TY - JOUR
T1 - Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis
AU - Willems, Anke P.
AU - Sun, Lingbo
AU - Schulz, Morten Alder
AU - Tian, Weihua
AU - Ashikov, Angel
AU - van Scherpenzeel, Monique
AU - Hermans, Esther
AU - Clausen, Henrik
AU - Yang, Zhang
AU - Lefeber, Dirk J.
PY - 2019
Y1 - 2019
N2 - 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 acid and 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 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 13C6-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 acid production and point towards an alternative phosphatase activity, bypassing NANP. General significance: This report contributes to a better understanding of sialic acid biosynthesis in humans.
AB - 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 acid and 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 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 13C6-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 acid production and point towards an alternative phosphatase activity, bypassing NANP. General significance: This report contributes to a better understanding of sialic acid biosynthesis in humans.
KW - Carbohydrate metabolism
KW - Glycosylation
KW - Inborn errors of metabolism
KW - Nucleotide sugars
KW - Sialic acid
U2 - 10.1016/j.bbagen.2019.05.011
DO - 10.1016/j.bbagen.2019.05.011
M3 - Journal article
C2 - 31121216
AN - SCOPUS:85066955353
SN - 0304-4165
VL - 1863
SP - 1471
EP - 1479
JO - B B A - General Subjects
JF - B B A - General Subjects
IS - 10
ER -