Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV−/− mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

To determine the molecular basis of eukaryotic polysialylation, the function of a structurally unique polybasic motif of 32 amino acids (pI approximately 12) in the polysialyltransferases (polySTs), ST8Sia II (STX and ST8Sia IV (PST) was investigated. This motif, designated the "polysialyltransferase domain" (PSTD), is immediately upstream of the sialylmotif S (SM-S). PolyST activity was lost in COS-1 mutants in which the entire PSTD in ST8Sia IV was deleted, or in mutants in which 10 and 15 amino acids in either the N- or C- terminus of PSTD were deleted. Site-directed mutagenesis showed that Ile(275), Lys(276) and Arg(277) in the C-terminus of PSTD in ST8Sia IV, which is contiguous with the N-terminus of sialylmotif-S, were essential for polysialylation. Arg(252) in the N-terminus segment of the PSTD was also required, as was the overall positive charge. Thus, multiple domains in the polySTs can influence their activity. Immunofluorescent microscopy showed that the mutated proteins were folded correctly, based on their Golgi localization. The structural distinctness of the conserved PSTD in the polySTs, and its absence in the mono- oligoSTs, suggests that it is a "polymerization domain" that distinguishes a polyST from a monosialyltransferases. We postulate that the electrostatic interaction between the polybasic PSTD and the polyanionic polySia chains may function to tether nascent polySia chains to the enzyme, thus facilitating the processive addition of new Sia residues to the non-reducing end of the growing chain. In accord with this hypothesis, the polyanion heparin was shown to inhibit recombinant human ST8Sia II and ST8Sia IV at 10 microM.

␣2,8-Linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal ␣2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-␤-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs ϳ50 -60 or less, a subpopulation of chains with DPs ϳ150 to ϳ180 and extending to DP ϳ400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-␤-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

No abstract