Differential biosynthesis of polysialic or disialic acid Structure by ST8Sia II and ST8Sia IV.

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 relationship between ST6Gal I Golgi retention and its cleavage-secretion.

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.

Molecular cloning and functional expression of two members of mouse NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2,6-sialyltransferase family, ST6GalNAc III and IV.

Two cDNA clones encoding NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 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 NeuAcalpha2, 3Galbeta1, 3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta1,3GalNAc 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.

Developmental expression and characterization of the alpha2,8-polysialyltransferase activity in embryonic chick brain.

The alpha2,8-polysialyltransferases (polySTs) from embryonic chick brain catalyze the alpha2,8-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 KMand Vmaxfor CMP-Neu5Ac were 133 microM and 0.13 microM/h, respectively, at pH 6.1, 33 degrees C. CMP-Neu5Gc was not a donor substrate. PolyST activity was increased 5- to 6-fold in the presence of 10 mM MnCl2,the preferred divalent cation, and 1 mM dithiothreitol (DTT). Heparin (3 kDa) was a noncompetitive inhibitor of polysialylation with a Kiof 9 microM. Based on the affinity of the enzyme for heparin, the polyST activity was partially purified ( approximately 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 alpha2,3-, alpha2,6-, and alpha2,8-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.

Identification of sulfated oligosialic acid units in the O-linked glycan of the sea urchin egg receptor for sperm.

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 alpha2(-->5-O(glycolyl)Neu5Gc alpha2-->)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.