Fiber optic biosensor for the detection of C-reactive protein and the study of protein binding kinetics

Application of a fiber optic biosensor (FOB) to the real-time investigation of the interaction kinetics between FITC-conjugated monoclonal sheep anti-human C-reactive protein (CRP) antibody and CRP isoforms on the surface of optical fiber is described. Recently, both the native pentameric CRP (pCRP), an acute phase protein belonging to pentraxin family, and an isoform of pCRP, modified CRP (mCRP), have been suggested to have proinflammation effects on vascular cells in acute myocardial infarction (AMI). In current studies, we generate mCRP from pCRP, and use several methods including fluorescence spectral properties, circular dichroism, analytical ultracentrifuge, and Western blotting to demonstrate their differences in physical and chemical properties as well as the purity of pCRP and mCRP. In addition, we design and implement an FOB to study the real-time qualitative and quantitative biomolecular recognition of CRP isoforms. Specifically, the association and dissociation rate constants of the reaction between FITC-conjugated monoclonal sheep anti-human CRP antibody and the pCRP and mCRP are determined. The feasibility of our current approach to measure the association and dissociation rate constants of the reaction between tested CRP isoforms was successfully demonstrated.     

Proinflammatory changes in human umbilical cord vein endothelial cells can be induced neither by native nor by modified CRP

The role of C-reactive protein (CRP) in atherosclerosis is controversial. It is not clear, either, if the presumed endothelium-activating effect of CRP resides in native CRP (nCRP) or in a conformational isoform of CRP known as modified CRP (mCRP). In the present study we evaluated and compared the effect of nCRP, recombinant modified CRP (rmCRP) and urea-modified CRP (umCRP) on human umbilical vein endothelial cells (HUVECs). CRP preparations were carefully analyzed by biochemical, immunological and cell biological methods in order to avoid endotoxin or sodium azide contamination as well as inappropriate conformational changes, which together had possibly been the main reason for the previously published controversial results. Neither nCRP nor mCRP showed significant cytotoxicity up to 100 microg ml(-1) at 24 h but high concentrations of CRPs induced cell death at 48 h. rmCRP but not nCRP nor umCRP showed membrane binding to HUVEC by confocal microscopy. However, none of the CRP forms induced intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin expression or IL-8 production. Monocyte chemotactic protein-1 production was weakly inhibited by high concentration of both nCRP and rmCRP, analyzed by sandwich ELISA. Neither nCRP nor mCRP could induce pro-inflammatory changes in the phenotype of HUVECs. Therefore, our present findings do not support the notion that different isoforms of CRP alone have significant effects on inflammation of the vessel wall via an interaction with endothelial cells (ECs), although one cannot exclude the possibility that there may be significant differences among various types of ECs in the response to CRP.     

Modified C-reactive protein selectively binds to immunoglobulins

Modified C-reactive protein (mCRP) has been reported to non-specifically bind to immunoglobulins; notwithstanding, the nature of these interactions is not clear. The aim of this study was to investigate the binding of antibodies directed against HSA and IgG to mCRP, fibrinogen (Fg), IgG, fibronectin (Fn) and C1q and its contaminants. We also studied the binding of mCRP to the antibodies directed towards receptors involved in CRP signalling (anti-CD32, anti-CD16). For the analysis of such interactions, a combination of ELISA and Western immunoblotting has been applied. The tested antibodies powerfully bound to either the contaminations of purified proteins (Fg, IgG, Fn and mCRP) or interacted directly with some of these proteins (C1q, mCRP, Fg). The effectiveness of anti-HSA binding to immobilized proteins was influenced by the antigenic specificity of the antibody, the content of various protein fractions in the contaminants of a given protein (albumin augmented the interactions), overall protein purity and a natural avidity of a given protein towards immunoglobulins. The relative binding of anti-HSA or anti-IgG to immobilized mCRP was considerably lower than that observed for plasma proteins. Furthermore, the strength of the direct interaction between immunoglobulins and mCRP varied from the lack of response (anti-HSA) or a negligible response (anti-IgG) to the relatively high signal (human IgG, anti-CD16, anti-CD32), as compared to the control. Based on these observations, we conclude that the binding of mCRP to immunoglobulins cannot be easily generalized as a kind of some universal phenomenon.     

