DEVELOPMENTAL EXPRESSION AND CHARACTERIZATION OF THE ALPHA-2,8-POLYSIALYLTRANSFERASE ACTIVITY IN EMBRYONIC CHICK BRAIN

The alpha 2,8-polysialyltransferases (polySTs) from embryonic chick br ain catalyze the alpha 2,8-specific polysialylation of endogenous neur al cell adhesion molecules (N-CAMs), This posttranslation glycosylatio n decreases N-CAM-dependent cell adhesion and migration. The enzymatic properties of the membrane-bound form of the polyST activity was inve stigated in vitro. Our results show that the polyST activity was devel opmentally expressed with maximum specific activity appearing about 12 days after fertilization. This time shortly precedes maximal expressi on of the cognate polysialylated N-CAMs. Kinetic studies showed the K- M and V-max for CMP-Neu5Ac were 133 mu M and 0.13 mu M/h, respectively , at pH 6.1, 33 degrees C. CMP-Neu5Gc was not a donor substrate. PolyS T 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 K-i o f 9 mu M. Based on the affinity of the enzyme for heparin, the polyST activity was partially purified (similar to 30-fold) by heparin-Sephar ose affinity chromatography, after differential solubilization with th e 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 alpha 2,3-, alpha 2,6-, and alpha 2,8-monosialyltransferases (monoSTs), A su lfhydryl residue is implicated in chain initiation. Two important stru ctural differences between the mono- and polySTs were revealed by sequ ence analyses. First, the polySTs contain heparin-like, positively cha rged amino acid clusters upstream of both sialylmotif L and S, Second, the polySTs contain a uniquely extended basic amino acid region (pI 1 1.6-12.0) of 31 residues immediately upstream of sialylmotif S, This e xtended, positively charged region may function in the processive mech anism of polymerization by allowing nascent polySia chains to remain b ound to the polyST during the repetitive addition of each new Sia resi due to the nonreducing termini of the growing chain. The importance of these studies is that they provide new information on the enzymatic b asis 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 import ant distinguishing features between the classes of distributive (monoS Ts) and processive polysialyltransferases, which have not been previou sly described.