Abstract. A novel form of protein-saccharide linkage consisting of single N-acetylglucosamine (GIcNAc) residues attached in O-linkages directly to the polypeptide backbone has been described (Holt, G. D., and G. W. Hart, 1986, J. Biol. Chem., 261:8049-8057). This modification was found on proteins distributed throughout the cell, although proteins bearing O-linked GIcNAc moieties were particularly abundant in the cytosolic and nuclear envelope fractions of rat liver. In the accompanying article (Snow, C. M., A. Senior, and L. Gerace, 1987, J. Cell. Biol., 104: 1143-1156, the authors describe monoclonal antibodies directed against eight proteins localized to the nuclear pore complex. These proteins occur on the cytoplasmic and nucleoplasmic (but not lumenal) sides of nuclear membranes. In this report, we demonstrate that all members of this group of pore complex proteins bear multiple O-linked GIcNAc residues. Further, we show that the O-linked GIcNAc moieties are linked via serine (and possibly threonine) side chains to these proteins. Perturbing the O-linked GIcNAc residues either by covalently attaching galactose to them or by releasing them with 13-N-acetylglucosaminidase strongly diminishes the immunoreactivity of the proteins with all of the monoclonal antibodies. However, the O-linked GIcNAc moieties are only part of the epitopes recognized, since O-GlcNAc-containing limit pronase fragments of nuclear pore complex proteins cannot be immunoprecipitated by these antibodies. These findings, taken together with those in the accompanying article, are a direct demonstration that proteins of the cytoplasm and nucleoplasm bear O-linked GIcNAc residues. p REVAILING evidence suggests that the bulk of the wellstudied types of carbohydrate moieties on glycoconjugates are either localized to the cell surface or within lumenal compartments of intracellular organelles (e.g., lysosomes, Golgi, and endoplasmic reticulum). In contrast, relatively little evidence has been obtained suggesting the existence of glycoproteins in the cytoplasmic and nucleoplasmic compartments of cells. Early studies involving carbohydrate analyses of nuclei and nuclear matrix fractions indicated the presence of glycoconjugates in these fractions (reviewed in Furukawa and Terayanm, 1979;Stein et al., 1981;Stoddart, 1979), and the presence of cytoplasmic glycoproteins has also been suggested (Meyer and Burger, 1976). These data have received support in several more recent investigations (e.g., Kan and Pinto da Silva, 1986;Fedarko and Conrad, 1986;Nagakura et al., 1986). However, attempts to identify and characterize nucleoplasmic and cytoplasmic glycoproteins have been severely hampered by cross-contamination with other cellular compartments, and detailed biochemical analyses of most putative cytoplasmic and nucleoplasmic glycoproteins is lacking.Recently the subcellular distribution of glycoproteins bearing terminal N-acetylglucosamine (GIcNAc) ~ residues was surveyed (Holt and Hart, 1986) by employing galactosyltransferase, an enzyme that trans...

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Abstract. Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A-or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin.The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]/~l-lceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.

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