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Protein carbamylation has been linked with diseases commonly associated with patients with reduced kidney function. Carbamylated human serum albumin (cHSA), which has been proven to be nephrotoxic and associated with heart failure for chronic kidney disease (CKD) patients, was chosen for our study. Through phage display against cHSA, one specific peptide sequence (cH2-p1) was identified with higher selectivity toward cHSA over native HSA. The cH2-p1 peptide was synthesized, and its target binding was analyzed through isothermal titration calorimetry (ITC). The result showed that cH2-p1 was able to bind cHSA of different levels of carbamylation with a similar dissociation constant of ∼1.0 × 10-4 M. This peptide also showed a binding specificity to carbamylated fibrinogen (cFgn), while not binding to native Fgn at all. For better understanding of the binding mechanism of cH2-p1, competitive binding of cH2-p1 and anti-homocitrulline to cHSA was performed, and the result revealed that cH2-p1 may bind to homocitrulline residues in a similar manner to the antibody. A molecular docking study was further performed to investigate the favored binding conformation of homocitrulline residue to cH2-p1. This work demonstrates the potential of peptides as a specific binding element to carbamylated proteins.

Cardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis patients. This morbidity is due to the accelerated atherosclerosis observed in these patients. Atherosclerosis is a chronic inflammatory disease characterized by key players such as monocytes, macrophages, or oxidized LDLs. Monocytes-macrophages are classified into subsets of polarized cells, with M1 and M2 macrophages considered, respectively, as pro- and anti-inflammatory. (1) Methods: The monocyte subsets and phenotypes were analyzed by flow cytometry. These data were completed by the quantification of plasma M-CSF, IL-8, CRP, Mox-LDLs, Apo-B, Apo-AI, chloro-tyrosine, and homocitrulline concentrations. The statistical differences and associations between two continuous variables were assessed using the Mann&ndash, Whitney U test and Spearman&rsquo, s correlation coefficient, respectively. (2) Results: Hemodialyzed patients showed a significant increase in their concentrations of CRP, M-CSF, and IL-8 (inflammation biomarkers), as well as chloro-tyrosine and homocitrulline (myeloperoxidase-associated oxidative stress biomarkers). Moreover, we observed a higher percentage of M2 monocytes in the plasma of hemodialysis patients as compared to the controls. (3) Conclusions: Our data suggest that oxidative stress and an inflammatory environment, which is amplified in hemodialysis patients, seems to favor an increase in the concentration of circulating M-CSF, therefore leading to an increase in M2 polarization among circulating monocytes.

Abnormal gut microbiota and blood metabolome profiles have been reported both in children and adults with uncomplicated type 1 diabetes as well as in adults with type 1 diabetes and advanced stages of diabetic nephropathy. In this study we aimed to investigate the gut microbiota and a panel of targeted plasma metabolites in individuals with type 1 diabetes of long duration without and with different levels of albuminuria. In a cross-sectional study we included 161 individuals with type 1 diabetes and 50 healthy control individuals. Individuals with type 1 diabetes were categorised into three groups according to historically measured albuminuria: (1) normoalbuminuria (

Die Immunantwort gegen posttranslational modifizierte Antigene, insbesondere die Entwicklung von Autoantikörpern gerichtet gegen citrullinierte Proteine („anti citrullinated protein antibodies“, [ACPA]), ist ein sehr spezifisches Phänomen der rheumatoiden Arthritis. Bis heute ist unklar, wie es zur Entwicklung dieser Immunantwort kommt und welche Faktoren dazu beitragen, dass aus dieser zunächst asymptomatischen Autoimmunreaktion eine Autoimmunerkrankung entsteht. Analysen zu genetischen Risikofaktoren legen nahe, dass T‑Helfer-Zellen hierbei eine wichtige Rolle zukommt. Unter ihrem Einfluss kommt es zu einer Reifung der citrullinspezifischen B‑Zell-Immunantwort im Vorfeld des Erkrankungsbeginns. Welche Trigger diese Entwicklung stimulieren ist nicht bekannt. Neue Daten zeigen, dass ACPA nicht nur citrullinierte Antigene erkennen. Auch andere Eiweißmodifikationen wie Homocitrullin und Acetyllysin werden spezifisch erkannt. Diese Kreuzreaktivität konnte für verschiedene monoklonale ACPA nachgewiesen werden. Hierdurch erweitert sich das Spektrum der Antigene, durch die ACPA-exprimierende B‑Zellen stimuliert und aktiviert werden können. Auch T‑Zellen, die selbst nicht autoreaktiv sind, sondern Fremdantigene erkennen, treten als mögliche Helfer in den Vordergrund. Die vorliegende Übersichtsarbeit gibt einen Einblick in die Bedeutung dieser neuen Erkenntnisse für das Krankheitsbild der rheumatoiden Arthritis.

