Cellular homeostasis of N -acetylneuraminic acid and non-canonical sialic acids is mediated by human N -acetylneuraminate lyase

Sialic acids are important for cellular communication, with N-acetylneuraminic acid (Neu5Ac) being the canonical form of sialic acid in humans. Presence of non-canonical sialic acids, derived from dietary intake or as metabolic side product, has been linked to immune disorders and cancer. As homeostasis of different sialic acids remains poorly understood in humans, we studied the role of N-acetylneuraminate lyase (NPL) in their catabolism. In vitro expression of NPL in different biological systems revealed broad substrate specificity towards sialic acids and related 2-keto-3-deoxy metabolites. In agreement with the broad substrate specificity, NPL-deficient red blood cells accumulated Neu5Ac and 3-deoxy-d-glycero-d-galacto-nonulosonic acid (KDN). Interestingly, endogenous levels of non-canonical sialic acids, including N-glycolylneuraminic acid (Neu5Gc) and KDN, were depleted in HEK293T cells upon NPL overexpression, while Neu5Ac and CMP-Neu5Ac levels remained stable. This was further confirmed by supplementation with different sialic acids. Detailed analysis of sugar phosphate intermediates of the hexosamine and sialic acid biosynthesis pathways showed strongly elevated ManNAc-6P (N-acetyl-d-mannosamine 6-phosphate) and Neu5Ac-9P, indicating efficient recycling of ManNAc to increase de novo Neu5Ac biosynthesis. However, this recycling was not efficient for Neu5Gc and KDN. While GlcNAc-6P (N-acetyl-d-glucosamine 6-phosphate) levels were slightly elevated, no evidence was found for further metabolism towards GlcN-6P (glucosamine 6-phosphate) and energy production via glycolysis as shown for bacterial neuraminate lyases. In conclusion, human NPL catabolizes a broad range of sialic acids. However, depending on the cellular context, NPL contributes to a net cellular reduction in non-canonical sialic acids, such as KDN, due to a lack of efficient recycling.