Autoantibodies against monomeric C-reactive protein in sera from patients with lupus nephritis are associated with disease activity and renal tubulointerstitial lesions

Serum levels of C-reactive protein (CRP) often remain low despite high disease activity in systemic lupus erythematosus (SLE). Sera from 96 patients with renal biopsy-proven active lupus nephritis, 24 of 96 patients in remission, and 49 patients with SLE with negative urinalysis (nonrenal SLE) was collected. Immunoglobulin G autoantibodies against monomeric CRP (mCRP) were screened by enzyme-linked immunosorbent assay with purified human CRP. Associations with clinical features, pathological data, and laboratory findings were investigated. The prevalence of mCRP autoantibodies in active lupus nephritis (57/96, 59.4%) was significantly higher than that in patients with SLE without clinical evidence of kidney involvement (20/49, 40.8%, p = 0.034). For the 13 patients with positive mCRP autoantibodies and sequential sera, their positive mCRP autoantibodies in active phase turned negative in remission (13/13, 100%). Patients with mCRP autoantibodies had significantly higher SLEDAI scores than patients without mCRP autoantibodies (18.3 +/- 5.2 vs 15.8 +/- 4.0, p = 0.013), who were more likely to experience acute renal failure (14/55 vs 2/33, p = 0.022), oral ulcer (15/57 vs 3/39, p = 0.022), and delayed activated partial thromboplastin time (18/52 vs 2/38, p = 0.001). Positive correlations between levels of mCRP autoantibodies and semiquantitative scores of renal histologic features were first observed in lupus nephritis as follows: interstitial inflammation (r = 0.328), tubular atrophy(r = 0.276), interstitial fibrosis (r = 0.211), and chronicity index score (r = 0.243). Autoantibodies against mCRP are prevalent in patients with lupus nephritis and are associated with disease activity and renal tubulointerstitial lesions.     

Amyloid Plaques Dissociate Pentameric to Monomeric C‐Reactive Protein: A Novel Pathomechanism Driving Cortical Inflammation in Alzheimer's Disease?

Beta-amyloid (Aβ) plaques and local inflammation are central to the pathogenesis of Alzheimer's disease. Although an association between circulating pentameric C-reactive protein (pCRP) and Alzheimer's disease has been reported no pathomechanistic link has been established. We hypothesized that Aβ plaques induce the dissociation of pCRP to individual monomers (mCRP), which possess strong pro-inflammatory properties not shared with pCRP and localizing inflammation to Alzheimer's plaques. pCRP was incubated with Aβ plaques generated in vitro and with non-aggregated Aβ(42) peptide. pCRP dissociation to mCRP was found only when co-incubated with Aβ plaques. Furthermore, sections of frontal cortex from brains of patients with and without Alzheimer's disease were stained with antibodies specific for mCRP and pCRP. There was significantly more mCRP in the cortex of Alzheimer's disease patients (P ≤ 0.01). In contrast, there was no significant difference in pCRP staining. These findings establish that Aβ plaques possess a previously unrecognized potential to dissociate pentameric CRP to monomeric CRP. The existence of mCRP but not pCRP in the brains of Alzheimer's disease patients strongly indicates that this newly described biological effect of Aβ plaques is relevant in Alzheimer pathobiology; potentially localizing and amplifying inflammation via the strong pro-inflammatory effects of locally generated mCRP.     

Monomeric C‐reactive protein and inflammation in age‐related macular degeneration

Age‐related macular degeneration (AMD) is a devastating disease characterized by central vision loss in elderly individuals. Previous studies have suggested a link between elevated levels of total C‐reactive protein (CRP) in the choroid, CFH genotype, and AMD status; however, the structural form of CRP present in the choroid, its relationship to CFH genotype, and its functional consequences have not been assessed. In this report, we studied genotyped human donor eyes (n = 60) and found that eyes homozygous for the high‐risk CFH (Y402H) allele had elevated monomeric CRP (mCRP) within the choriocapillaris and Bruch's membrane, compared to those with the low‐risk genotype. Treatment of choroidal endothelial cells in vitro with mCRP increased migration rate and monolayer permeability compared to treatment with pentameric CRP (pCRP) or medium alone. Organ cultures treated with mCRP exhibited dramatically altered expression of inflammatory genes as assessed by RNA sequencing, including ICAM‐1 and CA4, both of which were confirmed at the protein level. Our data indicate that mCRP is the more abundant form of CRP in human choroid, and that mCRP levels are elevated in individuals with the high‐risk CFH genotype. Moreover, pro‐inflammatory mCRP significantly affects endothelial cell phenotypes in vitro and ex vivo, suggesting a role for mCRP in choroidal vascular dysfunction in AMD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The yin-yang of long pentraxin PTX3 in inflammation and immunity

