Protein kinase C and a calcium-independent phospholipase are required for IgG-mediated phagocytosis by Mono-Mac-6 cells.

Mono-Mac-6 (MM6) human monocytes ingest IgG-opsonized particles better than other human cell lines. We compared the phagocytic signaling pathway in MM6 with human monocytes. MM6 expressed FcgammaRI at levels similar to monocytes, whereas FcRgammaII expression was approximately double. MM6 ingested IgG-opsonized erythrocytes (EIgG) in a calcium-independent manner. Incubation of MM6 with bromoenol lactone, an inhibitor of the phagocytic phospholipase (pPL), coordinately decreased phagocytosis and pPL activity. This inhibition was overcome by exogenous arachidonic acid, suggesting that phagocytosis requires pPL activation and arachidonic acid release. MM6 phagocytosis was inhibited with staurosporine and activated with diacylglycerol, supporting a role for protein kinase C (PKC) in this process. The pPL activators mastoparan and melittin restored phagocytosis to PKC-inhibited cells, suggesting that pPL lies downstream from PKC. These results suggest that the MM6 signal transduction pathway for IgG-mediated phagocytosis is similar to that of monocytes (PKC-->pPL-->arachidonic acid-->phagocytosis). The results are discussed in the context of the finding that MM6 exhibit low phagocytosis relative to monocytes and thus may represent an attractive cell line for molecular manipulation in "recovery of function" studies.

Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides.

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B). The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B). The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal. Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.

Overview of N- and O-linked oligosaccharide structures found in various yeast species.

Yeast and most higher eukaryotes utilize an evolutionarily conserved N-linked oligosaccharide biosynthetic pathway that involves the formation of a Glc3Man9GlcNAc2-PP-dolichol lipid-linked precursor, the glycan portion of which is co-translationally transferred in the endoplasmic reticulum (ER) to suitable Asn residues on nascent polypeptides. Subsequently, ER processing glycohydrolases remove the three glucoses and, with the exception of Schizosaccharomyces pombe, a single, specific mannose residue. Processing sugar transferases in the Golgi lead to the formation of core-sized structures (Hex<15GlcNac2) as well as cores with an extended poly-alpha1,6-Man 'backbone' that is derivatized with various carbohydrate side chains in a species-specific manner (Hex50-200GlnNAc2). In some cases these are short alpha1,2-linked Man chains with (Saccharomyces cerevisiae) or without (Pichia pastoris) alpha1,3-Man caps, while in other yeast (S. pombe), the side chains are alpha1,2-linked Gal, some of which are capped with beta-1,3-linked pyruvylated Gal residues. Charged groups are also found in S. cerevisiae and P. pastoris N-glycans in the form of mannose phosphate diesters. Some pathogenic yeast (Candida albicans) add poly-beta1,2-Man extension through a phosphate diester to their N-glycans, which appears involved in virulence. O-Linked glycan synthesis in yeast, unlike in animal cells where it is initiated in the Golgi using nucleotide sugars, begins in the ER by addition of a single mannose from Man-P-dolichol to selected Ser/Thr residues in newly made proteins. Once transported to the Golgi, sugar transferases add one (C. albicans) or more (P. pastoris) alpha1,2-linked mannose that may be capped with one or two alpha1,3-linked mannoses (S. cerevisiae). S. pombe is somewhat unique in that it synthesizes a family of mixed O-glycans with additional alpha1,2-linked Man and alpha1,2- and 1, 3-linked Gal residues.

All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-[(R)-(1-carboxyethylidine)]-Galbeta1,3Galalpha1-.

