Effects of amino acid substitutions in the sialylmotifs on molecular expression and enzymatic activities of α2,8-sialyltransferases ST8Sia-I and ST8Sia-VI

Abstract

Mouse sialyltransferases are grouped into four families according to the type of carbohydrate linkage they synthesize: -galactoside α2,3- sialyltransferases (ST3Gal-I-VI), -galactoside α2,6-sialyltransferases (ST6Gal-I and ST6Gal-II), N-acetylgalactosamine α2,6-sialyltransferases (ST6GalNAc-I-VI) and α2,8-sialyltransferases (ST8Sia-I-VI). These sialyltransferases feature a type II transmembrane topology and contain highly conserved motifs termed sialylmotifs L, S, III and VS. Sialylmotifs L and S are involved in substrate binding, whereas sialylmotifs III and VS are involved in catalytic activity. In addition to the conventional sialylmotifs, family and subfamily specific sequence motifs have been proposed. In this study, we analyzed the properties and functions of sialylmotifs in characterizing the enzymatic activity of mouse ST8Sia-I and ST8Sia-VI, both of which are α2,8-sialyltransferases involved in the synthesis of either ganglioside GD3 or disialic acid structures on O-glycans, respectively. The ST8Sia-VI-based chimeric enzymes, whose sialylmotif L sequences were replaced with those of ST8Sia-I and ST8Sia-IV (polysialic acid synthetase), were still active toward O-glycans. However, ST8Sia-VI-based chimeric enzymes lost expression or activity when their sialylmotif L sequences were replaced with those of ST3Gal-I and ST6GalNAc-II, suggesting the existence of an ST8Sia family specific motif in the sialylmotif L. The ST8Sia-I- and ST8Sia-VI-based chimeric enzymes lost enzymatic activity when their sialylmotif S sequences were interchanged. Amino acid substitutions in the sialylmotif S of ST8Sia-I and ST8Sia-VI also affected the enzymatic activity in many cases, indicating the crucial and functional importance of the sialylmotif S in substrate binding, which determines the substrate specificity of sialyltransferase. © 2013 The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.