Posttranslational modifications of proteins like citrullination and carbamylation are associated with several diseases. Detailed analytical characterization of citrullinated and carbamylated proteins or peptides could be difficult due to the low concentration of the analytes in complex biological samples. High structural similarity and chemical behavior of citrullinated and carbamylated residues also pose a challenge. We previously reported the "citrulline effect" phenomenon that is manifested in the generation of intense y type ions originating from Cit-Zzz amide bond scissions in collision-induced dissociation tandem mass spectra of citrullinated tryptic peptides. In this study, we created a rigorous tryptic-like model system of both citrulline and homocitrulline-containing peptides that included appropriate and well-defined controls and fragment analogues to quantify the citrulline effect and investigate whether there is an effect for homocitrulline residues as well. Our results show that citrulline residues significantly increased fragmentation at their C-terminus relatively independent of the identity of the following amino acid. In comparison, homocitrulline residues displayed inconclusive results at the same energies. However, the strength of effects was dependent on collision energy and the position of citrulline and homocitrulline in the sequences. As newer software algorithms tend to observe structure-intensity relationships during annotation, this finding increases reliable identification of modified proteins/peptides.

Backgroud Homocitrulline (Hcit), is involved in the pathological processes of some diseases. However, the role and function of Hcit (CBL) in human skin remains largely obscure. Objective To investigate the correlation of the level of Hcit in seborrheic keratosis, skin aging, and its clinical significance. Methods Immunohistochemistry was used to analyze the level of Hcit in skin lesions of seborrheic keratosis (SK), unaffected skin (distant 0.5 centimeters from SK lesion), and normal skin of healthy subjects in the control group. ELISA test was used to detect the serum level of CBL in SK patients and healthy subjects of different ages. Results Hcit was mainly localized in the nucleus of epidermal cells. In healthy control skin, the expression of Hcit increased with age and showed a positive correlation with age (the correlation coefficient was 0.806, p = 0.0002). The expressional level of Hcit in SK lesions was higher than that in healthy control skin (Z = −3.703, p = 0.0002). The serum level of CBL in healthy subjects and in SK patients increased with age (the correlation coefficient were 0.5763, p = 0.0032; 0.682, p = 0.004. respectively). The serum level of CBL in SK patients was higher than that in healthy subjects (Z = −2.19, p = 0.030). Study limitations The small serum sample size in the study. Conclusion The high expressional level of Hcit is correlated with seborrheic keratosis and skin aging. HCit may be one of the potential biomarkers of skin aging.

Osteoarthritis of the hand is the second most common location of symptomatic osteoarthritis after the knee. Several studies have suggested that the properties of cartilage depend on its anatomical location. We compared cartilage from proximal interphalangeal (PPI), metacarpophalangeal (MCP) and knee joints from recently deceased human donors. Cell density was inversely correlated with cartilage thickness. The levels of homocitrulline (HCit) and carboxymethyllysine (CML), post-translational modifications of proteins related to ageing, varied according to anatomical location. Non-arthritic cartilages had different baseline secretion profiles depending on their location: PPIs secreted less MMP-3 than PCMs. Under the influence of a pro-inflammatory stimulus (IL-1β), the increase in IL-6 secretion was 6-fold greater in knees than in PPIs, that of MMP-3 greater in PPIs than in PCMs and knees. IL-1β-stimulated OA cartilage showed greater increases in IL-6 and PGE2 secretion at the knees than at the PPIs. Finally, serum HCit and CML levels did not correlate with the severity of radiographic damage in a cohort of patients with hand OA. Overall, there are differences in biochemical composition, and biological functional differences between digital and knee cartilage. The development of new therapeutic approaches must take this variability into account., L'arthrose de la main est la deuxième localisation d'arthrose symptomatique après le genou. Plusieurs études ont suggéré que les propriétés du cartilage dépendaient de sa localisation anatomique. Nous avons comparé des cartilages d’articulations inter-phalangiennes proximales (IPP), métacarpo-phalangiennes (MCP) et de genoux issus de donneurs humains récemment décédés. La densité cellulaire était inversement corrélée à l’épaisseur du cartilage considéré. Les taux d’homocitrulline (HCit) et de carboxymethyllysine (CML), modifications post-traductionnelles des protéines liées au vieillissement, variaient suivant la localisation anatomique. Les cartilages non arthrosiques avaient des profils de sécrétion de base différents selon leur localisation: les IPP sécrétaient moins de MMP-3 que les MCP. Sous l’influence d’un stimulus pro-inflammatoire (IL-1β), l’accroissement de sécrétion de l’IL-6 était 6 fois plus importante aux genoux qu’aux IPP, celui de la MMP-3 plus important aux IPP qu’aux MCP et qu’aux genoux. Les cartilages arthrosiques stimulés par l’IL-1β montraient des accroissements de sécrétion de l’IL-6 et de la PGE2 plus importants aux genoux qu’aux IPP. Enfin, les taux sériques d’HCit et de CML n’étaient pas corrélés à la sévérité de l’atteinte radiolographique dans une cohorte de patients souffrant d’arthrose des mains. Au total, il existe des différences de composition biochimique, et des différences fonctionnelles biologiques entre les cartilages digitaux et de genoux. Le développement de nouvelles approches thérapeutiques doit prendre en compte cette variabilité.

