CMLase removed age-related chemical damage from lysine residues in proteins

CMLase removed age-related chemical damage from lysine residues in proteins

On July 14, Nature Communications published a study of the enzyme CMLase. The enzyme removes CML, a common form of age-related chemical damage to lysine, from proteins and restores the original amino acid. The researchers demonstrated the reaction in model proteins and samples of human lens, skin, and aorta.

Lens proteins and collagen in the skin and blood vessel walls remain in place for years or decades. Reactive compounds derived from sugars and lipids bind to amino acids in these proteins, producing glycation and lipoxidation products. One such product is Nε-carboxymethyllysine, or CML. Its carboxymethyl group attaches to the amino group in the lysine side chain and changes its charge from positive to negative.

CML affects both the modified protein and nearby cells. It binds to RAGE, a cellular receptor for glycation products, and activates inflammatory and oxidative signaling. Cellular enzymes neutralize some of these reactive molecules before they reach proteins. CMLase acts after CML has formed, when the modification is already attached to a long-lived protein.

The authors started with a bacterial glycine oxidase and screened more than 500 million variants over five rounds of directed evolution. The resulting CMLase recognizes CML within an intact protein and converts the damaged residue back into lysine. In model proteins, overnight treatment reduced the CML signal by 52–97% in an antibody assay.

Mass spectrometry showed that CMLase reduced the total amount of CML in soluble lens proteins from a 64-year-old donor by 45%. The antibody assay showed a reduction of 78%. Mass spectrometry measures the total amount of CML after the protein has been chemically digested, whereas the antibody assay detects only modifications accessible to the antibody. The authors suggest that CMLase removes modifications from protein surfaces more readily than those buried within the protein.

In thin, fixed sections of aorta from a 75-year-old donor, CML staining decreased by more than 70%. In skin from older donors, it decreased by more than 55%. An inactivated control enzyme did not change the original signal. Soluble lens proteins and thin tissue sections give the enzyme direct access to its target. In a living blood vessel, the enzyme would need to penetrate the dense extracellular matrix, the network of proteins that supports the tissue. Researchers will also need to measure the immune response to repeated administration of the bacterial enzyme.

CMLase makes it possible to test whether removing a single chemical modification changes RAGE signaling and blood vessel elasticity. Glucosepane, an age-related crosslink between matrix proteins, will require a separate enzyme. An analysis of the matrix glycation hypothesis links the accumulation of damage in collagen and elastin to the loss of elasticity in aging tissues.