Glycobiology Advance Access originally published online on October 19, 2005
Glycobiology 2006 16(2):165-172; doi:10.1093/glycob/cwj050
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Identification of the sequence encoding N-acetylneuraminate-9-phosphate phosphatase
2 Laboratory of Physiological Chemistry, Université Catholique de Louvain and the Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels, Belgium; and 3 Hormone and Metabolism Unit, Université Catholique de Louvain and the Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels, Belgium
1 To whom correspondence should be addressed; e-mail: vanschaftingen{at}bchm.ucl.ac.be
Received on September 2, 2005; revised on October 10, 2005; accepted on October 12, 2005
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 [EC] ) 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.
Key words: glycosylation / haloacid dehalogenase family / N-acetylmannosamine 6-phosphate / sialic acid
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