Research Reports

Overview

What is carbamylation?

Urea is synthesized from ammonia for detoxification in the body and is generally considered less toxic. However, when urea dissociates, isocyanic acid is formed and binds to proteins. This protein modification reaction is called carbamylation (Figure 1). Lysine (Lys), one of the amino acids that make up proteins, is the main target of carbamylation and is converted to homocitrulline (HCit) by the addition of a carbamoyl group (-CONH2) upon reaction with isocyanic acid. This carbamylation reaction has been reported to cause changes in protein structure and function and to be a risk factor for various diseases, including fibrosis, rheumatoid arthritis, and anemia (Reference 2). Renal function is most relevant to the carbamylation reaction. As renal function declines and urea cannot be excreted into the urine, the concentration of urea in the blood increases, resulting in an acceleration of carbamylation. Recently, carbamylation has been reported to be associated with cardiovascular and other complications of kidney disease, and blood HCit and carbamylated proteins have been validated as biomarkers of mortality and cardiovascular risk in patients with kidney disease (Reference 2). Other factors contributing to carbamylation include high-protein diet, which increases urea; smoking, which increases thiocyanate, the source of another isocyanic acid-generating pathway; and inflammation.

Figure 1. Schematic representation of carbamylation reaction of collagen.

Carbamylation of collagen

Collagen is a very stable protein that forms a triple-helical structure with three chains and is present in tissues for long periods of time without being degraded. However, due to its slow metabolism, it has been reported that carbamylated collagen accumulates with age and that the amount of HCit formed in skin collagen is higher than that of an advanced glycation end products (AGEs) of Lys generated by “glycation”, a well-known aging factor (Reference 3). In addition, the effects of carbamylation on collagen have been shown to include destabilization of the triple helix structure and decreased collagen fibril formation capacity (Reference 4). Collagen contains hydroxylysine (Hyl), an amino acid formed by the addition of a hydroxyl group (-OH) to Lys. We hypothesized that this collagen-specific amino acid might also be a target of the carbamylation reaction. In our experiments, we found that a Hyl-derived carbamylation product named hydroxyhomocitrulline (HHCit) is present in collagen (Figure 1; Reference 1). Similar to HCit, HHCit was found to accumulate in skin and bone collagens in proportion to age (Figure 2; Reference 1). HHCit accumulates more in skin than in bone, suggesting that the effect of carbamylation differs among tissues.

Figure 2. Accumulation of carbamylation products in rat tissue collagens with aging. HCit, homocitrulline; HHCit, hydroxyhomocitrulline; Lys, lysine; Hyl, hydroxylysine.

In the blood of patients with kidney disease, the concentration of HHCit was greatly increased compared to healthy subjects, and the degree of increase differed from that of HCit (Figure 3; Reference 1). HCit in blood is mainly derived from fast-metabolizing proteins other than collagen, indicating the degree of carbamylation in recent weeks, whereas HHCit is derived from collagen and would indicate the degree of carbamylation in recent months to years. Therefore, HHCit is expected to be a new biomarker for kidney disease and its complications.

Figure 3. Increased blood levels of carbamylation products in patients with kidney disease. HCit, homocitrulline; HHCit, hydroxyhomocitrulline; Lys, lysine; Hyl, hydroxylysine.

Inhibition of collagen cross-linking by carbamylation

Bridges, called cross-links, are formed between collagen molecules to increase tissue strength (Figure 4). However, since collagen cross-links are mainly formed through Lys and Hyl, it was speculated that cross-link formation would be impaired when they are converted to HCit and HHCit, respectively, by carbamylation. In fact, while collagen forms an elastic gel under physiological conditions, carbamylated collagen forms a soft and collapsed gel (Figure 5), suggesting that the effect of carbamylation on cross-link formation is also involved in this decrease in gelling ability.

Figure 4. Schematic representation of collagen cross-link formation and carbamylation-induced inhibition of the cross-linking

Figure 5. Effect of carbamylation on collagen gelation.  The gelation was performed by incubating bovine skin collagen with neutral salt.

We therefore conducted an experiment in which carbamylation was accelerated by ingestion of cyanate water (15 mM sodium cyanate for 8 weeks) and found that mature cross-links, the end products of cross-link formation, were reduced in mouse bone and tendon (Reference 1). More recently, it was observed that even under milder conditions (0.1 mM sodium cyanate for 10 months), similar to carbamylation that occurs in vivo, the formation of collagen cross-links, including immature cross-links that are precursors of mature cross-links, was inhibited in bone (Figure 6). Since the risk of fractures is high in patients with kidney disease, carbamylation may be one of the contributing factors to bone fragility. We are continuing to study the effects of carbamylation on cross-link formation and tissue strength.

Figure 6. Reduction of collagen cross-links by accelerating carbamylation in mouse bone. HCit, homocitrulline; HHCit, hydroxyhomocitrulline; Lys, lysine; Hyl, hydroxylysine; d-Pyr, deoxy-pyridinoline; Pyr, pyridinoline; LNL, lysinonorleucine; HLNL, hydroxylysinonorleucine; DHLNL, dihydroxylysinonorleucine.

Conclusion

References