Pentraxins are a family of multimeric proteins characterized by the presence of a pentraxin signature in their C-terminus region. Based on the primary structure, pentraxins are divided into short and long pentraxin: C-reactive protein (CRP) is the prototype of the short pentraxin subfamily while pentraxin 3 (PTX3) is the prototypic long pentraxin. Despite these two molecules exert similar fundamental actions in the regulation of innate immune and inflammatory responses, several differences exist between CRP and PTX3, including gene organization, protein oligomerization and expression pattern. The pathophysiological roles of PTX3 have been investigated using genetically modified mice since PTX3 gene organization and regulation are well conserved between mouse and human. Such in vivo studies figured out that PTX3 mainly have host-protective effects, even if it could also exert negative effects under certain pathophysiologic conditions. Here we will review the general properties of CRP and PTX3, emphasizing the differences between the two molecules and the regulatory functions exerted by PTX3 in innate immunity and inflammation.     

CRP-Associated Determinant Expressed by Human Monocytes

Reports of a large number of biological activities of the acute phase reactant C-reactive protein (CRP) (1,2) and its association with a variety of blood borne cells (3,4), led us to a study of the expression of CRP by human peripheral blood cells (PBL) using two kinds of CRP-reactive monoclonal antibodies. We had previously described a reaction of CRP with a monoclonal antiidiotype antibody to a murine phosphocholine (PCh) binding myeloma protein (5). Of another group of monoclonal antibodies obtained by immunizing mice with human CRP, three were shown to react with human CRP in a Ca⁺⁺-dependent manner while a fourth reacted with CRP independently of calcium and binding of this antibody was also not blocked with PCh (6). Initial experiments showed that one of these site specific antibodies also reacted with a proportion of normal human peripheral blood cells. We then showed that this subpopulation included 100% of human monocytes suggesting that a CRP-like determinant exists on the surface of human monocytes but not on other lymphoid or NK cells or granulocyte populations.     

Serum CRP-like protein profile in common carp Cyprinus carpio challenged with Aeromonas hydrophila and Escherichia coli lipopolysaccharide

The potential of C-reactive protein (CRP)-like proteins to be used as a biomarker of health status in cultured carp obtained from various European fish lines has been assessed. Varying CRP-like protein levels in the serum of carp were monitored using an indirect competitive enzyme-linked immunosorbent assay. CRP-like protein basal levels in normal fish varied between carp lines, ranging on average from 2.9+/-0.15 to 12.57+/-1.19mugml(-1). Serum levels of CRP-like protein in carp were observed to increase several fold in fish infected with the pathogen Aeromonas hydrophila. However, carp injected with Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 did not exhibit an increase in CRP-like proteins levels.     

Modified C-Reactive Protein Is Expressed by Stroke Neovessels and Is a Potent Activator of Angiogenesis In Vitro

Native C-reactive protein (nCRP) is a pentameric oligo-protein and an acute phase reactant whose serum expression is increased in patients with inflammatory disease. We have identified by immunohistochemistry, significant expression of a tissue-binding insoluble modified version or monomeric form of CRP (mCRP) associated with angiogenic microvessels in peri-infarcted regions of patients studied with acute ischaemic stroke. mCRP, but not nCRP was expressed in the cytoplasm and nucleus of damaged neurons. mCRP co-localized with CD105, a marker of angiogenesis in regions of revascularisation. In vitro investigations demonstrated that mCRP was preferentially expressed in human brain microvessel endothelial cells following oxygen-glucose deprivation and mCRP (but not column purified nCRP) associated with the endothelial cell surface, and was angiogenic to vascular endothelial cells, stimulating migration and tube formation in matrigel more strongly than fibroblast growth factor-2. The mechanism of signal transduction was not through the CD16 receptor. Western blotting showed that mCRP stimulated phosphorylation of the key down-stream mitogenic signalling protein ERK1/2. Pharmacological inhibition of ERK1/2 phosphorylation blocked the angiogenic effects of mCRP. We propose that mCRP may contribute to the neovascularization process and because of its abundant presence, be important in modulating angiogenesis in both acute stroke and later during neuro-recovery.     

