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Microsomal membranes from mouse lymphoma BW5147 cells were fractionated on a continuous sucrose gradient and assayed for two enzymes involved in the synthesis of O-linked oligosaccharides . Both enzymes were recovered in membranes that were less dense than the membranes containing the endoplasmic reticulum marker enzymes, glucosidase I and II. UDP-Gal :Nacetylgalactosamine-ß1,3-galactosyltransferase had a distribution that coincided with that of the galactosyltransferase that acts on asparagine-linked oligosaccharides . This latter enzyme has been immunolocalized to the trans Golgi elements. The UDP-GaINAc :polypeptide N-acetylgalactosaminyltransferase was recovered in a membrane fraction of intermediate density, between the endoplasmic reticulum and trans Golgi markers . These findings are consistent with the assembly of O-linked oligosaccharides occurring in at least two different Golgi compartments .Many membranes and secretory proteins contain oligosaccharide units linked O-glycosidically to serine and/or threonine residues (1-8). In most instances, the linkage sugar is Nacetylgalactosamine, which is then substituted by galactose and/or N-acetylglucosamine . A number ofdifferent structures can then be formed from this basic core by the stepwise addition of monosaccharides that are donated directly from nucleotide sugars (9) . This is in contrast to the synthesis of N-linked oligosaccharides that are formed by the en bloc transfer of an oligosaccharide from a lipid carrier to the nascent protein (10, 11) . This oligosaccharide is then processed as the protein passes through the endoplasmic reticulum and the Golgi apparatus (12).The subcellular location of the initial reactions in the synthesis of O-linked oligosaccharides is not yet firmly established . Strous (13) concluded that the attachment of the Nacetylgalactosamine residues to Ser/Thr takes place while the nascent peptide is still associated with ribosomes, indicating that this reaction is a co-translational event . In contrast to this conclusion, there is an increasing body of evidence that the initial step in O-linked glycosylation occurs posttranslationally in the smooth endoplasmic reticulum or the Golgi apparatus (3,5,(14)(15)(16)(17)(18)(19)(20). Studies of the biosynthesis of the low density lipoprotein receptor in A431 cells have revealed that Ga1NAc is added to the protein before processing of the receptor's asparagine-linked high mannose oligosaccharide (8) . Subsequently the high mannose oligosaccharide is converted to a complex-type unit and the assembly of the 0-linked units is completed by the addition of galactose and THE JOURNAL OF CELL BIOLOGY " VOLUME 98 JULY 1984 327-331 0 The Rockefeller University Press -0021-9525/84/07/0327/05 $1 .00 sialic acid residues. This indicates that the synthesis of 0-linked oligosaccharides occurs in more then one subcellular compartment .One approach for analyzing the subcellular localization of oligosaccharide processing enzymes is to fractionate total microsomal membranes on linear sucrose gr...

The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor–globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm– egg interactions.

The natural product aureobasidin A (AbA) is a potent, well-tolerated antifungal agent with robust efficacy in animals. Although native AbA is active against a number of fungi, it has little activity against Aspergillus fumigatus, an important human pathogen, and attempts to improve the activity against this organism by structural modifications have to date involved chemistries too complex for continued development. This report describes novel chemistry for the modification of AbA. The key step involves functionalization of the phenylalanine residues in the compound by iridium-catalyzed borylation. This is followed by displacement of the pinacol boron moiety to form the corresponding bromide or iodide and substitution by Suzuki biaryl coupling. The approach allows for synthesis of a truly wide range of derivatives and has produced compounds with A. fumigatus minimal inhibitory concentrations (MIC) of <0.5 μg/mL. The approach is readily adaptable to large-scale synthesis and industrial production.

Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW) 3 repeat sequence at the C terminus that consists of three tandem repeats (␣, ␤, and ␥). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp 444 in the ␣ repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp 444 and the corresponding aspartate residues in the ␤ and the ␥ repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.O-Glycosidically linked oligosaccharides, called mucin-type oligosaccharides, are linked to polypeptides through an ␣-linkage (GalNAc␣1 3 Ser (or Thr)) and are known to occur on mucins as well as on other secretory and membrane glycoproteins (1). The initial step in the biosynthesis of these structures is catalyzed by a group of enzymes known as the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNActransferases).