The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Galbeta1,3- (PvGal) caps on a portion of the Galalpha1,2- residues in their outer chains (Gemmill, T. R., and Trimble, R. B. (1998) Glycobiology 8, 1087-1095). PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S. pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues. Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy. Restoration of the cell surface negative charge by complementation with an S. pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1-pvg5. Pvg1p may be a pyruvyltransferase, since NMR of pvg1(-) mutant N-glycans revealed the absence of only the pyruvyl moiety. Pvg2p-Pvg5p are crucial for attachment of the Galbeta1,3- residue that becomes pyruvylated. Pvg3p is predicted to be a member of the beta1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization. Predicted Pvg1p and Pvg3p functions imply that Galbeta1,3- is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGalbeta1,3- caps to the outer chain. Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADS-box DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes. Pvg2p and/or Pvg5p may contribute to an enzyme complex. Whereas a functional role for the PvGal epitope in S. pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions.

Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.

The Saccharomyces cerevisiae alg12Delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12Delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12Delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9Delta invertase was more severely hypoglycosylated than the alg12Delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12Delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9Delta invertase is underglycosylated in comparison to the alg12Delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12Delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.

N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man(5)GlcNAc(2)-PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man(3)GlcNAc(2)-PP-dolichol and Man(4)GlcNAc(2)-PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm alpha1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif.

A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-β-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N4-(N-acetyl-β-glucosaminyl) Asn amidase (EC 3.5.1.52)-released complex, and β-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at −20°C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.

This communication describes the use of two-dimensional relayed (TOCSY)-ROESY experiments for the rapid and selective identification of α/β1,2-glycosidic linkages in polysaccharides. The method assists in the identification of cross-peaks in crowded regions of ROESY spectra by moving them to less congested areas. In addition, the appearance of the spectra provides information relating the location of the glycosidic linkage within the sequence of the glycan under study. Selection of solely the 1,2- linkages is achieved by appropriately tuning the duration of the TOCSY mixing period. The method is demonstrated both theoretically and experimentally for a variety of test case polysaccharides.

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.

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.

Schizosaccharomyces pombe synthesizes very large N-linked galactomannans, which are elongated from the Man9GlcNAc2 core that remains after the trimming of three Glc residues from the Glc3Man9GlcNAc2 originally transferred from dolichyl pyrophosphate to nascent proteins in the endoplasmic reticulum. Prior to elongation of the galactomannan outer chain, the Man9GlcNAc2 core is modified into a family of Hex10-15GlcNAc2 structures by the addition of both Gal and Man residues (Ziegler et al. (1994) J. Biol. Chem., 269, 12527-12535). To understand the pathway of Man9GlcNAc2 modification, the Hex10GlcNAc-sized pool was isolated by Bio-Gel P-4 gel filtration from the endo H-released N-glycans of S.pombe glycoproteins. This pool yielded four major fractions, a, b, c, and g, on preparative high pH, anion exchange chromatography, that represented 10, 29, 46, and 13% of the total Hex10GlcNAc present, respectively. Structures of the glycan isomers present in each fraction were determined by one- and two-dimensional 1H NMR spectroscopy techniques. Fraction a is principally (approximately 93%) a Man10GlcNAc with a new alpha1,2-linked Man cap on the upper-arm of Man9GlcNAc. Fraction b contained two isomers of GalMan9GlcNAc in which an alpha1,2-linked terminal Gal had been added either to the upper (b1, 30%) or middle-arm (b2, 70%) of Man9GlcNAc. The gma12 - alpha1,2-galactosyltransferase-negative S. pombe strain (Chappell et al. (1994) Mol. Biol. Cell., 5, 519-528) did not make fraction b implying that the gma12p galactosyltransferase is responsible for synthesis of both isomers b1 and b2. Isomer c is Man10GlcNAc in which a new branching alpha1, 6-linked Man had been added to the lower-arm alpha1,3-linked core residue as found earlier in Saccharomyces cerevisiae and Pichia pastoris. Fraction g had less than molar stoichiometry of both Gal and Glc. The major isomer (g1, 85%) is the Man9GlcNAc core with an alpha1,3-linked branching Gal on the penultimate 2-O-substituted Man of the lower arm. This residue is also found on a novel O-linked oligosaccharide recently described in S.pombe; Manalpha1,2(Galalpha1, 3)Manalpha1,2Mannitol (Gemmill and Trimble (1999) Glycobiology, 9, 507-515). The second isomer (g2, 15%) is the partially processed Glc2Man9GlcNAc intermediate. Defining these Hex10GlcNAc structures provides a starting point for understanding the enzymology of N-linked galactomannan core heterogeneity seen on S.pombe glycoproteins.

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.

The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively charged pyruvylated 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 H-1 NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta 1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Ga(57)Man(64)GlcNAc N-glycans. The pyruvate-2,(4,6)Gal-beta 1,3Gal epitope is chemically similar to acetaldehyde-Gal beta 1,3Gal 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 beta 1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha 1,2-linked Gal residues in the N-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-alpha 1,6Man backbone 2-O-substituted with either Gal or the pyruvylated galactobiose and contains little aid-linked or 2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain, which is poly-alpha 1,6Man substituted with alpha 1,2-linked Man sidechains (Ballou,C.E.,1990, Methods Enzymol, 185, 440-470).

The large N-linked oligosaccharides released by endo-β-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 Nlinked oligosaccharides in yeast and appears only to be the second documentation of this sugar modification in eukaryotes.