Backgrounds Nonalcoholic fatty liver disease (NAFLD) has multiple causes, is triggered by individual genetic susceptibility, environmental factors, and metabolic disturbances, and may be triggered by acquired metabolic stress. The metabolic profiles of NAFLD show significant ethnic differences, and the metabolic characteristics of NAFLD in Chinese individuals are unclear. Our study aimed to identify the metabolites and pathways associated with NAFLD in a Chinese cohort. Methods One hundred participants, including 50 NAFLD patients and 50 healthy controls, were enrolled in this retrospective observational study at Jinling Hospital in Nanjing; serum samples were collected from the patients and healthy subjects. The metabolome was determined in all samples by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). Univariate and multivariate statistical analyses were used to compare the metabolic profiles between the two groups. Results The comparison indicated that the levels of 89 metabolites were different between the two groups. The glycerophospholipid family of metabolites was the most abundant family of metabolites that demonstrated significant differences. L-acetylcarnitine, L-homocitrulline, and glutamic acid were the top three metabolites ranked by VIP score and had favorable effective functions for diagnosis. Moreover, pathway enrichment analysis suggested 14 potentially different metabolic pathways between NAFLD patients and healthy controls based on their impact value. Biological modules involved in the lipid and carbohydrate metabolism had the highest relevance to the conditions of NAFLD. Glycerophospholipid metabolism had the strongest associations with the conditions of NAFLD. Conclusions Our data suggest that the serum metabolic profiles of NAFLD patients and healthy controls are different. L-Homocitrulline was remarkably increased in NAFLD patients.

We have deployed a multi-omics approach in large cohorts of patients with existing type 2 diabetes to identify biomarkers for disease progression across three molecular classes, metabolites, lipids and proteins. A Cox regression analysis for association with time to insulin requirement in 2,973 patients in the DCS, ANDIS and GoDARTS cohorts identified homocitrulline, isoleucine and 2-aminoadipic acid, as well as the bile acids glycocholic and taurocholic acids, as predictive of more rapid deterioration. Increased levels of eight triacylglycerol species, and lowered levels of the sphingomyelin SM 42:2;2 were also predictive of disease progression. Of ∼1,300 proteins examined in two cohorts, levels of GDF-15/MIC1, IL-18RA, CRELD1, NogoR, FAS, and ENPP7 were associated with faster progression, whilst SMAC/DIABLO, COTL1, SPOCK1 and HEMK2 predicted lower progression rates. Strikingly, identified proteins and lipids were also associated with diabetes incidence and prevalence in external replication cohorts. Implicating roles in disease compensation, NogoR/RTN4R improved glucose tolerance in high fat-fed mice and tended to improved insulin signalling in liver cells whilst IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. Conversely, high NogoR levels led to islet cell apoptosis. This comprehensive, multi-disciplinary approach thus identifies novel biomarkers with potential prognostic utility, provides evidence for new disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Derivatization of amino acids by 2 M HCl/CH3OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)m-(PFP)n. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N5-carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N6-carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)4 and hCit-(PFP)4 are esterified on their C1-carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)m-(PFP)n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.

Carbamylation is an ubiquitous process in which cyanate (OCN−) reacts with the N-terminal amino or e-amino moiety and generates α-carbamyl amino acids and e-carbamyl-lysine (homocitrulline). The process leads to irreversible changes in protein charge, structure and function. In this study, we have investigated the effect of carbamyl (generated from potassium cyanate) on human serum albumin (HSA) structure and function. The carbamylated-HSA (c-HSA) showed various modifications when examined by UV, fluorescence, FT-IR and far-UV CD spectroscopies. c-HSA exhibited hypochromicity, loss in α-helical content, changes in the amide I and amide II band, etc. Native-PAGE showed increase in the mobility of c-HSA compared to native-HSA. Aggregate(s) formation in c-HSA was detected by thioflavin T dye. The biochemical investigations carried out on c-HSA suggested increase in carbonyl content and decreased binding of TNBS (trinitrobenzenesulphonic acid) and Sakaguchi reagent. The attachment of the carbamyl moiety to HSA was confirmed from MALDI-TOF results. The functional defects in c-HSA were confirmed from the low binding of bilirubin. Taken together, carbamylation of albumin caused changes in the structural and functional properties of HSA. To the best of our knowledge, this is the first report on detailed biophysical characterization of carbamylated-HSA.

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase, which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine and low-density-lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high fat diet and carrying human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homo-citrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques., SCOPUS: ar.j, info:eu-repo/semantics/published

