Fetal lung C4BPA induces p100 processing in human placenta.

The non-canonical NF-κB signaling may be a central integrator of a placental clock that governs the length of human pregnancy. We sought to identify fetal signals that could activate this NF-κB pathway in the placenta, and in turn, contribute to the onset of labor. Proteomics analysis of exosomes purified from fetal cord arterial blood revealed a total of 328 proteins, among which 48 were more significantly abundant (p < 0.01) in samples from women who delivered following elective Cesarean-section at term (39 to 40 weeks of estimated gestational age, EGA) compared to those who had elective Cesarean deliveries near term (35 to 36 weeks of EGA). Computational, crystal structural, and gene functional analyses showed that one of these 48 proteins, C4BPA, binds to CD40 of placental villous trophoblast to activate p100 processing to p52, and in turn, pro-labor genes. These results suggest that fetal C4BPA-induced activation of non-canonical NF-κB in human placenta may play a critical role in processes of term or preterm labor.

UPLC-MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4-binding protein.

Serum levels of fully sialylated C4-binding protein (FS-C4BP) are remarkably elevated in patients with epithelial ovarian cancer (EOC) and can be used as a marker to distinguish ovarian clear cell carcinoma from endometrioma. This study aimed to develop a stable, robust and reliable liquid chromatography-hybrid mass spectrometry (UPLC-MS/MS) based diagnostic method that would generalize FS-C4BP as a clinical EOC biomarker. Glycopeptides derived from 20 µL of trypsin-digested serum glycoprotein were analyzed via UPLC equipped with an electrospray ionization time-of-flight mass spectrometer. This UPLC-MS/MS-based diagnostic method was optimized for FS-C4BP and validated using sera from 119 patients with EOC and 127 women without cancer. A1958 (C4BP peptide with two fully sialylated biantennary glycans) was selected as a marker of FS-C4BP because its level in serum was highest among FS-C4BP family members. Preparation and UPLC-MS/MS were optimized for A1958, and performance and robustness were significantly improved relative to our previous method. An area under the curve analysis of the FS-C4BP index receiver operating characteristic curve revealed that the ratio between A1958 and A1813 (C4BP peptide with two partially sialylated biantennary glycans) reached 85%. A combination of the FS-C4BP index and carbohydrate antigen-125 levels further enhanced the sensitivity and specificity.

Crystal Structure of an Invasivity-Associated Domain of SdrE in S. aureus.

The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with ß2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes.

Fully-sialylated alpha-chain of complement 4-binding protein: Diagnostic utility for ovarian clear cell carcinoma.

OBJECTIVE: While a certain fraction of endometriomas can develop de novo epithelial ovarian cancer (EOC) such as clear cell carcinoma (OCCC), there is currently no useful biomarker available for early detection of OCCC from endometriomas. The aim of this study was to describe the diagnostic utility of a novel biomarker for EOC especially for OCCC to distinguish from endometrioma. METHODS: More than 100,000 glycan structures of serum glycoproteins obtained from 134 pretreatment all stage EOC patients (including 45 OCCCs) and 159 non-cancer control women (including 36 endometriomas) were explored for a mass spectrum approach. Diagnostic accuracy of identified biomarker was compared to the one of CA-125 by comparing area under curve (AUC) and positive/negative predictive values (PPV and NPV). RESULTS: A2160, a fully-sialylated alpha-chain of complement 4-binding protein, was identified as a candidate target marker. A2160 was significantly elevated in all stages of OCCC compared to with endometriomas. Diagnostic accuracy of A2160 (cutoff 1.6U/mL) to distinguish early stage OCCC from endometrioma is significantly higher than that of CA-125 (cutoff 35IU/L): AUC for A2160 versus CA-125, 0.92 versus 0.67; PPV 95% versus 64%; and NPV 85% versus 58%. In addition, fully-sialylated glycans had a higher accuracy for diagnosing EOC as compared to partially-sialylated glycans of alpha-chain of complement 4-binding protein. CONCLUSION: Our study suggested that A2160 may be a useful biomarker to distinguish early-stage OCCC from endometrioma. This new biomarker can be potentially applied for the monitoring of endometrioma patients, making possible the early diagnosis of OCCC.

