Soluble collectin-12 mediates C3-independent docking of properdin that activates the alternative pathway of complement.

Properdin stabilizes the alternative C3 convertase (C3bBb), whereas its role as pattern-recognition molecule mediating complement activation is disputed for decades. Previously, we have found that soluble collectin-12 (sCL-12) synergizes complement alternative pathway (AP) activation. However, whether this observation is C3 dependent is unknown. By application of the C3-inhibitor Cp40, we found that properdin in normal human serum bound to Aspergillus fumigatus solely in a C3b-dependent manner. Cp40 also prevented properdin binding when properdin-depleted serum reconstituted with purified properdin was applied, in analogy with the findings achieved by C3-depleted serum. However, when opsonized with sCL-12, properdin bound in a C3-independent manner exclusively via its tetrameric structure and directed in situ C3bBb assembly. In conclusion, a prerequisite for properdin binding and in situ C3bBb assembly was the initial docking of sCL-12. This implies a new important function of properdin in host defense bridging pattern recognition and specific AP activation.

Complement activation is associated with crescent formation in IgA nephropathy.

IgA nephropathy (IgAN) is common chronic glomerulonephritis with variable prognosis, ranging from minor urinary abnormalities to end-stage renal disease. The revised Oxford classification of IgAN explains that cellular/fibrocellular crescents are associated with poor renal prognosis, proposing an extension to the MEST-C score. C3 immunofluorescent staining follows a distribution similar to IgA staining. Therefore, complement activation was reported to play a pivotal role in IgAN pathogenesis. This study included 132 IgAN patients diagnosed by renal biopsies. The clinical parameters at the time of the biopsies were obtained from patient data records. We classified the patients into C ≥ 1 and C0 groups, and compared clinical, light microscopic, and immunofluorescent features. In the C ≥ 1 group, 2 (1.5%) and 31 (23.5%) patients were assigned to C2 and C1, respectively. The remaining 99 patients (75%) were classified as C0. The C ≥ 1 group had lower average age and rate of hypertension, and higher score of urinary occult blood and E score. The C ≥ 1 group had significantly higher average immunofluorescence scores for IgA, C5b-9, mannose-associated serine protease (MASP) 1/3, MASP2, properdin, factor B, and kappa. The steroid use rate was significantly higher in the C ≥ 1 group. During the follow-up period of 2.90 years on average, the rate of renal dysfunction was not significantly different between groups. Crescent formation in IgAN was associated with activation of the lectin and alternative pathways. The C ≥ 1 group had significantly increased use of steroids, which probably caused comparable renal function during the follow-up period.

Prognostic Value of Complement Properdin in Cancer.

The complement system is readily triggered by the presence of damage-associated molecular patterns on the surface of tumor cells. The complement alternative pathway provides rapid amplification of the molecular stress signal, leading to complement cascade activation to deal with pathogens or malignant cells. Properdin is the only known positive regulator of the alternative pathway. In addition, properdin promotes the phagocytic uptake of apoptotic T cells by macrophages and dendritic cells without activating the complement system, thus, establishing its ability to recognize "altered-self". Dysregulation of properdin has been implicated in substantial tissue damage in the host, and in some cases, chronic unresolved inflammation. A corollary of this may be the development of cancer. Hence, to establish a correlation between properdin presence/levels in normal and cancer tissues, we performed bioinformatics analysis, using Oncomine and UALCAN. Survival analyses were performed using UALCAN and PROGgeneV2 to assess if properdin can serve as a potential prognostic marker for human lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), cervical squamous cell carcinoma (CESC), and pancreatic adenocarcinoma (PAAD). We also analyzed levels of tumor-infiltrating immune cells using TIMER, a tool for characterizing immune cell composition in cancers. We found that in LUAD and LIHC, there was a lower expression of properdin in the tumors compared to normal tissues, while no significant difference was observed in CESC and PAAD. Survival analysis demonstrated a positive association between properdin mRNA expression and overall survival in all 4 types of cancers. TIMER analysis revealed that properdin expression correlated negatively with tumor purity and positively with levels of infiltrating B cells, cytotoxic CD8+ T cells, CD4+ helper T cells, macrophages, neutrophils and dendritic cells in LUAD, CESC and PAAD, and with levels of B cells, CD8+ T cells and dendritic cells in LIHC. Immunohistochemical analysis revealed that infiltrating immune cells were the most likely source of properdin in the tumor microenvironment. Thus, complement protein properdin shows promise as a prognostic marker in cancer and warrants further study.

