with IC-IgG opsonized with RAHS. In the case of active RA patients, neutrophils presented significantly higher CR1 and CR3 expression, as well as slight elevation in CD32 and CD16 expression. Positive correlations between Fc R and CR, complement alternative pathway activation, and increased RA serum chemotaxic activity were only detected in active RA patients. Taken together, these results indicate that several abnormalities of the complement system exist at the systemic level, namely impaired membrane receptor cooperation, alternative pathway activation, and presence of pre-existing chemoattractant factors in the serum, as reflected by the neutrophil function in the particular case of active RA patients. All, these abnormalities may synergistically contribute to RA pathogenesis.

show abstract

“…Methods to purify CR1 to homogeneity at the 1-to 10-nM level have been described by ourselves (16) and others (17,18). The NH2 terminus of CR1 is blocked (16,17).…”

Section: Introductionmentioning

confidence: 99%

“…The structural basis for this polymorphism is incompletely understood but appears to be due to peptide differences rather than posttranslational modifications (15). This conclusion is based on results (15) that demonstrated the following: (i) deglycosylation with endoglycosidase F decreases the molecular weight by -20,000 for all four variants, (ii) biosynthetic labeling of all four allotypic CR1 variants in Epstein-Barr virus (EBV)-transformed cell lines identifies pro-CR1 forms that retain the molecular weight differences of the mature CR1 molecules, and (iii) CR1 contains only N-linked carbohydrates and no sulfate, phosphate, or lipids.Methods to purify CR1 to homogeneity at the 1-to 10-nM level have been described by ourselves (16) and others (17,18). The NH2 terminus of CR1 is blocked (16,17).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Human complement C3b/C4b receptor (CR1) mRNA polymorphism that correlates with the CR1 allelic molecular weight polymorphism.

Holers¹,

Chaplin²,

Leykam³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The human C3b/C4b receptor (CR1) is a (5)(6)(7)(8). Initial identification of a partial CR1 cDNA has allowed localization of this complement regulatory gene complex to the q32 region of chromosome 1 (9). More recently, the complement receptor for C3d (CR2) was shown to be structurally related to CR1 (10).An interesting and unusual allelic polymorphism of CR1 based on molecular weight differences has been described (11-14). The four allelic variants have molecular weights of 190,000 (CR1-A), 220,000 (CR1-B), 160,000 (CR1-C), and 250,000 (CR1-D) and are found with a gene frequency of 0.83, 0.16, 0.01, and 0.002, respectively. The structural basis for this polymorphism is incompletely understood but appears to be due to peptide differences rather than posttranslational modifications (15). This conclusion is based on results (15) that demonstrated the following: (i) deglycosylation with endoglycosidase F decreases the molecular weight by -20,000 for all four variants, (ii) biosynthetic labeling of all four allotypic CR1 variants in Epstein-Barr virus (EBV)-transformed cell lines identifies pro-CR1 forms that retain the molecular weight differences of the mature CR1 molecules, and (iii) CR1 contains only N-linked carbohydrates and no sulfate, phosphate, or lipids.Methods to purify CR1 to homogeneity at the 1-to 10-nM level have been described by ourselves (16) and others (17,18). The NH2 terminus of CR1 is blocked (16,17). In order to obtain sequence of internal tryptic peptides, a method was developed to rapidly isolate and sequence individual peptides. A 2.5-kilobase (kb) cDNA clone for CR1 was identified and characterized. The molecular nature of the CR1 allelic molecular weight polymorphism was assessed by an analysis of mRNA of cells expressing the CR1 phenotypes. MATERIALS AND METHODSPurification of CR1 and Tryptic Peptide Fragments. Four nanomoles of erythrocyte CR1 (16) was reduced in 0.1 M NH4HCO3/1% NaDodSO4/20 mM dithiothreitol at 370C for 90 min and then alkylated in the dark by the addition of50 mM iodoacetic acid at 370C for 60 min. After precipitation overnight at -20'C with a 10-fold excess (vol/vol) of methanol and a 2% (wt/wt) amount of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (TPCKtrypsin), the preparation was centrifuged at 25,000 x g at 40C for 30 min. The supernatant was removed, the tube was dried under N2, and CR1 was resolubilized in 0.5 ml of 0.1 M NH4HC03/0.001% NaDodSO4/2 mM CaCl2. TPCK-trypsin (2%, wt/wt) was added and then again at 4 hr. After incubation at 370C for a total of 16 hr, the sample was lyophilized.A "two-dimensional" HPLC separation of tryptic fragments was performed, using a Waters liquid chromatograph.Abbreviation: EBV, Epstein-Barr virus. 2459The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

show abstract

Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

Cited by 31 publications

References 30 publications

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Activation of Complement Alternative Pathway in Rheumatoid Arthritis: Implications in Peripheral Neutrophils Functions

Human complement C3b/C4b receptor (CR1) mRNA polymorphism that correlates with the CR1 allelic molecular weight polymorphism.

Contact Info

Product

Resources

About