Enhanced activation of and increased production of matrix metalloproteinase-9 by human blood monocytes upon adhering to carbamylated collagen

AbstractCarbamylation refers to chemical modification of protein side chains by cyanate derived e.g. from urea. It alters their structural and functional properties. We have studied the influence of the carbamylation of type I collagen in vitro on its interactions with elutriated human monocytes, and its potential role in atherosclerosis. Adhesion of monocytes onto carbamylated collagen was significantly enhanced compared to native collagen. There was no change in superoxide anion production. On the other hand, there was an increase in the production and the activation of matrix metalloproteinase-9. No effect was found on tissue inhibitor of metalloproteinase-1 production. Thus, the presence of carbamylated collagen may stimulate the remodelling of extracellular matrix mediated by activated monocytes. Such alterations may contribute to enhanced atherosclerosis in renal insufficiency, a pathological condition associated with elevated levels of carbamylation

Labile glycated haemoglobin and carbamylated haemoglobin are still critical points for HbA1c measurement

IntroductionHaemoglobin A1c (HbA1c) is a key analyte for the monitoring of glycemic balance in diabetic patients and is used for diabetes diagnosis in many countries. The potential interference of carbamylated haemoglobin (cHb) and labile glycated haemoglobin (LA1c) on HbA1c assays must remain a matter of vigilance. Such a situation has occurred in our laboratory with a kit replacement on the Bio-Rad Variant™ II testing system, a cation-exchange high performance liquid chromatography (HPLC) system. With this method, LA1c and cHb coeluted in a same peak which may have different consequences on HbA1c values. Materials and methodsThe influence of increasing LA1c and cHb values on HbA1c results was studied with in vitro glycation and carbamylation of samples. Samples from patients with high and normal blood urea concentrations were assayed by HPLC and immunological assay. ResultsWe observed that the degree of interference greatly varied depending on the nature of the interfering Hb fractions found under the so-called “LA1c peak”. Thus, we have decided to apply a decision tree using “LA1c” thresholds depending on: (i) the retention time, (ii) the shape of the peak, (iii) other analytes, like urea. If the peak recognized as “LA1c” is mainly formed by LA1c, we consider that there is no interference until 4%. If the peak is mainly formed by cHb, we consider an interference threshold equal to 2%. ConclusionsThis situation reminds that cHb and LA1c remain critical issues in chromatography-based HbA1c assays and that adapted criteria must be set up for result interpretation

Evaluation of the analytical performances of the Cobas c513 analyser for HbA1c assay

Introduction: Haemoglobin A1c (HbA1c) is considered to be the gold standard for the follow-up of glycaemic control in patients with diabetes mellitus and is also a diagnostic tool. Accordingly, reliable and efficient methods must be used for its quantification. Roche Diagnostics have recently adapted the Tina-quant® HbA1c Third Generation immunoassay on a fully dedicated analyser, the Cobas c513, which allows a high throughput of up to 400 samples per hour. The present article deals with the evaluation of the analytical performances of this system which has been recently introduced to the market. Materials and methods: Precision, comparison with two ion-exchange high-performance liquid chromatography (HPLC) methods (Variant II and D-100 systems, BioRad Laboratories) using Passing Bablok and Bland-Altman analyses, accuracy and interference of the most frequent haemoglobin (Hb) variants on HbA1c measurement were evaluated. Results: Precision was high, with coefficients of variation lower than 1.1% (HbA1c values expressed in National Glycohemoglobin Standardization Program units, 1.7% for values expressed in International Federation of Clinical Chemistry and Laboratory Medicine [IFCC] units). The comparison study showed similar results with the two HPLC systems. The analysis of samples with IFCC-assigned values showed high methodological accuracy. Finally, no interference of bilirubin, triglycerides and common Hb variants (Hb AC, AD, AE, AS) was observed. Conclusions: This evaluation showed that the analytical performance of the Cobas c513 analyser for HbA1c assay makes it suitable for a routine use in clinical chemistry laboratories

Influence de la carbamylation sur les propriétés structurales du collagène de type I et ses interactions avec les polynucléaires neutrophiles humains

La @carbamylation est une modification post-traductionnelle caractérisée par la fixation non enzymatique de cyanate, un dérivé réactif de l'urée, sur les groupements NH2 libres des protéines, affectant plus particulièrement les résidus de lysine, transformés en homocitrulline. Les modifications post-traductionnelles conduisent dans la majorité des cas à une altération des propriétés structurales et fonctionnelles des protéines et de leurs interactions avec les cellules. Au cours de l'insuffisance rénale chronique, contexte pathologique favorisant la réaction de carbamylation, les patients présentent des troubles infectieux et inflammatoires importants, d'origine multifactorielle. Notre travail traite de l'influence de la carbamylation sur les caractéristiques structurales et fonctionnelles du collagène de type I, protéine de la matrice extracellulaire impliquée dans l'architecture du tissu conjonctif mais également dans la régulation des fonctions cellulaires, notamment celles des polynucléaires neutrophiles. Après carbamylation in vitro (au cours de laquelle seulement 1 % des acides aminés sont transformés), le collagène conserve une structure en hélice de type polyproline II, mais présente, par analyse spectroscopique et polarimétrique, une légère fragilisation de sa conformation dans certaines régions. Ces changements conformationnels, même mineurs, sont responsables d'une incapacité du collagène carbamylé à s'associer sous forme de fibres, ainsi que d'une altération de sa sensibilité à la dégradation enzymatique : le collagène carbamylé est plus résistant aux collagénases et plus sensible aux gélatinases et à la pepsine. Par ailleurs, la carbamylation entraîne une inhibition de l'activation des polynucléaires neutrophiles par le collagène de type I. La recherche du mécanisme impliqué dans ce processus a permis de montrer que le peptide a1CB6, contenant la séquence activatrice des polynucléaires neutrophiles, constituait une cible préférentielle de carbamylation. D'autre part, des expériences de mutagenèse dirigée ont révélé l'importance du résidu de lysine en position 1208, situé à proximité de cette séquence, pour l'activation des polynucléaires neutrophiles par le collagène de type I. Ces résultats montrent que la carbamylation du collagène peut être considérée comme un évènement favorisant la progression de l'insuffisance rénale chronique. En effet, les modifications des propriétés structurales du collagène observées après carbamylation peuvent entraîner un trouble du renouvellement du collagène au sein de la matrice extracellulaire et une désorganisation architecturale de différents tissus comme la paroi vasculaire ou le rein. Par ailleurs, l'incapacité du collagène carbamylé à stimuler les fonctions oxydatives des polynucléaires neutrophiles peut contribuer à l'apparition d'infections, une des causes majeures de morbidité et de mortalité des patients urémiques.REIMS-BU Santé (514542104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Carbamylated Proteins in Renal Disease: Aggravating Factors or Just Biomarkers?