Thromboinflammatory changes in plasma proteome of pregnant women with PCOS detected by quantitative label-free proteomics.

Polycystic ovary syndrome (PCOS) is the most common endocrinological disorder of fertile-aged women. Several adverse pregnancy outcomes and abnormalities of the placenta have been associated with PCOS. By using quantitative label-free proteomics we investigated whether changes in the plasma proteome of pregnant women with PCOS could elucidate the mechanisms behind the pathologies observed in PCOS pregnancies. A total of 169 proteins with ≥2 unique peptides were detected to be differentially expressed between women with PCOS (n = 7) and matched controls (n = 20) at term of pregnancy, out of which 35 were significant (p-value < 0.05). A pathway analysis revealed that networks related to humoral immune responses, inflammatory responses, cardiovascular disease and cellular growth and proliferation were affected by PCOS. Classification of cases and controls was carried out using principal component analysis, orthogonal projections on latent structure-discriminant analysis (OPLS-DA), hierarchical clustering, self-organising maps and ROC-curve analysis. The most significantly enriched proteins in PCOS were properdin and insulin-like growth factor II. In the dataset, properdin had the best predictive accuracy for PCOS (AUC = 1). Additionally, properdin abundances correlated with AMH levels in pregnant women.

Complement activation products in the circulation and urine of primary membranous nephropathy.

BACKGROUND: Complement activation plays a substantial role in the pathogenesis of primary membranous nephropathy (pMN). C5b-9, C3c, MBL, and factor B have been documented in the subepithelial immune deposits. However, the changing of complement activation products in circulation and urine is not clear. METHODS: We measured the circulating and urinary levels of C1q, MBL, C4d, Bb, properdin, C3a, C5a, and sC5b-9, in 134 patients with biopsy-proven pMN, by enzyme-linked immunosorbent assay. All the plasma values were corrected by eGFR and all the urinary values were corrected by urinary creatinine and urinary protein excretion. Anti-PLA2R antibodies were measured in all patients. RESULTS: The plasma complement activation products were elevated both in the patients with and without anti-PLA2R antibodies. C3a levels were remarkably increased in the circulation and urine, much higher than the elevated levels of C5a. C5b-9 was in normal range in plasma, but significantly higher in urine. The urinary C5a had a positive correlation with anti-PLA2R antibody levels and urinary protein. The plasma level of C4d was elevated, but C1q and MBL were comparable to healthy controls. Positive correlations were observed between plasma C4d/MBL and urinary protein, only in the patients with positive anti-PLA2R antibodies but not in those without. The plasma level of Bb was elevated and had positive correlation with urinary protein only in the patients without anti-PLA2R antibodies. CONCLUSION: Complement activation products were remarkable increased in pMN and may serve as sensitive biomarkers of disease activity. The complement may be activated through lectin pathway with the existence of anti-PLA2R antibodies, while through alternative pathway in the absence of antibody.

Parsimonious Charge Deconvolution for Native Mass Spectrometry.

Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new "parsimonious" charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.

Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS.

AIM: Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). RESULTS: In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. CONCLUSION: The validated method was robust and implemented in support of a preclinical PK/PD study.

Elevated properdin and enhanced complement activation in first-degree relatives of South Asian subjects with type 2 diabetes.

OBJECTIVE: Emerging data implicate activation of the complement cascade in the pathogenesis of type 2 diabetes. The objective of the current study was to evaluate the relationships between components of the complement system, metabolic risk factors, and family history of type 2 diabetes in healthy South Asians. RESEARCH DESIGN AND METHODS: We recruited 119 healthy, first-degree relatives of South Asian subjects with type 2 diabetes (SARs) and 119 age- and sex-matched, healthy South Asian control subjects (SACs). Fasting blood samples were taken for measurement of complement factors and standard metabolic risk factors. RESULTS: SARs were characterized by significantly higher properdin (mean concentration 12.6 [95% CI 12.2-13.1] mg/L vs. SACs 10.1 [9.7-10.5] mg/L, P < 0.0001), factor B (187.4 [180.1-195.0] mg/L vs. SACs 165.0 [158.0-172.2] mg/L, P < 0.0001), and SC5b-9 (92.0 [86.1-98.3] ng/mL vs. SACs 75.3 [71.9-78.9] ng/mL, P < 0.0001) and increased homeostasis model assessment of insulin resistance (2.86 [2.61-3.13] vs. SACs 2.31 [2.05-2.61], P = 0.007). C-reactive protein did not differ between SARs and SACs (P = 0.17). In subgroup analysis of 25 SARs and 25 SACs with normal oral glucose tolerance tests, properdin, factor B, and SC5b-9 remained significantly elevated in SARs. CONCLUSIONS: Increased properdin and complement activation are associated with a family history of type 2 diabetes in South Asians independent of insulin resistance, and predate the development of impaired fasting glucose and impaired glucose tolerance. Properdin and SC5b-9 may be novel biomarkers for future risk of type 2 diabetes in this high-risk population and warrant further investigation.

