Developmental expression and characterization of the  2,8-polysialyltransferase activity in embryonic chick brain

Abstract

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.

Unique Disulfide Bond Structures Found in ST8Sia IV Polysialyltransferase Are Required for Its Activity

“…This cysteine is also involved in the enzymatic activity, because the mutation of this cysteine 169 to alanine reduced the enzymatic activity. This result is consistent with a previous report that at least one free cysteine residue is critical for polysialylation (43).…”

Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules ofEscherichia coli K1 and K92 and Neisseria meningitidis Groups B or C

“…Indeed, disulfide-reducing agents inhibit the mammalian monosialyltransferases (43), whereas they either weakly stimulate or have no effect on the bacterial polySTs, indicating the possibility of distinctly different architectures for substrate binding. Interestingly, mammalian polyST is not inhibited by disulfidereducing agents (40), suggesting a possible similarity between bacterial and mammalian PSA biosynthetic mechanisms despite their lack of obvious primary structural homology.…”

Degree of Polymerization (DP) of Polysialic Acid (PolySia) on Neural Cell Adhesion Molecules (N-CAMs)

“…Reconstituted in Vitro PolyST Assay-Incorporation of Neu5Ac from CMP-Neu5Ac into FcN-CAM as the exogenous acceptor substrate was carried out by a modification of the polyST assay developed earlier in our laboratory (29). All incubation mixtures contained 50 mM MES buffer (pH 6.1), 10 mM MnCl 2 , 1 mM CMP-Neu5Ac, purified FcN-CAM (0.6 g of protein), and STX(s) (3 g of protein) in a total volume of 15 l. After incubation at 37°C for 2 h, polysialylation of N-CAM was qualitatively assessed by SDS-PAGE/Western blot analysis both before and after treatment with endo-N-acylneuraminidase (Endo-N) or Endo-␤-Galase using either anti-polySia or anti-N-CAM mAbs.…”