Two cDNA clones encoding NeuAc␣2,3Gal␤1,3GalNAc GalNAc␣2,6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAc␣2, 3Gal␤1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Gal␤1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14 C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[ 14 C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAc␣2,6-sialyltransferase, which requires sialic acid residues linked to Gal␤1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.Sialic acids are key determinants of carbohydrate structures that play important roles in a variety of biological functions, like cell-cell communication, cell-substrate interaction, adhesion, and protein targeting. The transfer of sialic acids from CMP-Sia 1 to the terminal positions of the carbohydrate groups of glycoproteins and glycolipids is catalyzed by a sialyltransferase. Although roles of sialic acids have been proposed in the regulation of many biological phenomena, the purpose of this structural diversity remains largely obscure. To determine the meaning of the diversity of and the regulatory mechanism for the sialylation of glycoconjugates, it is necessary to obtain information on the enzymes themselves and the gene structure of sialyltransferases. Each sialyltransferase exhibits strict specificity for acceptor substrates and linkages (3-6). Although three linkages, Sia␣2,6Gal, Sia␣2,3Gal, and Sia␣2,6GalNAc, are commonly found in glycoproteins (7), and two, Sia␣2,3Gal and Sia␣2,8Sia, occur frequently in gangliosides (8), each of these linkages has been found in both gangliosides and glycoproteins (8 -10).So far, the cloning of three members of the ␣2,6-sialyltransferase family (ST6GalNAc I, II and III) has been reported (11-14). The cDNAs of ST6GalNAc I and II were cloned from both chick (11, 12) and mouse (13,62).2 The overall amino acid sequence identity of chick ST6GalNAc I is 30.5% to chick ST6GalNAc II, 43.2% to mouse ST6GalNAc I, and 33.6% to mouse ST6GalNAc II, and that of mouse ST6GalNAc I is 29.6% to mouse ST6GalNAc II and 28.3% to chick ST6GalNAc II, and that of chick ST6GalNAc II is 57.3% ...

ST8Sia II (STX) and ST8Sia IV (PST) are polysialic acid (polySia) synthases that catalyze polySia formation of neural cell adhesion molecule (NCAM) in vivo and in vitro. It still remains unclear how these structurally similar enzymes act differently in vivo. In the present study, we performed the enzymatic characterization of ST8Sia II and IV; both ST8Sia II and IV have pH optima of 5.8 -6.1 and have no requirement of metal ions. Because the pH dependence of ST8Sia II and IV enzyme activities and the pK profile of His residues are similar, we hypothesized that a histidine residue would be involved in their catalytic activity. There is a conserved His residue (cf. His 348 in ST8Sia II and His 331 in ST8Sia IV, respectively) within the sialyl motif VS in all sialyltransferase genes cloned to date. Mutant ST8Sia II and IV enzymes in which this His residue was changed to Lys showed no detectable enzyme activity, even though they were folded correctly and could bind to CDP-hexanolamine, suggesting the importance of the His residue for their catalytic activity. Next, the degrees of polymerization of polySia in NCAM catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger polySia formation of NCAM than ST8Sia II. We also analyzed the (auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II or IV itself was sialylated under conditions for polysialylation, the disialylated compound was the major product, even though polysialylated compounds were also observed. These results suggested that both ST8Sia II and IV catalyze polySia synthesis toward preferred acceptor substrates such as NCAM, whereas they mainly catalyze disialylation, similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.

The α2,8-polysialyltransferases (polySTs) from embryonic chick brain catalyze the α2,8-specific polysialylation of endogenous neural cell adhesion molecules (N-CAMs). This posttranslation glycosylation decreases N-CAM-dependent cell adhesion and migration. The enzymatic properties of the membrane-bound form of the polyST activity was investigated in vitro. Our results show that the polyST activity was developmentally expressed with maximum specific activity appearing about 12 days after fertilization. This time shortly precedes maximal expression of the cognate polysialylated N-CAMs. Kinetic studies showed the K M and V max for CMPNeu5Ac were 133 µM and 0.13 µM/h, respectively, at pH 6.1, 33_C. CMP-Neu5Gc was not a donor substrate. PolyST activity was increased 5-to 6-fold in the presence of 10 mM MnCl 2, the preferred divalent cation, and 1 mM dithiothreitol (DTT). Heparin (3 kDa) was a noncompetitive inhibitor of polysialylation with a K i of 9 µM. Based on the affinity of the enzyme for heparin, the polyST activity was partially purified (∼30-fold) by heparin-Sepharose affinity chromatography, after differential solubilization with the zwitterionic detergent, CHAPS. DTT and chemical modification studies using the thiol-directed alkylating reagents, N-ethylmaleimide (NEM) and iodoacetamide (IAA), were used to show that at least one cysteinyl residue in the polyST was of critical importance for polysialylation, but of lesser importance for monosialylation, catalyzed by the α2,3-, α2,6-, and α2,8-monosialyltransferases (monoSTs). A sulfhydryl residue is implicated in chain initiation. Two important structural differences between the mono-and polySTs were revealed by sequence analyses. First, the polySTs contain heparin-like, positively charged amino acid clusters upstream of both sialylmotif L and S. Second, the polySTs contain a uniquely extended basic amino acid region (pI 11.6-12.0) of 31 residues immediately upstream of sialylmotif S. This extended, positively charged region may function in the processive mechanism of polymerization by allowing nascent polySia chains to remain bound to the polyST during the repetitive addition of each new Sia residue to the nonreducing termini of the growing chain. The importance of these studies is that they provide new information on the enzymatic basis of polysialylation. They also reveal that sulfhydryl residues and extended basic amino acid domains are two structural features unique to polysialylation, in contrast to monosialylation. Both may be important distinguishing features between the classes of distributive (monoSTs) and processive polysialyltransferases, which have not been previously described.