Reduced skeletal muscle density in older adults is associated with insulin resistance, decreased physical function, and an increased all-cause mortality risk. To elucidate mechanisms that may underlie the maintenance of skeletal muscle density, we conducted a secondary analysis of previously published muscle composition and serum metabolomic data in 73 older adults (average age, 78 y). Multivariable-adjusted linear regression was used to examine associations between 321 metabolites with muscle composition, defined as the ratio between normal density (NDM) with low density (LDM) thigh muscle cross sectional area (NDM/LDM). Sixty metabolites were significantly (p ≤ 0.05 and q < 0.30) associated with NDM/LDM. Decreased renal function and the immune response have been previously linked with reduced muscle density, but the mechanisms underlying these connections are less clear. Metabolites that were significantly associated with muscle composition were then tested for their association with circulating markers of renal function (blood urea nitrogen, creatinine, uric acid), and with the immune response (neutrophils/lymphocytes) and activation (kynurenine/tryptophan). 43 significant NDM/LDM metabolites (including urea) were co-associated with at least 1 marker of renal function; 23 of these metabolites have been previously identified as uremic solutes. The neutrophil/lymphocyte ratio was significantly associated with NDM/LDM (β ± SE: −0.3 ± 0.1, p = 0.01, q = 0.04). 35 significant NDM/LDM metabolites were co-associated with immune activation. Carbamylation (defined as homocitrulline/lysine) was identified as a pathway that may link renal function and immune activation with muscle composition, as 29 significant NDM/LDM metabolites were co-associated with homocitrulline/lysine, with at least 2 markers of renal function, and with kynurenine/tryptophan. When considering that elevated urea and uremic metabolites have been linked with an increased systemic microbial burden, that antimicrobial defense can be reduced in the presence of carbamylation, and that adipocytes can promote host defense, we propose the novel hypothesis that the age-related increase in adipogenesis within muscle may be a compensatory antimicrobial response to protect against an elevated microbial burden.

Objective.Antibodies that target citrullinated protein/peptide (ACPA) and homocitrullinated/carbamylated protein/peptide (AHCPA) are associated with rheumatoid arthritis (RA). The relationship between ACPA and AHCPA remains unclear. We examined the expression and cross-reactivity of these antibodies using citrulline- and homocitrulline-containing synthetic peptides, CitJED and HomoCitJED, respectively, which have equal numbers of citrulline or homocitrulline residues on the same peptide backbone.Methods.Serum from healthy subjects (n = 51) and patients with RA (n = 137), systemic lupus erythematosus (SLE; n = 37), and psoriatic arthritis (PsA; n = 37) were screened for IgG anti-CitJED and anti-HomoCitJED antibodies by ELISA. Cross-reactivity of these antibodies was examined by inhibition with various concentrations of CitJED and HomoCitJED.Results.Out of 137 patients with RA, antibodies to CitJED and HomoCitJED were detected in 69 (50%) and 78 (57%), respectively. Anti-CitJED and HomoCitJED antibodies were 77% concordant and their levels were strongly correlated [Spearman correlation coefficient (rs) = 0.6676]. Sera from 25/27 patients (93%) with RA were inhibited by both CitJED and HomoCitJED with equal or higher affinity for the cognate (homologous) peptide.Conclusion.Antibodies to CitJED and HomoCitJED frequently occurred in RA, but were not found in SLE or PsA, suggesting that these antibodies are specific to RA. Cross-reactivity between anti-HomoCitJED and anti-CitJED antibodies suggests that ACPA and AHCPA are derived from the same B cell population and both may contribute to the pathogenesis of RA.

Background Brain degenerative protein modifications (DPMs) are associated with the apparition and progression of dementia, and at the same time, Alzheimer’s disease with cerebrovascular disease (AD + CVD) is the most prevalent form of dementia in the elder population. Thus, understanding the role(s) of brain DPMs in this dementia subtype may provide novel insight on the disease pathogenesis and may aid on the development of novel diagnostic and therapeutic tools. Two essential DPMs known to promote inflammation in several human diseases are the ureido DPMs (uDPMs) arginine citrullination and lysine carbamylation, although they have distinct enzymatic and non-enzymatic origins, respectively. Nevertheless, the implication of uDPMs in the neuropathology of dementia remains poorly understood. Methods In this study, we use the state-of-the-art, ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC)-coupled mass spectrometry technology to undertake a comparative characterization of uDPMs in the soluble and particulate postmortem brain fractions of subjects diagnosed with AD + CVD and age-matched controls. Results An increase in the formation of uDPMs was observed in all the profiled AD + CVD brains. Citrulline-containing proteins were found more abundant in the soluble fraction of AD + CVD whereas homocitrulline-containing proteins were preferentially abundant in the particulate fraction of AD + CVD brains. Several dementia-specific citrulline residues were also identified in soluble proteins previously categorized as pro-immunogenic, which include the receptor P2X7, alpha-internexin, GFAP, CNP, MBP, and histones. Similarly, diverse dementia-specific homocitrulline residues were also observed in the particulate fractions of AD + CVD in proteins that have been vastly implicated in neuropathology. Intriguingly, we also found that the amino acids immediately flanking arginine residues may specifically influence the increase in protein citrullination. Conclusions Taken together, these results indicate that uDPMs widely contribute to the pathophysiology of AD + CVD by promoting neuroinflammation and proteinopathy. Furthermore, the obtained results could help to identify disease-associated proteins that can act as potential targets for therapeutic intervention or as novel biomarkers of specific neuropathology. Electronic supplementary material The online version of this article (10.1186/s12974-017-0946-y) contains supplementary material, which is available to authorized users.