The α7ß0 isoform of the complement regulator C4b-binding protein induces a semimature, anti-inflammatory state in dendritic cells.

The classical pathway complement regulator C4b-binding protein (C4BP) is composed of two polypeptides (α- and ß-chains), which form three plasma oligomers with different subunit compositions (α7ß1, α7ß0, and α6ß1). We show in this article that the C4BP α7ß0 isoform (hereafter called C4BP[ß(-)] [C4BP lacking the ß-chain]), overexpressed under acute-phase conditions, induces a semimature, tolerogenic state on human monocyte-derived dendritic cells (DCs) activated by a proinflammatory stimulus. C4BP isoforms containing ß-chain (α7ß1 and α6ß1; C4BP[ß(+)]) neither interfered with the normal maturation of DCs nor competed with C4BP(ß(-)) activity on these cells. Immature DCs (iDCs) treated with C4BP(ß(-)) retained high endocytic activity, but, upon LPS treatment, they did not upregulate surface expression of CD83, CD80, and CD86. Transcriptional profiling of these semimature DCs revealed that treatment with C4BP(ß(-)) prevented the induction of IDO and BIC-1, whereas TGF-ß1 expression was maintained to the level of iDCs. C4BP(ß(-))-treated DCs were also unable to release proinflammatory Th1 cytokines (IL-12, TNF-α, IFN-γ, IL-6, IL-8) and, conversely, increased IL-10 secretion. They prevented surface CCR7 overexpression and, accordingly, displayed reduced chemotaxis, being morphologically indistinguishable from iDCs. Moreover, C4BP(ß(-))-treated DCs failed to enhance allogeneic T cell proliferation, impairing IFN-γ production in these cells and, conversely, promoting CD4(+)CD127(low/neg)CD25(high)Foxp3(+) T cells. Deletion mutant analysis revealed that the complement control protein-6 domain of the α-chain is necessary for the tolerogenic activity of C4BP(ß(-)). Our data demonstrate a novel anti-inflammatory and immunomodulatory function of the complement regulator C4BP, suggesting a relevant role of the acute-phase C4BP(ß(-)) isoform in a number of pathophysiological conditions and potential applications in autoimmunity and transplantation.

Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation.

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic ß-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in ß-cell death in type 2 diabetic patients. Because Aß-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.

Differences in protein distribution between human plasma preparations, EDTA-plasma and heparin-plasma, analyzed by non-denaturing micro-2-DE and MALDI-MS PMF.

The effects of plasma preparation methods on the status of human plasma proteins were analyzed by non-denaturing micro-2-DE followed with polypeptide assignment with MALDI-TOF MS and PMF. In order to facilitate the separation of high-molecular-mass plasma proteins (up to ca. 2x10(3) kDa) in short separation time, agarose micro-IEF gels were employed. The comparisons of the 2-DE patterns between EDTA-plasma (1.0 mg EDTA/mL blood) and heparin-plasma (0.025 mg heparin/mL blood) revealed the differences in protein distribution around pI 5.4-6.5 and apparent molecular mass of ca. 1x10(3) kDa, which were mainly attributed to the presence of the complexes of complement C4b and C4b-binding protein in heparin-plasma and their absence in EDTA-plasma. The distribution of several spots around pI 5.0-5.6 and apparent molecular mass 1.2-1.5x10(2) kDa was also found to be different; the fragments of complement C3 and C4 were detected in heparin-plasma but not in EDTA-plasma. The 2-DE pattern of high-heparin-plasma (0.50 mg heparin/mL blood) showed pI changes of three plasma proteins, fibronectin, complement factor B, and pre-alpha-inhibitor when compared with that of heparin-plasma, suggesting the interactions between heparin and the proteins. These results demonstrated that subtle changes in plasma proteins, caused by the different plasma preparation procedures, can be analyzed by non-denaturing agarose-IEF/micro-2-DE followed by MALDI-MS and PMF.