Longevity Briefs: Using Engineered Enzymes To Repair Damaged Proteins

Longevity briefs provides a short summary of novel research in biology, medicine, or biotechnology that caught the attention of our researchers in Oxford, due to its potential to improve our health, wellbeing, and longevity.

The problem:

Our proteins are constantly exposed to various sources of damage, and one particularly problematic source of damage is glycation. This is when sugar molecules like glucose and fructose react with proteins to eventually form advanced glycation end-prodcucts, or AGEs. This an issue for a few reasons – glycation can interfere with protein function, and AGEs also trigger an inflammatory response from the immune system. AGEs can also cross-link with other proteins, binding two proteins together. This is especially problematic for proteins that don’t get replaced very often, such as collagen and elastin – the structural proteins that hold tissues together and give them their elasticity. AGEs are a major cause of fragile, stiffened skin in old age.

AGE formation is irreversible, meaning that once they form, the only way to remove them is to degrade the entire protein. Fortunately, we now live in a world where it is conceivable to engineer new proteins for specific purposes. In this study, researchers use directed evolution to produce an enzyme that can undo specific AGEs, showing that in principal, this form of age-related damage could be reversed.

The discovery:

In order to engineer the desired enzyme, researchers used an approach called directed evolution – essentially creating millions of enzyme variants and selecting the best performers, then creating variants of those and so on. This mimics the refinement that occurs by natural selection, but at an accelerated pace, and researchers control the selective pressure.

They started with an existing enzyme produced by a bacteria called Bacillus subtilis. This enzyme showed weak activity against a well-studied form of glycation: CML (Nε-carboxymethyl-lysine), in which a lysine amino acid (protein building block) is glycated. However, it only worked on free CML, not CML that was part of a protein. To address this problem, researchers first screened over 44,000 enzymes from databases in search of variants that looked like they had the right structure to attack CML in larger proteins. This led them to an enzyme called CrGO (Calidithermus roseus Glycine Oxidase) from the Calidithermus roseus bacteria. This enzyme had the first detectable (but low) activity against CML as part of a peptide chain.

Next came the selected evolution, which was carried out by bacteria. For this to work, researchers needed to tie CML-cleaving ability to bacterial survival. In order to do this, they introduced millions of CrGO enzyme variants to a strain of E.coli bacteria that needs lysine to survive, and then provided them with CML as their only source of lysine. This meant that E.coli that were better at reversing lysine glycation would have a selective advantage. Researchers would then take the most promising surviving E.coli, introduce new mutations into CrGO, and then culture those bacteria again. After 5 rounds of mutation and selection, the researchers obtained a CrGO enzyme in which 15 amino acids had been replaced and 2 had been deleted compared to the starting point. This enzyme was over 10 times better at cleaving CML than the original. Researchers named this enzyme CMLase.

Now came the true test of success – would this new enzyme be able to reverse glycation of human AGEs? The answer: yes. In human lens proteins from a 64-year-old donor, CMLase reduced total CML content by between 45% and 78% (glycation of cyrstallin proteins in the lens is a driver of cataracts). In aged human arterial tissue from a 75-year-old donor, CMLase treatment reduced CML by more than 70% (down to levels below those observed in 31-year-old tissue), while in skin tissue it achieved more than 55% reduction in CML. A different variant of CrGO was used as a control and produced no detectable reduction in CML.

The implications:

This research demonstrates that protein damage previously considered irreversible can actually be repaired using engineered enzymes. If something like this could be safely delivered to tissues in living organisms, it could potentially reduce the burden of AGEs, lowering chronic inflammation, reducing tissue stiffness and delaying age-related disease.

There are still some significant hurdles remaining before this could become a reality. As a bacterial product, CMLase might produce an immune response if given to a living organism. CMLase was also tested on very thin tissue slices in this study, but to be effective in humans it would need to penetrate deep into tissues. Even if these problems are overcome, CML is just one type of AGE and is not responsible for all AGE-related damage. Glucosepane crosslinks, for example, are primarily responsible for cross-linking collagen and elastin, and will probably be more challenging to reverse than CML.

While clinical use of these enzymes might not yet be possible, limiting sugar in your diet is an effective way of reducing AGEs, since they form as a consequence of reactions with sugar molecules. Certain foods, particularly grilled or fried meat and caramelised sugars, contain pre-formed AGEs, and a proportion of these are absorbed into the blood when consumed (though there has been some debate as to whether this has a meaningful impact on health). There is also some evidence that exercise can promote AGE degradation.