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Glycobiology Advance Access published online on October 19, 2005
Glycobiology, doi:10.1093/glycob/cwj050
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.
Received August 25, 2005
Revised October 7, 2005
Accepted October 11, 2005

Article

Identification of the sequence encoding N-acetylneuraminate-9-phosphate phosphatase

Pushpa Maliekal 1, Didier Vertommen 2, Ghislain Delpierre 1, and Emile Van Schaftingen 1*

1 Laboratory of Physiological Chemistry, Université Catholique de Louvain and the Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels
2 Hormone and Metabolism Unit, Université Catholique de Louvain and the Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels

* To whom correspondence should be addressed.
Emile Van Schaftingen, E-mail: vanschaftingen{at}bchm.ucl.ac.be


  Abstract

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 the present 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 ~?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 orthologue of this protein is encoded by a 2-exon gene present on chromosome 20p11. The human protein was overexpressed in E. 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.

Keywords: glycosylation/haloacid dehalogenase family/N-acetylmannosamine 6-phosphate/sialic acid.
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