Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.

In the induction of an immune response, IL-15Rα on APCs transpresents IL-15 to NK and CD8+/CD44high T cells that express the IL-2/15Rβ and γc subunits only. In this study, we show data mimicking this transpresentation by using IL-15 preassociated with a chimeric protein that is comprised of the extracellular domain of murine IL-15Rα and the Fc portion of human IgG1. When tested in vitro, IL-15Rα-IgG1-Fc strongly increased the IL-15-mediated proliferation of murine NK and CD8+/CD44high T cells. The effect of IL-15Rα-IgG1-Fc was dependent on the presence of both IgG1-Fc and IL-15Rα. When injected into mice, IL-15Rα-IgG1-Fc enhanced the capacity of IL-15 to expand the number of NK and CD8+/CD44high T cells. The effect on cell numbers in vivo also depended on Fc receptor binding because reduced expansion was observed in FcRγ−/− mice. NK cells cultured in IL-15/IL-15Rα-IgG1-Fc complex gained cytotoxic activity toward a number of NK-sensitive targets. When mice bearing the NK-sensitive syngeneic tumor B16 were treated, the presence of IL-15Rα-IgG1-Fc increased the antitumor activity of IL-15. Thus, a preassociation with IL-15Rα-IgG1-Fc enhances the activities of IL-15 in vivo and in vitro that may be useful in the treatment of tumors.

Mammalian mucin-type O-glycosylation is initiated by a large family of ϳ20 UDP-GalNAc:polypeptide ␣-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer ␣-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the ؊1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.Mucin-type O-glycosylation is one of the most common post-translational modifications of secreted and membrane-associated proteins. Glycoproteins containing O-glycosylated mucin domains serve many important biological roles chiefly because of their unique biophysical and structural properties that include an extended peptide conformation and robust resistance to proteases. Consequently, glycoproteins containing O-glycosylated mucin domains function in the protection of the cell surface, the modulation of cell-cell interactions, in the inflammatory and immune response, in metastasis and tumorigenesis, and in protein sorting, targeting, and turnover (for examples see Refs.