Objective: Anti-carbamylated protein antibodies (ACarPA) are associated with rheumatoid arthritis (RA) risk and severity and are primarily directed against fibrinogen. The lack of understanding of ACarPA reactivity has limited analysis of their immunopathogenic associations in RA. To address this shortcoming, we mapped ACarPA epitope reactivity in RA patient sera. Methods: Immunoblotting identified a patient serum with specific reactivity to carbamylated human fibrinogen β-chain. Liquid chromatography/mass spectrometry (LC/MS) identified sites of homocitrullines (carbamylated lysines) present in the human fibrinogen β-chain. The reactivity of an ACarPA+ cohort to specific peptides containing carbamylated lysines was determined by ELISA through direct binding (n=63) and by competition assays (n=40). Results: Serum from an RA patient with specific reactivity to carbamylated, but not citrullinated, fibrinogen β-chain was identified. LC/MS identified carbamylation of 9/34 lysines in the human fibrinogen β-chain. Mapping of immunoreactivity against tryptic peptide fragments demonstrated several candidate carbamylated epitopes that were confirmed by competition experiments. Peptides containing a homocitrulline at position 83 appeared to be an immunodominant epitope in some RA patient sera, with additional reactivity to peptides containing homocitrullines at positions 52, 264, 351, 367, and 374. Conclusion: ACarPA appear to preferentially target specific regions of the human fibrinogen β-chain that contain homocitrullines. Interestingly, humoral immunoreactivity appears relatively restricted in some patients, which may enable detection of specific relationships with disease phenotype. This article is protected by copyright. All rights reserved.

Background Environmental enteric dysfunction (EED), a condition characterized by small intestine inflammation and abnormal gut permeability, is widespread in children in developing countries and a major cause of growth failure. The pathophysiology of EED remains poorly understood. Methods We measured serum metabolites using liquid chromatography-tandem mass spectrometry in 400 children, aged 12–59 months, from rural Malawi. Gut permeability was assessed by the dual-sugar absorption test. Findings 80.7% of children had EED. Of 677 serum metabolites measured, 21 were negatively associated and 56 were positively associated with gut permeability, using a false discovery rate approach (q, Highlights • Environmental enteric dysfunction (EED) affects millions of children in developing countries. • We used metabolomics to gain insight into the pathogenesis of EED. • EED is characterized by secondary carnitine deficiency and abnormal fatty acid oxidation. • Alterations in sulfur amino acids, tryptophan, urea cycle, and polyphenols were also present. Environmental enteric dysfunction (EED), an asymptomatic condition of small intestine inflammation and increased gut permeability, is widespread in developing countries and a major cause of growth failure in children. Clinical trials to ameliorate EED have been disappointing. The pathogenesis of EED is poorly understood. We examined the relationship between circulating metabolites and EED in young children in Africa. Children with EED had a serum metabolite profile consistent with secondary carnitine deficiency and impaired β-oxidation of fatty acids. Carnitine is a conditionally essential nutrient primarily found in animal source foods or synthesized endogenously from lysine and methionine. This study suggests that secondary carnitine deficiency may contribute to the altered metabolic profile related to energy balance in young children with EED.

Objectives Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA. Methods The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA. Results Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV. Conclusions Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease. Trial registration number ISRCTN36745608; EudraCT Number: 2006-003146-41.

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. METHODS Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). RESULTS The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides. CONCLUSION ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

International audience; Carbamylation, which corresponds to the binding of isocyanic acid to the amino groups of proteins, is a nonenzymatic post-translational modification responsible for alterations of protein structural and functional properties. Tissue accumulation of carbamylation-derived products and their role in pathological processes such as atherosclerosis or chronic renal failure have been previously documented. However, few studies have focused on the carbamylation of intracellular proteins and their subsequent role in cellular aging. This study aimed to determine the extent of intracellular protein carbamylation, its impact on cell functions and the ability of cells to degrade these modified proteins. Fibroblasts were incubated with cyanate or urea and the carbamylation level was evaluated by immunostaining and homocitrulline quantification. The results showed that carbamylated proteins accumulated intracellularly and that all proteins were susceptible. The presence of intracellular carbamylated proteins did not modify cell proliferation or type I collagen synthesis nor did it induce cell senescence, but it significantly decreased cell motility. Fibroblasts were able to degrade carbamylated proteins through the ubiquitin-proteasome system. In conclusion, intracellular proteins are susceptible to carbamylation but their accumulation does not seem to deeply affect cell function, owing largely to their elimination by the ubiquitin-proteasome system.

