학술논문
Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis.
Document Type
Article
Author
Source
Subject
*SIALIC acids
*GLYCOLIPIDS
*METABOLIC flux analysis
*BIOSYNTHESIS
*CELL membranes
*INBORN errors of metabolism
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Language
ISSN
0304-4165
Abstract
Sialylation of glycoproteins and glycolipids is important for biological processes such as cellular communication, cell migration and protein function. Biosynthesis of CMP-sialic acid, the essential substrate, comprises five enzymatic steps, involving ManNAc and sialic acid and their phosphorylated forms as intermediates. Genetic diseases in this pathway result in different and tissue-restricted phenotypes, which is poorly understood. We aimed to study the mechanisms of sialic acid metabolism in knockouts (KO) of the sialic acid pathway in two independent cell lines. Sialylation of cell surface glycans was reduced by KO of GNE (UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase), NANS (sialic acid synthase) and CMAS (N -acylneuraminate cytidylyltransferase) genes, but was largely unaffected in NANP (N -acylneuraminate-9-phosphatase) KO, as studied by MAA and PNA lectin binding. NANP is the third enzyme in sialic acid biosynthesis and dephosphorylates sialic acid 9-phosphate to free sialic acid. LC-MS analysis of sialic acid metabolites showed that CMP-sialic acid was dramatically reduced in GNE and NANS KO cells and undetectable in CMAS KO. In agreement with normal cell surface sialylation, CMP-sialic acid levels in NANP KO were comparable to WT cells, even though sialic acid 9-phosphate, the substrate of NANP accumulated. Metabolic flux analysis with 13C 6 -labelled ManNAc showed a lower, but significant conversion of ManNAc into sialic acid. Our data provide evidence that NANP activity is not essential for de novo sialic acid production and point towards an alternative phosphatase activity, bypassing NANP. This report contributes to a better understanding of sialic acid biosynthesis in humans. • Knockout of sialic acid synthesis genes GNE and NANS disrupt sialic acid synthesis. • Sialylation of cell-surface glycans is largely unaffected after knockout of NANP. • De novo sialic acid synthesis is active in the absence of NANP. • Activity of NANP is not essential for sialic acid biosynthesis. [ABSTRACT FROM AUTHOR]