CRP-associated determinant expressed by human monocytes

Reports of a large number of biological activities of the acute phase reactant C-reactive protein (CRP) (1,2) and its association with a variety of blood borne cells (3,4), led us to a study of the expression of CRP by human peripheral blood cells (PBL) using two kinds of CRP-reactive monoclonal antibodies. We had previously described a reaction of CRP with a monoclonal antiidiotype antibody to a murine phosphocholine (PCh) binding myeloma protein (5). Of another group of monoclonal antibodies obtained by immunizing mice with human CRP, three were shown to react with human CRP in a Ca++-dependent manner while a fourth reacted with CRP independently of calcium and binding of this antibody was also not blocked with PCh (6). Initial experiments showed that one of these site specific antibodies also reacted with a proportion of normal human peripheral blood cells. We then showed that this subpopulation included 100% of human monocytes suggesting that a CRP-like determinant exists on the surface of human monocytes but not on other lymphoid or NK cells or granulocyte populations.     

Rapid recycling of cholesterol: the joint biologic role of C-reactive protein and serum amyloid A

Proteins that are highly conserved throughout evolution are presumed to have critical roles in the survival of the species. The two major acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) increase up to 1000-fold during inflammation. Both proteins have been highly conserved phylogenetically for at least the last 500 million years. Thus far the physiologic role and the evolutionary significance of each remains uncertain and their potential interactions have been totally ignored despite a vast and accelerating scientific literature on the involvement of each in human disease. CRP is known to bind to phosphocholine in dead eukaryote and some live bacterial cell walls suggesting that CRP facilitates the phagocytosis of fragmented or intact dead cells and/or enhances host bacterial defenses. SAA has recently been shown to increase the rate of export of cholesterol of phagocytosed cell membranes from macrophages fourfold. We postulate that their combined physiological role is to facilitate the rapid endogenous recycling of cell membrane cholesterol and phospholipids during acute inflammation. CRP promotes efficient phagocytosis of dying cells by macrophages; SAA enhances the export of their free cholesterol/phospholipid for reuse in the membranes of the hundreds of billions of new cells required daily during acute inflammation and repair. The evolutionary conservation of these proteins in species from the horseshoe crab and echinoderms to humans suggests that the rapid endogenous recycling of cholesterol and phospholipids during the highly vulnerable period of acute inflammation is critical for their continual survival.     

Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein

There are many controversies surrounding the biological activities of native C-reactive protein (nCRP) and its various modified forms such as monomerized and biotinylated CRP (mCRP and bCRP). No simple methods have been described to distinguish among these forms. By adapting established electrophoresis methods, we have developed a useful quality control method with which we have investigated the structural and functional characteristics of these forms of CRP. Under all electrophoresis conditions, biotinylation altered the electrophoretic mobility of CRP. nCRP was sensitive to sodium dodecyl sulphate (SDS)-induced monomerization, and only mCRP was susceptible to digestion by trypsin or neutrophil-derived serine proteases. bCRP and mCRP but not nCRP bound to cells, suggesting that chemical modification by biotin and denaturation had altered the structural integrity of CRP. Neither nCRP nor mCRP had the ability to induce secretion of chemokines, nor did they increase intracellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.     

Novel neuronal proteolipid protein isoforms encoded by the human myelin proteolipid protein 1 gene

The human myelin proteolipid protein 1 gene (hPLP1), which encodes the major structural myelin proteins of the central nervous system (CNS), is classically described as expressed in the oligodendrocytes, the CNS myelinating cells. We identified two new exons in the intron 1 of the hPLP1 gene that lead to the expression of additional mRNA and protein isoforms mainly expressed in neurons instead of oligodendrocytes. Those novel neuronal PLP isoforms are detected as soon as human fetal development and their concomitant expression is specific of the human species. As classical PLP proteins, the novel protein isoforms seem to be addressed to the plasma membrane. These results suggest for the first time that PLP may have functions in humans not only in oligodendrocytes but also in neurons and could be implicated in axono-glial communication. Moreover, this neuronal expression of the hPLP1 gene might explain the neuronal dysfunctions in patients carrying hPLP1 gene mutations.     

