Protein carbamylation is a hallmark of aging

Abstract

Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging.

Keywords: carbamylation; longevity; nonenzymatic posttranslational modifications; skin; tissue aging.

Fig. 1.

Carbamylation reaction.

Fig. 2.

Age-related increase of skin HCit content. HCit was assayed by liquid chromatography–MS/MS in (A) total skin extract and (B) skin type I collagen in human (n = 60), bovine (n = 20), and murine (n = 35) individuals. HCit content progressively increased with age in all studied species. All Spearman coefficients of correlation (r) showed significant HCit increase with P < 0.001.

Fig. S1.

Evaluation of carbamylation of human skin elastin with age. HCit concentrations were evaluated by LC-MS/MS in elastin extracted from human skin (n = 28). Spearman coefficient of correlation (r) showed a significant HCit increase with P < 0.001.

Fig. 3.

Localization of carbamylated proteins in human skin. HCit (green) and type I collagen (red) were stained using specific antibodies. Nuclei were stained with DAPI (blue). A poor labeling of carbamylated proteins was found in (A) young skin (20-y-old human) compared with (B) old skin (80-y-old human) showing intense staining (arrows). d, Dermis; e, epidermis. (Scale bar: 40 µm.)

Fig. S2.

Specificity of anti-HCit antibodies. Anti-HCit antibodies were submitted to a preliminary incubation with PBS (PBS preincubation), 5 mM free HCit (HCit preincubation), or 5 mM free citrulline (Cit preincubation). Specificity was characterized by (A) Western blotting using native (N), citrullinated (Cit), or carbamylated (Carb) type I collagen and (B) immunohistofluorescence on mice skin sections with a staining of HCit (green) and nuclei (blue). A negative control (Neg Ct) was performed using secondary antibodies only. For both techniques, antibodies did not show cross-reactivity with citrulline, showing their specificity against HCit. d, Dermis; e, epidermis. (Scale bar: 40 µm.)

Fig. S3.

Comparison of HCit accumulation of different tissues during aging in mice. Tissue carbamylation was evaluated by HCit assay in type I collagen extracted from tail tendons, skin, and bones. Each point represents mean ± SD (n = 5).

Fig. 4.

Age-related HCit accumulation rate in skin of human, bovine, and murine species. Accumulation rate was evaluated in skin total extracts and type I collagen. (A) Nonlinear regressions (95% confidence interval; shaded zones) and linear regressions (dotted lines) of HCit concentrations over time were calculated for each species. For better clarity, the graphs focus on a 30-y period. (B) Slopes obtained from equations of linear regression (Table S1) were plotted as a function of mean life expectancy.

Fig. S4.

Comparison of carbamylation and glycation rates in skin during aging in human, bovine, and murine species. HCit and CML were measured by LC-MS/MS to evaluate carbamylation and glycation, respectively, of proteins in (A) total skin extracts and (B) skin type I collagen. Nonlinear regressions (95% confidence intervals) and Spearman coefficients of correlations (r values) were calculated. HCit contents were higher than CML contents during aging in all studied species.

Fig. 5.

Carbamylation and glycation sites in human type I collagen. Carbamylated and glycated lysine residues were identified by Orbitrap liquid chromatography–MS in type I collagen extracted from four human skin samples; (A) 20 carbamyl-lysine residues and 15 CML residues were identified in α1(I)-chain, and (B) 9 carbamyl-lysine and 11 CML residues were identified in α2(I)-chain. Details are given in Tables S3 and S4.

Fig. 6.

Turnover of carbamylated proteins in mice. Mice received Cy-drink for 3, 6, 9, or 12 wk. (A) Each group was compared with the control group receiving Wa-drink for 3 wk. Other groups of mice received Cy-drink for 3 wk before receiving Wa-drink for 3, 6, or 9 wk. (B) Each group was compared with the control group given Wa-drink for 3 wk. HCit concentrations were evaluated in plasma, total skin extract, and skin type I collagen by liquid chromatography–MS/MS. Whiskers indicate the minimum and maximum values. ns, Nonsignificant (n = 5). *P < 0.05; **P < 0.01.