The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively chargedpyruvylated galactoses present (Gemmill,T.R., and Trimble,R.B., 1996, J. Biol. Chem ., 271, 25945-25949) were attached to the oligosaccharide chains. Binding of biotinylated human serum amyloid P and peanut agglutinin to native and depyruvylated S.pombe glycoproteins, respectively, indicated that the pyruvylated epitope was likely to be in the beta configuration. Examination by high-field 1H NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Gal57Man64GlcNAc N-glycans. The pyruvate-2,(4,6)Gal-beta1,3Gal epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm, P., Scheid,A. and Choppin,P.W. ,1979, J. Biol. Chem ., 254, 9669-9677). The 1:1 stoichiometry between pyruvate and beta-linked galactose in these S.pombe glycans indicates that either pyruvate addition to terminal beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha1,2-linked Gal residues in theN-linked chains. In contradiction to many galactomannan-producing fungi, which add substantial amounts of Gal in the furanose form to their glycoproteins, all detectable Gal in the large S.pombe galactomannans is in the pyranose form, as found in higher eukaryotes. The current work shows that the S.pombe outer chain structure is a poly-alpha1,6Man backbone 2-O-substituted with either Gal or the pyruvylated galactobiose and contains little alpha1,2-linked or 2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain, which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains (Ballou,C.E. ,1990, Methods Enzymol , 185, 440-470).

Schizosaccharomyces pombe produces novel pyruvate-containing N-linked oligosaccharides.

The large N-linked oligosaccharides released by endo-beta-N-acetylglucosaminidase H from Schizosaccharomyces pombe glycoproteins were analyzed for the presence of noncarbohydrate functional groups. No phosphate, sulfate, or acetate could be detected; however, approximately six molecules of pyruvic acid/molecule were found on 98% of the oligosaccharides. Pyruvate moieties were acetal (ketal)-linked to galactose residues in the R configuration to carbons 4 and 6. This is the first report of pyruvate functional groups being attached to N-linked oligosaccharides in yeast and appears only to be the second documentation of this sugar modification in eukaryotes.

Glycoprotein synthesis in yeast. Early events in N-linked oligosaccharide processing in Schizosaccharomyces pombe.

Oligosaccharide-lipid precursors and glycoprotein N-linked oligosaccharides isolated from the fission yeast, Schizosaccharomyces pombe, were compared with those from the budding yeast, Saccharomyces cerevisiae. Bio-Gel P-4 chromatography of oligosaccharide intermediates showed that Glc3Man9GlcNAc2-PP-dol synthesis, transfer of glycan to protein, and glucose removal to yield Man9GlcNAc2 proceeded in S. pombe as in S. cerevisiae. Two series of oligosaccharides were released from S. pombe glycoproteins by endo-beta-N-acetylglucosaminidase H; large "mannan-like" structures and smaller precursor or "core-filling" species. Unexpectedly, the smallest S. pombe N-linked glycan was Man9GlcNAc, confirmed by 500 MHz 1H NMR spectroscopy to be the lipid-linked isomer. No endoplasmic reticulum Man9-alpha 1,2-mannosidase activity was detected in S. pombe, thus identifying Man9GlcNAc as the minimum precursor for oligosaccharide elongation in contrast to the Man8GlcNAc2 intermediate identified in S. cerevisiae (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). S. pombe Hex10GlcNAc was at least four isomers by high pH anion-exchange chromatography with pulsed amperometric detection. Compositional analyses identified two of the major species as GalMan9GlcNAc and GlcMan9GlcNAc, the latter of which suggests that glycan trimming may be attenuated in the S. pombe endoplasmic reticulum. Hex13GlcNAc from S. pombe was homogeneous by mass spectrometry but yielded 12 species by high pH anion-exchange chromatography. Compositional analyses, alpha-galactosidase digestion, and lectin affinity chromatography on Griffonia simplicifolia lectin I-agarose indicated these to be a family of GalxMan13-xGlcNAc isomers (X = 1-4 residues). The absence of Man9GlcNAc2 to Man8GlcNAc2 trimming in S. pombe and elongation of the lipid precursor of Man9GlcNAc with both Man and Gal to form "galactomannans" provides a novel system for N-linked glycoprotein processing studies.