Detection and identification of plasma proteins that bind GlialCAM using ProteinChip arrays, SELDI-TOF MS, and nano-LC MS/MS.

In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS. The advantages of this new strategy are that the primary high-throughput screening of samples can be performed with small amounts of sample, no specific antibody is needed and the proteins represented on the SELDI-TOF MS spectra can be identified with high confidence. Furthermore, the method is faster and less labor-intensive than other current approaches. Using this novel method, we isolated and identified the interactions of two mouse plasma proteins, mannose binding lectin C and properdin, with GlialCAM, a type 1 transmembrane glycoprotein that belongs to the Ig superfamily.

Tracking down immune markers from alternative system pathway factors in a diabetic population.

Hyperglycemia associated with type 1 diabetes (T1D) alters the host immune system, resulting in a predisposition to infectious diseases. The high risk of infection in the diabetic population may lead to life-threatening situations. The early proteins of the alternative complement system pathway, constituting factors P, B, and D, have been shown to play an important role in preventing infection because they form a membrane attack complex (MAC)-C5-9, which debilitates the target microbes and/or molecules via cytotoxic and cytolytic reactions. Patients who are devoid of or contain low levels of these proteins may be susceptible to developing chronic infections. We have observed striking differences in partially fractionated serum proteins in diabetic patients (type 2) relative to controls, through single and two-dimensional gel electrophoresis. Our data, obtained from 50 diabetic patients in the age group of 25-45 years, who had the disease for fewer than 5 years, indicated patterns in low- and high-molecular-weight proteins, which could be grouped into five different categories with minor differences in their respective levels of protein expression. Immunoblot assay could barely detect the presence of properdin expression in diabetic patients. Quantization by ELISA in 99 patients indicated low levels of properdin expression in 70% of 50 diabetic patients (6.5 +/- 3 mug/mL) when compared to nondiabetic controls (19.5 +/- 8.5 mug/mL). This study concluded that patients with low expression of properdin should be advised to take extensive preventive measures and seek early management with appropriate treatments against infection.

Serum amyloid A, properdin, complement 3, and toll-like receptors are expressed locally in human sinonasal tissue.

BACKGROUND: There is a growing appreciation of the role that nasal mucosa plays in innate immunity. In this study, the expression of pattern recognition receptors known as toll-like receptors (TLRs) and the effector molecules complement factor 3 (C3), properdin, and serum amyloid A (SAA) were examined in human sinonasal mucosa obtained from control subjects and patients with chronic rhinosinusitis (CRS). METHODS: Sinonasal mucosal specimens were obtained from 20 patients with CRS and 5 control subjects. Messenger RNA (mRNA) was isolated and tested using Taqman real-time polymerase chain reaction with primer and probe sets for C3, complement factor P, and SAA. Standard polymerase chain reaction was performed for the 10 known TLRs. Immunohistochemistry was performed on the microscopic sections using antibodies against C3. RESULTS: Analysis of the sinonasal sample mRNA revealed expression of all 10 TLRs in both CRS samples and in control specimens. Expression of the three effector proteins was detected also, with the levels of mRNA for C3 generally greater than SAA and properdin in CRS patients. No significant differences were found in TLR or innate immune protein expression in normal controls. Immunohistochemical analysis of sinonasal mucosal specimens established C3 staining ranging from 20 to 85% of the epithelium present. CONCLUSION: These studies indicate that sinonasal mucosa expresses genes involved in innate immunity including the TLRs and proteins involved in complement activation. We hypothesize that local production of complement and acute phase proteins by airway epithelium on stimulation of innate immune receptors may play an important role in host defense in the airway and, potentially, in the pathogenesis of CRS.

Innate immunity of the sinonasal cavity: expression of messenger RNA for complement cascade components and toll-like receptors.

