New enzyme removes years of aging from skin and other human tissue

A chemical scar that accumulates on aging proteins has long been treated as permanent. Scientists have now engineered an enzyme that removed much of that damage from preserved human eye, artery and skin tissue.

The enzyme, called CMLase, targets Nε-carboxymethyl-lysine, or CML. This altered form of the amino acid lysine appears on long-lived proteins and becomes more common with age.

Researchers from Revel Pharmaceuticals, Calico and the University of Colorado developed the enzyme through computational screening and directed evolution. Their proof-of-concept study appeared in Nature Communications.

CML belongs to a broad class of compounds called advanced glycation and lipoxidation end products, commonly known as AGEs. They form when reactive molecules derived from sugars or fats bind to amino acids without help from enzymes.

The chemistry resembles the Maillard reaction, which browns food during cooking and helps create rich flavors and aromas. Inside the body, however, similar reactions can gradually alter proteins in skin, blood vessels, eyes and other tissues.

AGEs can crosslink collagen, reduce tissue flexibility and interfere with normal protein turnover. Some also activate immune signaling and promote chronic inflammation.

CML is especially stable and abundant in long-lived proteins. It can bind to the receptor for advanced glycation end products, known as RAGE, and trigger a cascade involving NF-κB, inflammatory cytokines and growth factors associated with fibrosis.

“The engagement of the CML-RAGE axis triggers a signaling cascade that activates NF-κB and stimulates the release of pro-inflammatory cytokines and profibrotic growth factors,” the authors wrote. “In the context of the central nervous system, CML accumulation has been linked to oxidative stress and mitochondrial damage in microglia, further disrupting brain homeostasis during aging.”

Existing defenses can limit the formation of some AGEs. The body’s glyoxalase system, for example, clears reactive precursors such as methylglyoxal. Experimental drugs have also been studied for their ability to trap those precursors or slow new AGE formation.

Those strategies do not repair proteins already carrying CML. Until now, the modification was generally considered stable and essentially irreversible once attached.

The team began by searching for a microbial enzyme capable of removing CML. Earlier work had suggested that a soil mold contained such activity, but the original organism was unavailable and the responsible enzyme had never been identified.

The researchers instead examined glycine oxidases. CML contains a chemical structure resembling glycine, which suggested that one of these enzymes might provide a useful starting point.

A glycine oxidase from Bacillus subtilis acted on free CML but could not process CML attached to a peptide. The team then searched tens of thousands of related protein structures for an enzyme with a more accessible active site.

That search led to a glycine oxidase from Calidithermus roseus. It showed weak activity on peptide-bound CML because a missing structural helix left more room for larger substrates.

The scientists then used directed evolution, a laboratory process that generates and selects improved enzyme variants. They developed a genetic system in E. coli that allowed cells to grow only when an enzyme successfully converted CML back into lysine.

More than 500 million variants were screened through five rounds of engineering. The final enzyme, also known as CrGO-897, contained 15 amino acid substitutions and a two-amino-acid deletion.

Its efficiency on peptide-bound CML improved more than tenfold compared with the starting enzyme. It also gained the ability to remove CML from complete proteins.

The researchers first tested CMLase on laboratory-modified proteins, including collagen, hemoglobin, casein, bovine serum albumin and a protein extract from sheep eyes.

Overnight treatment reduced detectable CML by between 52 and 97 percent, depending on the protein. The variation appeared to reflect how easily the enzyme could reach individual lysine sites and the surrounding protein structure.

Proteomic analysis identified 33 modified lysines on bovine serum albumin. CMLase reduced CML at 30 of those sites. Twenty-one showed reductions above 50 percent, while seven declined by more than 90 percent.

The enzyme did not work equally well everywhere. Some resistant sites sat near bulky or water-repelling amino acids, which may have limited access.

The reaction also left the underlying protein intact. Tests found no evidence that the enzyme removed CML simply by breaking the protein apart.

The team next moved to tissues where CML had accumulated naturally over decades.

In lens proteins from a 64-year-old donor, mass spectrometry measured a 45 percent reduction in total CML after overnight treatment. An antibody-based test showed a 78 percent decline in surface-exposed CML.

The researchers also applied the enzyme to preserved tissue sections from older human donors. CMLase reduced staining by more than 70 percent in arterial tissue from a 75-year-old donor.

In elderly skin, CML staining fell by more than 55 percent across the epidermal and dermal layers. After treatment, the remaining signal dropped below levels observed in skin from a 31-year-old donor.

“The ability of CMLase to reduce CML burden by over 70% and 55% in elderly human arterial and skin tissue, respectively, demonstrates that the enzyme functions effectively in the heterogeneous and sterically complex environment of the aging human extracellular matrix,” the authors wrote.

The findings do not show that CMLase reverses aging in people. All human tissue work involved isolated proteins or thin, preserved sections that gave the enzyme easy access to damaged sites.

Living tissues present much harder barriers. The enzyme would need to move through dense extracellular structures, remain active and reach CML without provoking harmful immune responses.

Its bacterial origin could make it immunogenic, especially if repeated dosing were required. Researchers would also need to determine whether CML removal restores tissue flexibility or reduces RAGE signaling and inflammation in living organisms.

CML represents only one form of age-related protein damage. Reversing it alone would not address the many biological processes involved in aging.

CMLase gives scientists a direct tool for testing whether accumulated CML causes specific tissue problems rather than merely accompanying them. Removing the modification could reveal whether it contributes to stiffness, inflammation or impaired cell signaling.

The engineering method may also be adaptable to other forms of protein damage. One possible target is glucosepane, an abundant crosslink in human tissue that resists existing attempts at removal.

“The successful engineering of CMLase suggests that other oxidative or hydrolytic enzymes could be evolved to target the chemically diverse landscape of age-related protein modifications,” the authors wrote. “By reversing a hallmark of aging, CMLase provides a powerful tool for dissecting molecular causality and offers a foundation for developing regenerative therapies that repair damaged tissues.”

Research findings are available online in the journal Nature Communications.

The original story "New enzyme removes years of aging from skin and other human tissue" is published in The Brighter Side of News.

Like these kind of feel good stories? Get The Brighter Side of News' newsletter.