Abstract
N-glycolyl chondroitin (Gc-CN) is a metabolite of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine (GalNGc) that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide.
Highlights
Sialic acids constitute a family of acidic sugars with a 9-carbon backbone found at the terminal end of glycan chains attached to many soluble glycoproteins (Schauer 2000; Wang and Brand-Miller 2003)
We exhaustively digested chondroitin samples with chondroitin lyase ABC to afford GAG disaccharides and remove any other minor impurities, such as residual lipopolysaccharides. These digests were purified and AMAC labeled for LC–mass spectrometry (MS)/MS-multiple reaction monitoring (MRM) to verify the presence of glycolyl chondroitin (Gc-CN)
From our chondroitin producing construct, serotype O5:K4:H4, only chondroitin was produced as signified by the absence of AMAC-labeled Gc-CN disaccharide (Fig. 4a)
Summary
Sialic acids constitute a family of acidic sugars with a 9-carbon backbone found at the terminal end of glycan chains attached to many soluble glycoproteins (Schauer 2000; Wang and Brand-Miller 2003). Their presence at the terminal end of sugars gives them an advantage in performing their biological roles, providing structure, serving as a ligand for intrinsic and extrinsic receptors, as a binding site for pathogens and toxins, and in molecular mimicry for host invasion (Varki 2008). An enzyme called cytidine monophosphate N-acetylneuraminic acid hydroxylase (Cmah), encoded by the CMAH gene, is responsible for converting CMP-Neu5Ac to CMP-Neu5Gc through the addition of an oxygen atom. A few million years ago, a mutation event that occurred in our last common ancestor with the apes, resulted in humans losing the CMAH gene and being unable to convert Neu5Ac to Neu5Gc (Chou et al 1998, 2002)
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