Probing the C4-binding site on C1s with monoclonal antibodies. Evidence for a C4/C4b-binding site on the gamma-domain.

Matsumoto, Misako; Nagaki, Kumiko; Kitamura, Hisayo; Kuramitsu, Seiki; Nagasawa, Shigeharu; Seya, Tsukasa

doi:10.4049/jimmunol.142.8.2743

Cited by 24 publications

(2 citation statements)

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“…A previous study had determined the active status of C1s exploiting the characteristic of C1s cleaved into C2 and C4 ( 23 ). Unfortunately, the procedure of this method is cumbersome to perform.…”

Section: Discussionmentioning

confidence: 99%

“…The development of homogeneous FRET-based immunoassays contributes to quick and highly specific on-site detection of protein biomarkers for clinical molecular diagnostics of cancers ( 20 , 21 ) and autoimmune diseases ( 22 ). Based on the specific cleavage of C2 and C4 proteins by the active C1s ( 23 ), we currently designed the FRET-based peptide that could be cleaved by the active C1s, which conjugated with magnetic microbeads. This study aimed to establish a quantitative immunoassay for the detection of active C1s in serum samples ( Figure 1 ) .…”

Section: Introductionmentioning

confidence: 99%

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Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s

Zhang

et al. 2023

Front. Immunol.

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ObjectivesC1s activation is associated with the pathogenesis of various diseases, indicating the potential value of C1s activation detection in clinic. Here we aimed to establish fluorescence resonance energy transfer (FRET)-based immunoassay for the quantitative detection of activated C1s in serum.MethodsFRET-based fluorogenic peptides, sensitive to the enzymatic activity of activated C1s, were prepared and labeled with the fluorophore ortho-aminobenzoic acid (Abz) and quencher 2,4-dinitrophenyl (Dnp), and then were further selected depending on its Kcat/Km value. C1s in the samples was captured and separated using anti-C1s-conjugated magnetic microbeads. Next, enzymatic activity of activated C1s in samples and standards was examined using fluorescent quenched substrate assays. Limit of detection (LOD), accuracy, precision, and specificity of FRET-based immunoassay were also investigated.ResultsThis method presented a linear quantification range for the enzymatic activity of activated C1s up to 10 μmol min-1 mL-1 and LOD of 0.096 μmol·min-1·mL-1 for serum samples. The recovery of the method was in the range of 90% ~ 110%. All CV values of the intra-analysis and inter-analysis of three levels in samples were less than 10%. The cross-reaction rates with C1r enzyme, MASP1, and MASP2 were less than 0.5%. No significant interferences were found with bilirubin (0.2 mg mL-1), Chyle (2000 FTU), and haemoglobin (5 mg mL-1), but anticoagulants (EDTA, citrate and heparin) inhibited the enzymatic ability of activated C1s. Thus, this established method can be used for the determination of active C1s in human serum samples in the concentration interval of 0.096-10.000 μmol min-1 mL-1.ConclusionsOne anti-C1s-based FRET immunoassay for activated C1s detection in serum samples were established, and it will be useful to explore the role of C1s activation in the pathogenesis, diagnosis and treatment in complement-related diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s

Zhang

et al. 2023

Front. Immunol.

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Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and “sterile” subjects

et al. 1993

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A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46). These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologous of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and "sterile" subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from "sterile" subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunoblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP. The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.

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Immunologicalization of complement C1s and matrix metalloproteinase 9 (92kDa gelatinase/type IV collagenase) in the primary ossification center of the human femur

Sakiyama¹,

Inaba

Toyoguchi

et al. 1994

Cell Tissue Res

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The first component of complement C1s has been shown to degrade type I and type II collagens (Yamaguchi et al. 1990), the latter of which is a major constituent of the cartilage matrix. In order to understand the physiological roles of C1s in cartilage resorption, the expression of C1s was examined by immunohistochemistry in the primary ossification center where the matrix is removed and replaced by bone marrow. Hypertrophic chondrocytes, endothelium and hematogenous elements in the capillary buds were intensely stained by a monoclonal antibody against C1s. Matrix metalloproteinase 9 (MMP-9, 92kDa gelatinase/type IV collagenase) was also immunolocalized in hypertrophic chondrocytes, mesenchymal cells in the primitive bone marrow and the cartilage matrix adjacent to the marrow. In addition, C1s was found to activate the zymogen of MMP-9. These observations suggest that C1s and MMP-9 coordinately participate in matrix degradation in cartilage.

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Probing the C4-binding site on C1s with monoclonal antibodies. Evidence for a C4/C4b-binding site on the gamma-domain.

Cited by 24 publications

References 0 publications

Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s

Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s

Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and “sterile” subjects

Immunologicalization of complement C1s and matrix metalloproteinase 9 (92kDa gelatinase/type IV collagenase) in the primary ossification center of the human femur

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