Mouse β-galactoside α2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression

Abstract

Four types of beta-galactoside alpha 2,3-sialyltransferase (ST3Gal I-IV) have been cloned from several animals, but some contradictory observations regarding their substrate specificities and expression have been reported. Therefore, it is necessary to concurrently analyze the substrate specificities of the four enzymes, of which the source should be one animal. Accordingly, the acceptor substrate specificities and gene expression of mST3Gal I-IV were analyzed. Since we had already cloned ST3Gal I and II, as previously reported (Lee, Y.-C. et al., Eur. J. Biochem., 216, 377-385 (1993); J. Biol. Chem., 269, 10028-10033 (1994)), the cDNAs of ST3Gal III and IV were cloned from mouse cDNA libraries. Each of the four enzymes was expressed in COS-7 cells as a recombinant enzyme fused with protein A, and applied on an IgG-Sepharose gel to eliminate endogenous sialyltransferase activity. ST3Gal I and II showed the highest activity toward Gal beta 1, 3 GalNAc (type III), very low activity toward Gal beta 1,3GlcNAc (type I), but none toward Gal beta 1,4GlcNAc (type II). ST3Gal III and IV exhibited high activity toward the type I and II disaccharides, but very low activity toward the type III one. On the other hand, asialo-GM1 (Gg4Cer) was as good a substrate for ST3Gal I and II as the type III disaccharide, though ST3Gal III and IV hardly utilized glycolipids as substrates, as indicated by in vitro experiments. Northern blot analysis revealed that enzymes of the ST3Gal-family are expressed mainly in a tissue-specific manner. The ST3Gal I gene was strongly expressed in spleen and salivary gland, and weakly in brain, liver, heart, kidney, and thymus. The ST3Gal II gene was strongly expressed in brain, and weakly in colon, thymus, salivary gland, and testis, and developmentally expressed in liver, heart, kidney, and spleen. The ST3Gal III and IV genes were expressed in a wide variety of tissues. These differences in tissue specific expression suggest the expression of each ST3Gal influences the distribution of sialyl-glycoconjugates in vivo.

Structure-Function Analysis of the Human Sialyltransferase ST3Gal I

“…These values are in good agreement with the data previously obtained with the same enzyme expressed in COS cells (21,22). Rather similar apparent K m values toward the donor substrate have been obtained for the four mutants tested thus confirming the behavior of these mutants in CDP binding tests (Fig.…”

“…ST3Gal I transfers Neu5Ac to the galactose residue of type 3 disaccharide found on glycolipids or O-glycosyl proteins. Kinetic studies have shown that ST3Gal I exhibits high transfer efficiency and high affinity (K m of 51 M) toward the core 1 mucin type disaccharide Gal␤1-3GalNAc␣- (21). Therefore, this enzyme was considered as a good candidate for structure-function studies and for further crystallization trials.…”

A Family of Human β3-Galactosyltransferases

“…However, it is equally likely that this high number of enzymes have evolved as a result of specific requirements for enzymes with different functions. Apparent redundancies in substrate specificities of glycosyltransferases have been found in sialyltransferases (28,29), fucosyltransferases (30 -32), ␤4-galactosyltransferases (2-4), and polypeptide GalNAc-transferases (14,33). There are differences in the kinetic parameters of some members of each of these families that relate to type and complexity of acceptor glycoconjugate or acceptor peptide sequence for the polypeptide GalNAc-transferases.…”

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

“…ST3Gal-3 2 HL-60s displayed ;35% reduction in CLA expression, consistent with the notion that this enzyme shows higher activity toward type I (Galb1,3GlcNAc) rather than type II (Galb1,4Glc-NAc) LacNAc. 20 Silencing ST3Gal-6 augmented CLA expression by .twofold, though the precise reason for this is unknown. Similar observations, as with HECA-452, were made when mAb CSLEX-1 measured cell-surface sLe X expression ( Figure 2E).…”

“…16,17 We sought to identify the human a2,3 sialyltransferases (sialylTs) that regulate myeloid cell rolling on selectins. In this regard, among the 6 mammalian a2,3sialyTs (ST3Gal-1-6), we choose to focus on ST3Gal-3, -4, and -6 because these enzymes transfer sialic acid (NeuAc in humans) to the 3-position of galactose on type II n-Acetyllactosamine/LacNAc (Galb1,4GlcNAc) structures to generate sLe X and related glycans [18][19][20][21][22] ( Figure 1). Among these, ST3Gal-3 also acts on type I lactosamine substrates (Galb1,3GlcNAc) to create sialyl Lewis-a (sLe a )-type epitopes.…”