OBJECTIVE: To study the expression of important elements of the innate immune responses in human sinonasal tissue to elucidate its potential role in mucosal inflammation. DESIGN: We studied human sinonasal tissue from patients with chronic rhinosinusitis and an immortalized epithelial cell line to detect the expression of innate immune effectors and the responses of these cells to stimulation with compounds associated with pathogenic organisms. PATIENTS: Nine individuals undergoing endoscopic sinus surgery for chronic rhinosinusitis. MAIN OUTCOME MEASURES: Expression of complement components and toll-like receptors. RESULTS: We found detectable levels of messenger RNA for all toll-like receptors in human sinonasal tissue and in the BEAS-2B epithelial cell line. Expression of several components of the alternate pathway of complement (factors B, H, and I and properdin) was constitutively present in unstimulated BEAS-2B cells and was readily detectable in human sinonasal tissue. Stimulation of BEAS-2B cells with the toll-like receptor 3 ligand double-stranded RNA resulted in increased expression of messenger RNA for factors B and H but not for properdin or factor I. CONCLUSIONS: Toll-like receptors and the alternate pathway of complement are important components of innate immunity that are expressed in human sinonasal epithelium in vivo and in cultured airway epithelial cells in vitro. The expression of some of these components can be significantly induced by stimulation via toll-like receptors, and epithelial expression of components of innate immunity may play a role in inflammation in chronic rhinosinusitis.

Ontogeny of complement regulatory proteins - concentrations of factor h, factor I, c4b-binding protein, properdin and vitronectin in healthy children of different ages and in adults.

Previous studies of human in vivo complement protein levels have only compared data for neonates with that from adult sera. Here, we establish the normal concentration ranges of the following complement regulatory proteins in healthy Brazilian children of different age groups (neonates: 1 month-1 year, 1-6 years and 6-13 years) and in adults: factor H (fH), factor I (fI), C4b-binding protein (C4 BP), properdin and vitronectin. We found that the concentrations of fH, fI, properdin and vitronectin in neonates are significantly lower than in adults. Remarkably, the concentration of C4 BP is below the method resolution (<50 micro g/ml) in 76% of the sera from neonates, while adults presented 199-532 microg/ml of C4 BP in their sera. The concentration of properdin in the sera from neonates and up to 1-year-old children was less than that observed in older children. Adults presented vitronectin levels significantly higher than all the other age groups in the study. No significant sex differences in the concentrations of all the studied regulatory proteins were detected. This study reveals the ontogeny of complement system in greater detail than previously available and may point to the reasons why neonates have higher susceptibility to develop life-threatening pyogenic infections. These reference values will be of use in clinical and laboratory investigations of disorders associated with low levels of these regulatory proteins.

Alternative pathway evaluation.

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.

Discordant renal histopathologic findings and complement profiles in membranoproliferative glomerulonephritis type III.

Patients with membranoproliferative glomerulonephritis (MPGN) type III and a low serum C3 concentration tend to have evidence for a nephritic factor of the terminal complement pathway (Nft). Complement profiles were studied in three patients with MPGN type III and low serum C3 concentrations. Serum C3 concentrations were 52, 21, and 14 mg/dL (normal range, 83 to 177 mg/dL). Serum Clq, C2, C4, properdin, and C5 concentrations were normal in all patients, whereas two had a slight decrease of C7 or C8. This pattern of complement activation resembles that seen with MPGN type II in which a nephritic factor activates the amplification loop (NFa). We conclude that in patients with MPGN type III the previously reported profile/presence of Nft is not always found, at least in the chronic stage of the disease, despite a low C3 value.

Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization.

Normal endochondral bone formation follows a temporal sequence: immature or resting chondrocytes move away from the resting zone, proliferate, flatten, become arranged into columns, and finally become hypertrophic, disintegrate, and are replaced by bone. The mechanisms that guide this process are incompletely understood, but they include programmed cell death, a stage important in development and some disease processes. Using immunofluorescence we have studied the distribution of various complement proteins to examine the hypothesis that this sequence of events, particularly cell disintegration and matrix dissolution, are complement mediated. The results of these studies show that complement proteins C3 and Factor B are distributed uniformly in the resting and proliferating zones. Properdin is localized in the resting and hypertrophic zone but not in the proliferating zone. Complement proteins C5 and C9 are localized exclusively in the hypertrophic zones. This anatomically segregated pattern of distribution suggests that complement proteins may be important in cartilage-bone transformation and that the alternate pathway is involved.