Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis

Abstract Background 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 acidand their phosphorylated forms as intermediates. Genetic diseases in this pathway result in different and tissue-restricted phenotypes, which is poorly understood. Methods and results We aimed to study the mechanisms of sialic acidmetabolism in knockouts (KO) of the sialic acidpathway in two independent cell lines. Sialylation of cell surface glycans was reduced by KO of GNE ( UDP- N- acetylglucosamine 2-epimerase/ N- acetylmannosaminekinase), NANS( sialic acidsynthase) 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 acidbiosynthesis and dephosphorylates sialic acid9-phosphate to free sialic acid. LC-MS analysis of sialic acidmetabolites showed that CMP- sialic acidwas dramatically reduced in GNE and NANSKO cells and undetectable in CMAS KO. In agreement with normal cell surface sialylation, CMP- sialic acidlevels in NANP KO were comparable to WT cells, even though sialic acid9-phosphate, the substrate of NANP accumulated. Metabolic flux analysiswith 13 C 6 -labelled ManNAc showed a lower, but significant conversion of ManNAc into sialic acid. Conclusions Our data provide evidence that NANP activity is not essential for de novo sialic acidproduction and point towards an alternative phosphatase activity, bypassing NANP. General significance This report contributes to a better understanding of sialic acidbiosynthesis in humans.