A Potential Approach to Tackling CEL and CML Advanced Glycation End Products

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Advanced glycation end-products (AGEs) form in tissues as a side-effect of the normal operation of cellular metabolism where it touches on the processing of sugars. There are many types of AGEs, most short-lived, but some persistent and challenging for our biochemistry to break down. These persistent AGEs lead to cross-links, binding together molecules in the extracellular matrix and thereby altering the structural properties of tissues. This is perhaps most harmful where it reduces tissue elasticity, and is thus an important contributing cause of skin and vascular aging.

While sugars are involved, it is much debated as to whether the contents of diet, either fully formed AGEs from certain cooked and processed foods, or precursors in the form of excessive amounts of sugar, has much influence at all over the generation of the types of AGE involved in aging. As mentioned, there are many types of AGE. One of the big questions in the small research community focused on AGEs is whether or not glucosepane AGEs are the only target worthy of attention in the matter of aging. There is certainly good evidence for cross-links in humans to be overwhelmingly made of glucosepane, but equally there is a faction who argue that the research community does not yet have sufficiently robust data to be able to ignore AGEs such as carboxymethyl-lysine (CML).

The challenge inherent to all work on AGEs, and why this part of the larger field has been a comparative backwater for decades despite its great importance to aging, is that the usual tools for cell, tissue, and molecular biochemistry work just don’t exist. AGEs are hard to work with. The usual recipes for making the molecule of interest, the standardized tests for assessing its presence, and so forth, just don’t exist or didn’t exist until comparatively recently. Most research groups take a look at this desert of tooling and move on to something easier – it is a self-reinforcing problem. This was the case until the SENS Research Foundation and allied philanthropists turned up to try to solve the missing tools problem. Those efforts have led to significant progress in the past five years or so, but there is still a fair way to go yet. Today’s paper is of interest for showing progress towards tooling for CML, rather than for glucosepane. It is not open access, but sufficiently interesting to note nonetheless.

Biocatalytic Reversal of Advanced Glycation End Product Modification

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of proteins derived from the condensation of reducing sugars and nucleophilic amino acid residues, such as lysine and arginine. Although AGEs are formed in the body as a part of normal metabolism, they can accumulate to high concentrations and contribute to the progressive decline of multiple organ systems. This process is accelerated in diabetics, owing to their hyperglycemic conditions. In addition to causing spontaneous damage by altering protein structure and function, AGEs also interact with the receptor for AGEs (RAGE), eliciting oxidative stress and activating the transcription factor NF-κB thought to be a major contributor of AGE-associated chronic inflammation and cellular damage.

Elevated levels of AGEs are linked to the pathology of many metabolic and degenerative diseases of aging, such as diabetic complications, atherosclerosis, and Alzheimer’s disease. This association is manifested by age-dependent increases in cross-linking, browning, fluorescence, and AGE content in long-lived proteins such as collagens and lens crystallins. Structural characterization and synthesis of some of the more prevalent AGEs (e.g., glucosepane) have allowed more focused investigations into their individual chemical properties and formation. Indeed, chemical studies have shown strong correlations between specific AGEs and the development of age-related illnesses; however, it has been difficult to unequivocally demonstrate that any AGEs are direct causal factors largely due to the lack of tools for investigating the reversal of mature AGE modifications at the molecular level.

Here, we show that MnmC, an enzyme involved in a bacterial tRNA-modification pathway, is capable of reversing the AGEs carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML) back to their native lysine structure. Combining structural homology analysis, site-directed mutagenesis, and protein domain dissection studies, we generated a variant of MnmC with improved catalytic properties against CEL in free amino acid form. We show that this enzyme variant is also active on a CEL-modified peptidomimetic and an AGE-containing peptide that has been established as an authentic ligand of the receptor for AGEs (RAGE).

To the best of our knowledge, this is the first biochemical demonstration of an enzyme that can reverse a mature AGE-functionalized peptide. While the kinetic parameters, which are similar to known Amadoriases, could be substantially improved, C-MnmC variants represent lead catalysts for further directed evolution and development. As MnmC natively acts on nucleic acids, glycated DNA (e.g., carboxyethyl/carboxymethyl-deoxyguanosine) may also be suitable substrates to test in future studies. Such improved AGE-reversal tools could in principle enable a better understanding of the biology of AGEs at the molecular level, elucidate their direct roles in the pathogenesis of age-related diseases, and serve as leads for recombinant enzyme therapies.

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