The ST6Gal I is a sialyltransferase that modifies N-linked oligosaccharides of glycoproteins. Previous results suggested a role for luminal stem and active domain sequences in the efficiency of ST6Gal I Golgi retention. Characterization of a series of STtyr isoform deletion mutants demonstrated that the stem is sensitive to proteases and that preventing cleavage in this region leads to increased cell surface expression. A mutant lacking amino acids 32-104 (STDelta4) is not active or cleaved and secreted like the wild type STtyr, but does exhibit increased cell surface expression. It is probable that the STDelta4 mutant lacks the stem region and some amino acids of the active domain because the STDelta5 mutant lacking amino acids 86-104 is also not active but is cleaved and secreted. In contrast, deletion of stem amino acids between residues 32 and 86 in the STDelta1, STDelta2, and STDelta3 mutants does not inactive these enzyme forms, eliminate their cleavage and secretion, or increase their cell surface expression. Surprisingly, cleavage occurs even though the previously identified Asn63-Ser 64 cleavage site is missing. Further evaluation demonstrated that a cleavage site between Lys 40 and Glu 41 is used in COS cells. Mutagenesis of Lys 40 significantly decreased, but did not eliminate cleavage, suggesting that there are additional secondary sites of cleavage in the ST6Gal I stem.

The Strongylocentrotus purpuratus sea urchin egg receptor for sperm is a cell surface glycoprotein with a molecular mass of 350 kDa. Recent studies indicate that the sulfated O-linked glycans isolated from the receptor bind to acrosome-reacted sperm. The purified receptor was analyzed with respect to amino acid and carbohydrate content and shown to be composed of 70% carbohydrate by weight. Compositional analysis indicated that both N-and O-linked oligosaccharide chains were present. After peptide:N-glycanase treatment of the receptor to remove most of the N-linked glycan chains, the majority of the sialic acid residues remained associated with the receptor and were shown by several types of experiments to be composed of sulfated oligosialic acid units attached to the O-linked glycan chains of the receptor. Chemical and physical studies on oligosialic chains discovered earlier in the Pronase-generated glycopeptide fraction isolated from the egg cell surface complex of another species of sea urchin, Hemicentrotus pulcherrimus, established that these molecules had the structure: (SO 4 ؊ )-9Neu5Gc␣2(35-O glycolyl Neu5Gc␣23) n . Based on comparative and analytical studies, it was concluded that this sulfated oligosaccharide is a component of a GalNAc-containing chain that is O-linked to the polypeptide chain of the sea urchin egg receptor for sperm. Using a competitive inhibition of fertilization bioassay it was shown that the sulfated oligosialic acid chains derived from the S. purpuratus egg cell surface complex inhibited fertilization; the nonsulfated form of this oligosialic chain had little inhibitory activity.In fertilization, cell surface molecules of the egg and sperm play a central role in the species-specific interactions that occur in many organisms (1, 2). In the case of the sea urchin, earlier studies showed that the sperm protein, bindin, which is a component of the acrosome granule, plays a key role in gamete recognition (3, 4). Following early work in which a high molecular weight glycoprotein on the egg cell surface was implicated as a receptor for sperm (5-7), it was shown that Pronase-generated glycopeptides prepared from this crude receptor preparation inhibited fertilization, but without species specificity (8, 9). Later, a cell surface glycoprotein with a molecular mass of 350 kDa was identified as the egg receptor in Strongylocentrotus purpuratus (10,11). This molecule, as well as a 70-kDa extracellular fragment generated by lysyl endoproteinase C digestion of intact eggs, were shown to contain sugars typical of both N-and O-linked oligosaccharides, as well as sulfate (10, 12). These oligosaccharide chains were fractionated, and the putative O-linked chains were shown to inhibit fertilization and bind without species specificity to acrosome-reacted, but not to unreacted sperm (13). In a subsequent study it was shown that attachment of these chains via a neoglycoprotein to beads mediated kinetically stable binding of sperm to such beads (14).In earlier studies, ␣235-O glycolyl -linked p...