Molecular cloning and functional analysis of sialyltransferases

To elucidate the regulatory mechanism for carbohydrate expression and to understand the meaning of the carbohydrate-structural diversity, we started to clone sialyltransferase (ST) genes based on two different strategies, i.e. expression and homology cloning. So far, 13 STs have been cloned in our laboratory, 7 of which turned out to be new ones. The primary enzyme structures deduced from all the cloned ST genes suggest a putative domain structure with a type II transmembrane topology. There are no significant amino acid sequence similarities among these cloned STs, except for in two sialyl motifs, L and S, which are proposed to be the CMP-sialic acid recognition and/or catalytic sites. Northern blot analysis revealed the developmental stage-dependent and/or tissue-specific expression of most of the cloned STs. The cloned STs are classified into four families according to the carbohydrate linkages they synthesize, i.e. the ST3Gal-, ST6Gal-, ST6GalNAc-, and ST8Sia-families. Generally, enzymes in these families exhibit strong activity toward certain acceptor groups but show very weak activity toward other acceptor groups, and the substrate specificities of the enzymes overlap one another, as indicated by in vitro experiments. Enzymes in the ST3Gal-family are expressed mainly in a tissue-specific manner. However, those in the ST6GalNAc- and ST8Sia-families are expressed in a tissue-as well as developmental stage-specific manner. In vivo conditions are supposed to be more complex. Therefore, it is quite important to examine their substrate specificities in vivo and the mechanism of their expression to elucidate the physiological role of each enzyme and the meaning of the diversity in carbohydrate structure. Using cloned cDNAs and expressed enzymes, we have been studying how sialylcarbohydrate expression is regulated and what the functions of sialylcarbohydrate chains are. Recently, we found that transfection of the GD3 synthase, an alpha 2,8-ST (ST8Sia I), gene triggers cholinergic neuritogenesis in Neuro2a cells through the de novo expression of GD3, suggesting that the GD3 synthase gene behaves as a neural differentiation inducer.