Rheumatoid arthritis (RA) is a prototype autoimmune disease, with the hallmark signs of synovial inflammation and the presence of autoantibodies. One of the most prominent examples of such autoantibodies are anti-citrullinated protein antibodies (ACPA), which are directed against a wide-array of citrullinated proteins. The immune response to citrullinated antigens is a dynamic response that expands before the onset of disease and generates antibodies that are extensively glycosylated in the variable domain. This feature of ACPAs is remarkable and might be involved in the breach of tolerance to citrullinated proteins as well as function as an additional biomarker to predict disease onset in subjects at risk. Next to ACPA, it has become clear that the autoantibody response in RA extends towards several other modified proteins, such as proteins modified by carbamylation or acetylation. Carbamylation leads to the formation of homocitrulline. Structurally, homocitrulline greatly resembles citrulline but is one methylene group longer. In contrast, acetylation is mediated by intracellular acetyltransferases and is structurally less related to citrulline or homo-citrulline. Although the presence of autoantibodies against these post-translationally modified proteins (Anti-Modified Protein Antibodies; AMPA) hallmark RA, at present, there is no conceptual framework explaining the concordant presence of different AMPA-responses in RA. In the context of this presentation, the latest insights into the development of humoral response against citrullinated-, carbamylated and acetylated proteins in relation to their role as biomarkers to predict the development of RA will be discussed. Disclosure of Interests: Rene Toes Grant/research support from: Sanofi

Bu tez çalışmasında; tekrarlayan oral aftöz ülserler, genital ülserler, üveit, epididimit, mukokutanöz, eklem, gastrointestinal, nörolojik ve vasküler tutulumlar ile karakterize çok sistemli ve enflamatuar bir hastalık olan behçet hastalığında ve sağlıklı kişilerde serum homositrulin seviyelerinin tespit edilmesi amaçlanmıştır.Araştırmaya, Selçuk Üniversitesi Tıp Fakültesi Hastanesi Romatoloji kliniğine başvuran Behçet hastaları ve sağlıklı gönüllü olmak üzere toplamda 60 birey katılmıştır. Bireyler kendi içlerinde 30 hasta ve 30 kontrol grubu olmak üzere iki gruba ayrılmıştır. Romatoloji kliniğine başvuran Behçet hastalarının rutin takip amaçlı alınan kan örneklerinden artan serumlarında ve sağlıklı gönüllülerin serumlarında homositrulin ve lizin düzeyleri geliştirilen LC-MS/MS metodu kullanılarak ölçülmüştür. Hasta ve kontrol grupları arasında istatistiksel analizler yapılmış ve değerlendirilmiştir.Hasta grubunun homositrulin düzeyleri (1.11±0.74 mmol/mL)kontrol grubuna göre (0.38±0.12 mmol/mL) anlamlı olarak (p=0.000) yüksek bulunmuştur. Homositrulin lizin oranları bakımından değerlendirildiğinde hasta grubunun homositrulin/lizin düzeyleri hasta grubunda (1.15±0.89 mmol/mol) kontrol grubuna (0.31±0.10 mmol/mol) göre istatistiksel olarak daha yüksek olduğu (p=0.000) tespit edilmiştir. Lizin analizlerinde ise gruplar arasında anlamlı bir fark bulunamamıştırSonuç olarak; ilk defa gerçekleştirilen bu çalışmada, behçet hastalığı ile homositrulin düzeyleri arasında pozitif bir ilişkinin olduğu tespit edilmiştir. Bu nedenle homositrulin düzeylerinin behçet hastalığında biyobelirteç olarak kullanılabileceği düşünülmektedir.Anahtar Sözcükler: Behçet Hastalığı; Homositrulin; İnflamasyon. In this thesis study; it was aimed that was to determine serum homocytulin levels in healthy people and behçet's disease which is a multisystemic and inflammatory disease characterized by recurrent oral aphthous ulcers, genital ulcers, uveitis, epididymitis, mucocutaneous, joint, gastrointestinal, neurological, and vascular involvement. A total of 60 people, Behçet's patients and healthy volunteers, who applied to the rheumatology clinic of Selçuk University Medical Faculty, participated in the study. Individuals were divided into two groups as 30 patients and 30 control groups.Homocitulline and lysine levels were measured in the serum samples, that taken from routine blood analysis of healthy volunteers and Behçet's patients appliying from rheumatology clinic, by using the developed LC-MS/MS method. Statistical analysis was performed between patient and control groups and evaluated.Homocitulline levels (1.11±0.74 mmol/mL) of the patient group were significantly higher (p=0.000) than the control group (0.38±0.12 mmol/mL). Homocitrulline/lysine ratios were higher in the patient group (1.15 ± 0.89 mmol/mol) compared to the control group (0.31 ± 0.10 mmol/mol) as a statistically (p=0.000). However, lysine analysis showed no significant difference between groups.As a result; In this study performed for the first time, it was determined that there was a positive relationship between behçet disease and homocitrulline levels. Therefore, it is thought that homocytulin levels may be used as biomarkers in behcet disease.Keywords: Behçet's disease; Homocitrullin; Inflammation. 63

Carbamylation corresponds to the non-enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaches to the e-NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This unit describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on pre-analytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2018 by John Wiley & Sons, Inc.

Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.

