Identification of the sequence encoding N-acetylneuraminate-9-phosphate phosphatase.
The synthesis of N-acetylneuraminate (Neu5Ac), the main form of sialic acid, proceeds in vertebrates through the condensation of N-acetylmannosamine 6-phosphate and phosphoenolpyruvate to Neu5Ac-9-phosphate, followed by the dephosphorylation of the latter by a specific phosphatase. The sequence encoding Neu5Ac-9-phosphate phosphatase (Neu5Ac-9-Pase; E.C. 3.1.3.29) has not been determined until now. In this work, we have purified Neu5Ac-9-Pase more than 1000-fold from rat liver. Its dependency on Mg2+ and the fact that it was inhibited by vanadate and Ca2+ suggested that it belonged to the haloacid dehalogenase family of phosphatases. Trypsin digestion and mass spectrometry analysis of a polypeptide of about 30 kDa that co-eluted with the activity in the last purification step indicated the presence of a protein designated "haloacid dehalogenase-like hydrolase domain containing 4." The human ortholog of this protein is encoded by a 2-exon gene present on chromosome 20p11. The human protein was overexpressed in Escherichia coli as a fusion protein with a polyHis tag and purified to homogeneity. The recombinant enzyme displayed a >230-fold higher catalytic efficiency on Neu5Ac-9-phosphate than on its second best substrate. Its properties were similar to those of the enzyme purified from rat liver. Neu5Ac inhibited the enzymatic activity by 50% at 15 mM, indicating that no significant inhibition is exerted at physiological concentrations of Neu5Ac. The identification of the gene encoding Neu5Ac-9-Pase will facilitate studies aimed at testing its potential implication in unexplained forms of glycosylation deficiency.
Maliekal
Pushpa
P
Laboratory of Physiological Chemistry, Université Catholique de Louvain and the Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
Vertommen
Didier
D
Delpierre
Ghislain
G
Van Schaftingen
Emile
E
D000595:Amino Acid Sequence; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; C043518:N-acylneuraminate-9-phosphatase