C-reactive protein (CRP) but not the related pentraxins serum amyloid P and PTX3 inhibits the proliferation and induces apoptosis of the leukemia cell line Mono Mac 6

Background

Pentraxins are a family of highly conserved secreted proteins that regulate the innate immune system, including monocytes and macrophages. C-reactive protein (CRP) is a plasma protein whose levels can rise to 1000 μg/ml from the normal <3 μg/ ml during inflammation.

Results

We find that CRP inhibits proliferation of the human myeloid leukemia cell line Mono Mac 6 with an IC50 of 75 μg/ ml by inducing apoptosis of these cells. The related proteins serum amyloid P (SAP) and pentraxin 3 (PTX3) do not inhibit Mono Mac 6 proliferation. CRP has no significant effect on the proliferation of other leukemia cell lines such as HL-60, Mono Mac 1, K562, U937, or THP-1, or the survival of normal peripheral blood cells. The effect of CRP appears to be dependent on the CRP receptor FcγRI, and is negatively regulated by a phosphatidylinositol ?3-kinase pathway.

Conclusion

These data reveal differential signaling by pentraxins on immune cells, and suggest that CRP can regulate the proliferation of some myeloid leukemia cells.

     

Calsequestrins in skeletal and cardiac muscle from adult <Emphasis Type="Italic">Danio rerio</Emphasis>

Calsequestrin (Casq) is a high capacity, low affinity Ca²⁺-binding protein, critical for Ca²⁺-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located.     

Topographical distribution of antimicrobial genes in the zebrafish intestine

The zebrafish is increasingly being utilized to study aspects of the conserved innate intestinal immunity of vertebrates. In mammals, some antimicrobial proteins are synthesised by specialised immune cells that appear to have no equivalent in zebrafish. To delineate foci of antimicrobial protein production along the zebrafish intestine, we examined the antero-posterior expression gradients of antimicrobial genes. Quantitative PCR revealed distinct expression gradient profiles, with the mid-intestine exhibiting elevated expression of several genes such as dual oxidase and the defensin beta-like and peptidoglycan recognition protein families. This region also presented with the most numbers of leukocytes and endocytic cells, supporting a specialised immunological role. Conversely, expression of the Dr-RNase family was prominent in the anterior intestine. Expression of the zebrafish β-defensin family was examined in adult zebrafish tissues. Strong expression of defensin beta-like 1 was detected in the swim bladder of zebrafish from the larval stage of development through to adults.     

Pulmonary innate immune proteins and receptors that interact with gram-positive bacterial ligands

The two major gram-positive bacterial (GPB) ligands are peptidoglycan (PGN) and lipoteichoic acid (LTA). These polymeric LTA and highly organized PGN contain repeating carbohydrate moieties, which are potential targets for pattern recognition molecules. The major pattern recognition proteins and receptors, which bind GPB, either have a lectin, PGN recognition, collagen or leucine-rich repeat (LRR) domain. The soluble innate immune proteins (IIPs) that bind to PGN and LTA include pulmonary collectins surfactant-associated proteins (SP-) A and D, lectin-like pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP), and sCD14. Membrane-anchored lectin or lectin-like group members include macrophage mannose receptor (MR), complement receptor 3 (CR3, or Mac-1, or integrin CD11b/CD18), scavenger receptor A (SRCL-1), lectin-like oxidized LDL receptor 1 (LOX-1), and GPI-anchored CD14. Although Toll-like receptor (TLR) 2 and 4, and CD14 contain extracellular LRR domains, only TLRs have a cytoplasmic domain for signal transduction. Three of the four recently discovered human PGN recognition proteins (PGRP) have a transmembrane domain, and hence, considered as true receptors for GPB. Since lysozyme is the only known pulmonary enzyme that can lyse bacterial cell wall PGN, other innate immune molecules appear to be responsible for signalling and enhancing the clearance of GPB infection from the lung. Interestingly, pulmonary collectins bind not only to GPB ligands but also to the receptors, CD14 and TLR, and antigen processing cells such as dentritic cells. These complex interactions appear to play major roles in linking innate and adaptive immunity, and maintaining a pathogen-free lung with minimal, or no inflammation.