Objectives Autophagy is a homeostatic and physiological process that promotes the turnover of proteins and organelles damaged in conditions of cellular stress. We previously demonstrated that autophagy represents a key processing event creating a substrate for autoreactivity, which is involved in post-translational changes and generation of citrullinated peptides, recognized by the immune system in RA. In this study, we analysed whether autophagy is involved in other post-translational changes that can generate autoantigens, focusing on carbamylation processes. Carbamylation is a nonenzymatic post-translational modification, in which homocitrulline is generated by the reaction of cyanate with the primary amine of lysine residues; carbamylated peptides may accumulate during inflammation conditions. Methods The role of autophagy in the generation of carbamylated proteins was evaluated in vitro in fibroblasts as well as in synoviocytes from RA patients, treated with 5 μM tunicamycin or 200 nM rapamycin; the correlation between autophagy and carbamylated proteins was analysed in mononuclear cells from 30 naive early-active RA patients. Results Our results demonstrated that cells treated with tunicamycin or rapamycin showed a significant increase of carbamylated proteins. Immunoblotting and immunoprecipitation experiments identified vimentin as the main carbamylated protein. Furthermore, a correlation was found between autophagy and carbamylation levels in mononuclear cells of naive RA patients. Conclusion These data indicate that autophagy is able to induce in vitro carbamylation processes, and in vivo appears to be related to an increase in carbamylation during RA. These observations introduce a new pathogenetic mechanism of disease, which could contribute to more accurate monitoring of patients.

Background Protein carbamylation is one of the non-enzymatic reactions involved in protein molecular ageing. We sought to investigate the relationship between urea levels and protein carbamylation, and whether a Mediterranean diet (MD) and a very low protein diet (VLPD) reduce protein carbamylation through reduction in urea levels in patients with chronic kidney disease (CKD). Methods This is a prospective, randomized, crossover controlled trial that investigated 60 patients with CKD grades 3B-4 (46 males, mean age of 67 years). The enrolled CKD patients were randomly assigned (1:1) to two different nutritional treatment arms: (i) 3 months of free diet (FD), 6 months of VLPD, 3 months of FD and 6 months of MD; and (ii) 3 months of FD, 6 months of MD, 3 months of FD and 6 months of VLPD. Blood levels of lysine (Lys) and homocitrulline (Hcit) and their ratio were used as markers of cyanate levels. Due to a lack of pre-existing data on the potential effects of different dietary regimens and in light of the exploratory nature of the study, no formal sample size estimation was carried out. Results At study completion, lower diastolic blood pressure and decreased serum levels of urea, sodium, phosphorus and parathyroid hormone, but higher serum levels of bicarbonate and haemoglobin, were noted with MD and VLPD. When compared with FD, both MD and VLPD were also associated with a decrease in serum Hcit levels and Hcit/Lys ratios (P

Background and objectives Causes of CKD differ in prognosis and treatment. Metabolomic indicators of CKD cause may provide clues regarding the different physiologic processes underlying CKD development and progression. Design, setting, participants & measurements Metabolites were quantified from serum samples of participants in the Modification of Diet in Renal Disease (MDRD) Study, a randomized controlled trial of dietary protein restriction and BP control, using untargeted reverse phase ultraperformance liquid chromatography tandem mass spectrometry quantification. Known, nondrug metabolites (n=687) were log-transformed and analyzed to discover associations with CKD cause (polycystic kidney disease, glomerular disease, and other cause). Discovery was performed in Study B, a substudy of MDRD with low GFR (n=166), and replication was performed in Study A, a substudy of MDRD with higher GFR (n=423). Results Overall in MDRD, average participant age was 51 years and 61% were men. In the discovery study (Study B), 29% of participants had polycystic kidney disease, 28% had glomerular disease, and 43% had CKD of another cause; in the replication study (Study A), the percentages were 28%, 24%, and 48%, respectively. In the discovery analysis, adjusted for demographics, randomization group, body mass index, hypertensive medications, measured GFR, log-transformed proteinuria, and estimated protein intake, seven metabolites (16-hydroxypalmitate, kynurenate, homovanillate sulfate, N2,N2-dimethylguanosine, hippurate, homocitrulline, and 1,5-anhydroglucitol) were associated with CKD cause after correction for multiple comparisons (P Conclusions Metabolomic profiling identified several metabolites strongly associated with cause of CKD.

Antibodies to proteins/peptides containing citrulline are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE). RA patients also generate antibodies to homocitrulline-containing proteins/peptides (also referred to as anti-carbamylated protein antibodies (Anti-CarP)). This study was undertaken to investigate the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express the human SE. C57BL/6 (B6) and DR4tg (on a B6 background) mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). Splenic T cell proliferation was evaluated by 3H-thymidine incorporation assay. Antibodies to homocitrullinated and citrullinated antigens were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with HomoCitJED and its citrullinated counterpart peptide, CitJED (the number of homocitrullines in HomoCitJED is equal to the number of citrullines in CitJED). HomoCitJED-immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to citrullinated antigens. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. In conclusion, DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice.