Carbamylation is a nonenzymatic post-translational modification resulting from the reaction between cyanate, a urea by-product, and proteins. In vivo and in vitro studies have demonstrated that carbamylation modifies protein structures and functions, triggering unfavourable molecular and cellular responses. An enhanced formation of carbamylation-derived products (CDPs) is observed in pathological contexts, especially during chronic kidney disease (CKD), because of increased blood urea. Significantly, studies have reported a positive correlation between serum CDPs and the evolutive state of renal failure. Further, serum concentrations of carbamylated proteins are characterized as strong predictors of mortality in end-stage renal disease patients. Over time, it is likely that these modified compounds become aggravating factors and promote long-term complications, including cardiovascular disorders and inflammation or immune system dysfunctions. These poor clinical outcomes have led researchers to consider strategies to prevent or slow down CDP formation. Even if growing evidence suggests the involvement of carbamylation in the pathophysiology of CKD, the real relevance of carbamylation is still unclear: is it a causal phenomenon, a metabolic consequence or just a biological feature? In this review, we discuss how carbamylation, a consequence of renal function decline, may become a causal phenomenon of kidney disease progression and how CDPs may be used as biomarkers

Chronic increase of urea leads to carbamylated proteins accumulation in tissues in a mouse model of CKD.

Carbamylation is a general process involved in protein molecular ageing due to the nonenzymatic binding of isocyanic acid, mainly generated by urea dissociation, to free amino groups. In vitro experiments and clinical studies have suggested the potential involvement of carbamylated proteins (CPs) in chronic kidney disease (CKD) complications like atherosclerosis, but their metabolic fate in vivo is still unknown. To address this issue, we evaluated protein carbamylation in the plasma and tissues of control and 75% nephrectomised C57BL/6J mice by LC-MS/MS assay of homocitrulline, the major carbamylation-derived product (CDP). A basal level of carbamylation was evidenced under all conditions, showing that carbamylation is a physiological process of protein modification in vivo. CP plasma concentrations increased in nephrectomized vs. control mice over the 20 weeks of the experiment (e.g. 335 ± 43 vs. 167 ± 19 μmol homocitrulline/mol lysine (p<0.001) 20 weeks after nephrectomy). Simultaneously, CP content increased roughly by two-fold in all tissues throughout the experiment. The progressive accumulation of CPs was specifically noted in long-lived extracellular matrix proteins, especially collagen (e.g. 1264 ± 123 vs. 726 ± 99 μmol homocitrulline/mol lysine (p<0.01) in the skin of nephrectomized vs. control mice after 20 weeks of evolution). These results show that chronic increase of urea, as seen in CKD, increases the carbamylation rate of plasma and tissue proteins. These results may be considered in the perspective of the deleterious effects of CPs demonstrated in vitro and of the correlation evidenced recently between plasma CPs and cardiovascular risk or mortality in CKD patients

Vieillissement moléculaire des protéines

Le vieillissement moléculaire des protéines correspond aux modifications non enzymatiques que subissent celles-ci au cours de leur vie biologique et qui conduisent à l’altération de leurs propriétés structurales et fonctionnelles. Ce phénomène participe aux vieillissements cellulaire et tissulaire et, par conséquent, au vieillissement général de l’organisme. Il est également accentué au cours de maladies chroniques comme le diabète ou l’insuffisance rénale chronique, où il participe au développement de complications à long terme. Cette synthèse décrit les principales réactions responsables du vieillissement moléculaire des protéines, leurs conséquences ainsi que les facteurs influençant ce phénomène. Enfin, un schéma général exposant son rôle en physiopathologie est proposé

Molecular identification of omega-amidase, the enzyme that is functionally coupled with glutamine transaminases, as the putative tumor suppressor Nit2.

Our purpose was to identify the sequence of omega-amidase, which hydrolyses the amide group of alpha-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative 'carbon-nitrogen hydrolase'. The closest mammalian homolog of this putative bacterial omega-amidase is 'nitrilase 2', whose size and amino acid composition were in good agreement with those reported for purified rat liver omega-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of alpha-ketoglutaramate and other known substrates of omega-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is omega-amidase

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes

International audienceBackground: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methods: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.Results: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.Conclusions: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis