Site-directed mutagenesis of human membrane-associated ganglioside sialidase - Identification of amino-acid residues contributing to substrate specificity

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hyd rolyzes only gangliosides. To cast light on the molecular basis of this nar row substrate preference, predicted active site aminoacid residues of the h uman membrane sialidase were altered by site-directed mutagenesis. When com pared with the active site amino-acid residues proposed for Salmonella typh imurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmemb rane region. Alteration of seven residues, including these five, was follow ed by transient expression of the mutant enzymes in COS-1 cells and charact erization of their kinetic properties using various substrates. Substitutio n of glutamic acid (at position 51) by aspartic acid and of arginine (at po sition 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caus ed a marked reduction. The mutant enzyme E51D exhibited an increase in hydr olytic activity towards GM2 as well as sialyllactose (which are poor substr ates for the wild-type) with change to a lower K-m and a higher V-max. R114 Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides CD3 and GD1a that a re effectively hydrolyzed by the wild-type. The inhibition experiments usin g 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the re sults in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute t o its substrate specificity.