Background Autoimmunity in Rheumatoid arthritis (RA) patients is characterized by a spectrum of anti-modified protein antibodies (AMPA) directed against post-translationally modified (PTM) proteins. The best-known AMPA in RA are anti-citrullinated protein antibodies (ACPA). Much less is known about the occurrence and aetiology of other AMPA responses in RA such as autoantibodies directed to malondialdehyde-acetaldehyde (MAA) adducts, acetylated antigens, and carbamylated proteins. Anti-carbamylated protein (anti-CarP) autoantibodies recognize carbamylated proteins containing a homocitrulline, a PTM structurally similar to citrulline. With the presence of various AMPA responses in RA, PTM proteins have been implicated in the breach of autoreactive B-cell tolerance leading to the formation of these autoantibodies. At present it is unkown how AMPA are generated and how autoreactive B-cell responses against PTM proteins are induced. Objectives To investigate how PTM proteins, more specifically carbamylated proteins, could contribute to a breach of B-cell tolerance. Methods Serum reactivity towards five different carbamylated proteins was determined for 160 RA-patients and 40 healthy individuals. Anti-CarP antibody cross-reactivity was studied by inhibition experiments. Mass spectrometry was performed to identify carbamylated self-proteins in human rheumatic and osteoarthritic joint tissue. Mice were immunized with carbamylated- or non-modified foreign (OVA) and self-antigens (mouse Albumin). Sera of immunized mice and monoclonal antibodies were analyzed for antigen reactivity. Results We show that anti-CarP antibodies in RA are highly cross-reactive towards multiple carbamylated proteins, including modified self- as well as modified non-self proteins. Immunization with carbamylated foreign proteins (Ca-OVA) induced a strong anti-CarP response against both carbamylated foreign- and self-proteins. Similar to murine serum anti-CarP antibodies, murine monoclonal anti-CarP antibodies were highly specific and cross-reactive to multiple carbamylated (auto)antigens. Although citrulline greatly resembles homocitrulline residues in structure, murine anti-CarP antibodies differ in antigen recognition profile from ACPA as they are able to discriminate between citrullinated and homocitrullinated forms of the same protein. Interestingly, we were able to identify carbamylated-albumin, in RA synovial tissue, indicating that albumin is present in carbamylated form locally in the inflamed joint. Conclusions Self-reactive AMPA-responses can be induced by exposure to foreign proteins containing PTM. Our findings show that autoreactive B-cell responses against PTM self proteins can be induced by exposure to PTM foreign proteins and provide new insights on the breach of autoreactive B-cell tolerance by foreign proteins. Disclosure of Interest None declared

Rheumatoid arthritis (RA) is a prototype autoimmune disease, with the hallmark signs of synovial inflammation and the presence of autoantibodies. Of the different autoantibody systems present in RA, rheumatoid factors (RF) are probably the best studied. Their presence was first detected >70 years and it was in the late 1950s when it was realized that RF reacted to gamma globulins. Since the discovery of RF several other autoantibody systems have been discovered in RA, many of them directed against post-translationally modified protein antigens. The most prominent example of such autoantibodies are anti-citrullinated protein antibodies (ACPA), which are directed against a wide-array of citrullinated proteins. Now RF and ACPA determination are the two major diagnostic laboratory tests for RA and part of the EULAR and ACR criteria for RA. In the past few years, it has become clear that the autoantibody response present in RA extends towards several other modified proteins, such as proteins modified by acetylation or carbamylation. As all these auto-antibodies recognize Post-Translationally Modified (PTM) proteins, these antibodies are collectively called Anti-Modified Protein Antibodies (AMPA). In the context of this presentation, I will focus on the auto-antibody response against citrullinated, carbamylated and acetylated proteins. Carbamylation leads to the formation of homocitrulline. Structurally, homocitrulline greatly resembles citrulline but is one methylene group longer. Citrulline is generated when PAD enzymes modify the amino acid arginine. In contrast, the amino acid homocitrulline is generated by a chemical reaction in which cyanate reacts with the amino acid lysine. Arginine and lysine are located at different positions in the amino acid sequence of proteins, and therefore these modifications occur at different positions in proteins with different flanking amino acids. Intriguingly, although homocitrulline residues can also be recognised by auto-antibodies, these auto-antibodies often do not crossreact with citrulline. Acetylation is a process where acetyl groups are added to free amines of lysine residues by acetyl transferases. Acetylated lysine does not resemble citrulline but bears similarity to homocitrulline except at the side chain terminal amine, which is replaced by a methyl moiety. By now it is clear that AMPA consist of different auto-antibody families that are largely distinct, but that can also display a certain degree of cross-reactivity. Therefore, the notion is emerging that, although cross-reactivity exist, different classes of AMPAs are generally seen as distinct auto-antibody families that target different antigens, but intriguingly often co-occur. As the AMPA-responses in RA are often found together, it indicates that –somehow- AMPA-reactivity has a commonality that is currently not understood. Although, the reason why an immune response starts against PTM proteins is not known, it appears crucial to obtain understanding on the breach of tolerance towards PTM proteins as the immune response against these proteins has been intimately implicated in disease-pathogenesis. Understanding the full AMPA response, the triggers that drive AMPA production, their mutual crosstalk and the pathways by which AMPA and/or AMPA-expressing B cells possibly contribute to RA will be important for the development of curative interventions in RA. In the context of this presentation, some of these aspects will be discussed. Disclosure of Interest None declared

Summary Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during aging and is a risk factor for various diseases. The most common product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group has reacted with urea-derived cyanate. HCit has recently been detected in collagen; however, given that 15%–90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and in vivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys 933 , a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation.