The fission yeast, Schizosaccharomyces pombe, has proven to be a useful model system for studying glycoprotein trafficking and processing (Ziegler et al., 1994). With the exception of the absence of an ER Man9-[alpha]1,2-mannosidase, and therefore the inability to trim Man9GlcNAc2 to Man8GlcNAc2, S.pombe synthesizes and processes lipid-linked and newly-transferred N-glycans in the ER as found in most eukaryotes studied to date. Like Saccharomyces cerevisiae, S.pombe elongates its N-linked oligosaccharides into a series of extended glycans in the Golgi apparatus, but, unlike S.cerevisiae and more like animal cells, S.pombe's Golgi apparatus is morphologically well-defined and contains several galactosyltransferases (Chappell and Warren, 1989), which add Gal to most of its N-linked oligosaccharides (Ziegler et al., 1994; Ballou and Ballou, 1995).
O-linked oligosaccharide synthesis and processing is not well characterized in this organism (Ballou et al., 1994; Ballou and Ballou, 1995). Like all fungi studied to date, S.pombe initiates O-glycan synthesis by the addition of Man to Ser or Thr residues, then elongates this monosaccharide with either Gal or Man to form a series of short oligosaccharide chains. Here we deduce the role of two galactosyltransferases, gma12p (Chappell and Warren, 1989; Chappell et al., 1994) and gth1p (Thomas G. Chappell, personal communication), in S.pombe O-glycan processing by defining the structures of ß-eliminated O-linked oligosaccharides from wild-type and galactosyltransferase-deficient S.pombe glycoproteins. Comparison of O-glycans from gma12, gth1, gma12/gth1 double mutant, and the wild-type strain revealed gma12p and gth1p in concert to be almost entirely responsible for the addition of [alpha]1,2-linked Gal to S.pombe O-linked oligosaccharides and do not appear involved in Gal addition to the outer chains of N-linked oligosaccharides. However, gma12p does appear responsible for addition of terminal [alpha]1,2-linked Gal to N-linked oligosaccharide cores in S.pombe (Ziegler et al., 1998). In addition, we identified a previously unreported Gal[alpha]1,3-linked epitope on disubstituted Man on these O-glycans, implying the presence of a novel 2-O-substituted-Man [alpha]1,3-galactosyltransferase in S.pombe. This [alpha]1,3-linked Gal is also found on a portion of the N-linked Hex10GlcNAc core oligosaccharides formed in S.pombe, suggesting the same enzyme is responsible for both N- and O-linked additions of this novel sugar epitope (Ziegler et al., 1998).
A preliminary report of some of the work in this article has appeared previously (Gemmill and Trimble, 1997).
Approximately 80 mg of glycoprotein was prepared from the wild-type, gma12, gth1, and the gma12/gth1 strains of S.pombe, ß-eliminated and desalted as described in Materials and methods. Negatively charged species were recovered from the QAE-Sephadex column as described previously (Gemmill and Trimble, 1996) and examined by HPAEC analysis. A single negatively charged component was found by HPAEC (not shown), which gave no phenol-sulfuric color, was unhydrolyzable by 2N HCl, and did not coelute with pyruvic acid or known pyruvylated saccharides, and therefore was inferred to be a PAD-active protein-degradation product. Aliquots of neutral oligosaccharide-alditols from all four S.pombe strains were then analyzed by HPAEC-PAD (Figure
Figure 1. HPAEC-PAD profiles of S.pombe O-linked oligosaccharides. O-Glycans released from the wild-type, gma12, gth1, and gma12/gth1 double mutant strains of S.pombe by ß-elimination were chromatographed by HPAEC on the analytical Dionex PA1 column with PAD as described in the text. The individual isomers are labeled as well as the elution position of 4U, a galactosidase product of 5AB, which was not found as a natural product in any of the strains examined.
Figure 2. Bio-Gel P-4 profiles of S.pombe O-linked oligosaccharides. O-glycans from the four S.pombe strains released by ß-elimination were sized by Bio-Gel P-4 chromatography as described in the text. Open circles indicate the number of nanomoles of hexose by phenol-sulfuric acid assay, and (solid circles) indicate the number of nanomoles of mannitol by HPAEC per aliquot.
The wild-type S.pombe O-glycans were pooled into the five (Hex1-5-ol) size classes, and each size class was separated into individual isomers by preparative HPEAC-PAD. The separation pattern on the PA-100 column (not shown) was similar to that shown on the PA1 column (Figure
Table I.
With the exception of 5AB, which required HMQC/HMBC NMR experiments, the remaining Hex2-4-ol pools were examined by 1D proton and 2D DQF COSY NMR spectroscopy with the results shown in Figure
Figure 3. NMR spectra of S.pombe O-glycans. The individual O-glycan isomers purified by HPAEC (Figure 1) were analyzed by 1D proton (top of each panel) and 2D DQF COSY (bottom of each panel) NMR spectroscopy as described in the text. The amounts of each isomer pool in Table I were examined by 1H NMR at 500 MHz in 500 µl D2O.
Figure 4. Permethylation analysis of S.pombe O-glycans. The individual O-glycans and authentic S.cerevisiae Man[alpha]1,2Man-ol (YE) were permethylated, hydrolyzed, developed on silica gel TLC plates and visualized as described in the text. The region where di-O-methylhexoses elute is indicated (dMan), as are the positions of known standards: tMan, 2,3,4,6-tetra-O-methylmannose; tGal, 2,3,4,6-tetra-O-methylgalactose; 2Man, 3,4,6-tri-O-methylmannose.
Figure 5. Glycosidase treatment of S.pombe O-glycans. The purified O-glycans were treated with jack bean [alpha]-mannosidase (M[prime]ase) and coffee bean [alpha]-galactosidase (G[prime]ase), as described in Materials and methods, followed by HPAEC-PAD using the analytical PA1 column. Monosaccharides
Mannitol was the only monosaccharide alditol found on ß-elimination of S.pombe glycoproteins by HPAEC/PAD analysis of the Hex1-ol pool using an MA1 column. This, combined with the finding that mannitol was the only monosaccharide alditol found on analysis of the Hex2-5-ol isomers, indicates that mannose is the only saccharide directly O-linked to proteins in S.pombe. Disaccharides
The 2D DQF COSY spectra of 2A (Figure
The NMR spectra for 2B (Figure Trisaccharides
The 1D proton spectra of the 3AB pool (Figure
Table II. Tetrasaccharides
The NMR spectra of 4A (Figure
Figure 6. NMR analysis of the Hex5-ol isomer. The Hex5-ol isomer was purified as described in the text and examined by NMR; some of the data is summarized in Table II. Labels are as described in Table II and Figure 8: Mx, 2-substituted Man; Md, 2,3-disubstituted Man; Ga, 2-linked Gal; Gb, 3-linked Gal; Mol, mannitol. Numbers after a residue, e.g., Md2, indicate the ring carbon proton location, in this case a di-substituted Man's C2-H. The atom numbers of the individual mannitol signals were not labeled. Correlations between atoms in each spectra are indicated by hyphens. (A) details the 1D proton NMR spectrum. (B) shows a slice of the 2D DQF COSY experiment. (C) shows a slice of the 2D TOCSY experiment. (D) shows a 2D ROESY experiment, with fortuitous TOCSY signals indicated by T. (E) shows a composite of an HMBC experiment (inside gray boxes) overlaid onto an HMQC experiment (outside of gray boxes).
NMR analysis of 4B (Figure Pentasaccharide
The 5A and 5B peaks from the preparative PA1 column coeluted when rechromatographed on the PA1 analytical column and had the same molecular weight by mass spectrometry (Table I). They had identical permethylation patterns (Figure
Figure
The HMQC experiment displays the resonance of each proton along the x-axis and the resonance of the carbon it is attached to along the y-axis. The type of HMBC experiment performed here, in essence, shows connectivities between the protons on carbons involved in glycosidic linkages (x-axis) and the carbon on the other side of the glycosidic bond (y-axis). The experiments in Figure Role of gma12p and gth1p in N-glycan modeling
To determine the effects of the gma12 and gth1 mutations on the production of large N-linked galactomannans, the Bio-Gel P-4 V0 glycan fraction from all four S.pombe strains was subjected to 1D proton NMR analysis (Figure
Figure 7. NMR analysis of S.pombe N-linked galactomannans from all four S.pombe strains. The large galactomannans purified from the S.pombe strains as described in the text were analyzed by 600 MHz 1D proton NMR spectroscopy. Selected signals are assigned as described elsewhere (Ziegler et al., 1998). The resonances of Gal's C1 proton (TG1), the 2,6-disubstituted Man backbone C1-H (DM1), The 3-substituted Gal C4 proton (3G4), the pyruvate CH3 protons (Pv), and the pyruvylated ß-Gal C1 (ßG1) and C4 (ßG4) protons are labeled.
In agreement with earlier work (Ballou and Ballou, 1995), mannose was the only sugar detectable in O-glycan linkage to protein, Gal[alpha]1,2Man-ol (2A) and Man[alpha]1,2Man-ol (2B) were the only Hex2-ol components, while a Gal[alpha]1,2Man[alpha]1,2Man-ol species (3A) was present in S.pombe. However, our study found no evidence for substituted Gal, by either NMR or permethylation analyses, in any of the O-glycans isolated. This stands in contrast to the tetrasaccharide species reported elsewhere as Gal[alpha]1,2Gal[alpha]1,2Man[alpha]1,2Man-ol (Ballou and Ballou, 1995), which had the same anomeric proton resonances as seen here for isomer 4A (Figure
Figure 8. Proposed pathway for O-glycosylation in S.pombe. Residues are labeled as in Table II and Figure 6: Mt, nonreducing terminal Man; Mx, 2-substituted Man; Md, 2,3-disubstituted Man; Ga, 2-linked Gal; Gb, 3-linked Gal; Mol, Mannitol. Arrows up, [alpha]1,2-linkage. Diagonal arrows, [alpha]1,3-linkage. Forward reaction arrows indicate the deduced processing pathway, with the enzymes involved indicated: Gma, gma12p; Gth/Gma, either gma12p or gth1p; Gtm, the unknown enzyme(s) which adds Gal to the 3-position of 2-O-substituted Man. Left reaction arrows indicate products obtained after digestion with either jack bean [alpha]-mannosidase (M[prime]ase) or coffee bean [alpha]-galactosidase (G[prime]ase) as described in Materials and methods. Structure 3U represents a theoretical glycosidase digestion product, and structure 4U represents a presumed biosynthetic O-linked intermediate not found on S.pombe glycoproteins in this current work.
In comparing the O-linked glycans from all the S.pombe strains (Figure
Although examination of large galactmannans from gma12, gth1, gma12/gth1, and wild-type S.pombe showed no clear-cut role for these encoded galactosyltransferases in their synthesis, gma12p has been shown to add [alpha]1,2-linked Gal to the Man9GlcNAc2 precursor to generate novel GalMan9GlcNAc2 core N-glycans (Ziegler et al., 1998). Additionally, the presence of branching Gal[alpha]1,3Man epitope on O-linked isomers 4A, 4B, and 5AB indicates the presence of an [alpha]1,3-galactosyltransferase activity previously unreported in S.pombe. The failure to find here any O-linked isomers with a lone [alpha]1,3-linked Gal terminus in the oligosaccharide structures, leads to the speculation that this galactosyltransferase introduces an [alpha]1,3-branch on an [alpha]1,2-linked mannose already 2-O-substituted by either Gal or Man. Interestingly, this enzyme also may be responsible for addition of an [alpha]1,3-linked Gal to the N-linked Man9GlcNAc2 core in S.pombe to form a GalMan9GlcNAc2 isomer in which the lower [alpha]1,3-linked arm has the structure Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1- (Ziegler et al., 1998) as found in O-linked isomer 4B (Figure Materials
The gma12-deficient TCP3001 (h-,leu1-32, ura4-D12, his3-327, gma12-D10::ura4), gth1-deficient TCP3017 (h-,leu1-32, ura4-D12, his3-327, ade 1-D25, ade6-M210, gth1-D9::ura4), and gma12/gth1-deficient TCP3019 (h-,leu1-32, ura4-D12, his3-327, ade 1-D25, ade6-M210, gth1-D9::ura4, gma12-D10::ura4) strains of S.pombe were gifts of Dr. Thomas G.Chappell of The MRC Laboratory for Molecular and Cell Biology, London, England. The wild-type (gma12+/gth1+) S.pombe strain, 972 (h-), was from the American Type Culture Collection (ATCC #24843).
QAE-Sephadex and Sephadex G-50 were from Pharmacia Biotech, Piscataway, NJ. Bio-Gel P-4 and Bio-Gel P-2 gel filtration resins were from Bio-Rad Laboratories, Hercules, CA. All solvents were reagent grade or the purest grade available from commercial sources. Endo H was a cloned Escherichia coli product from this laboratory (Trumbly et al., 1985). PNGase F was a kind gift of Drs. Plummer and Tarentino of the Wadsworth Center. Coffee bean [alpha]-galactosidase was from Boehringer Mannheim Corp., Indianapolis, IN, and jack bean [alpha]-mannosidase was purified as described previously (Li and Li, 1972). Sep-Pak C18 disposable cartridges were obtained from Millipore Corp., Bedford, MA. Man[alpha]1,2Man-ol was isolated from S.cerevisiae by ß-elimination and characterized by 1H NMR and permethylation analysis (Cohen and Ballou, 1980). Galß1,3Galß1-OCH3, Man[alpha]1,(2,3 and 6)Man methyl glycosides and 99.96% 2H2O were from Sigma Chemical Corp., St. Louis, MO. Cambridge Isotopes Lab Inc., Woburn, MA, supplied the 99.996% 2H2O. Monosaccharide standards and the MA1, PA1, and PA-100 HPAEC columns were procured from Dionex Corp., Sunnyvale, CA. V-Labs inc. of Covington, LA provided the 2[prime]-fucosyllactose and the Gal[alpha]1,3Galß1,4Gal[alpha]1,3Gal.
Cells were grown in YEA medium at 32°C, and whole-cell glycoproteins were extracted and purified as described previously (Ziegler et al., 1994; Gemmill and Trimble, 1996). N-glycans were removed from the glycoproteins as described elsewhere (Gemmill and Trimble, 1996) by sequential treatment with endo H and PNGase F. The endo H-released oligosaccharides were recovered and purified by Bio-Gel P-4 chromatography. Little oligosaccharide was removed by the PNGase F treatment, and it was assumed that this residual glycan was resistant to endo H because of inaccessibility due to protein conformation.
The residual glycoprotein was washed with 50% methanol three times to remove any remaining soluble carbohydrate, then dried and dissolved in 40 ml 0.1 M NaOH containing 1 M NaBH4 and allowed to stand 16 h at 45°C. The solutions were acidified by dropwise addition of acetic acid. Cations were removed by passage through two 20 ml columns of Dowex 50 H+. Borate was removed by three rotary evaporations with 1% acetic acid in methanol. The samples were then passaged through 5 ml QAE-Sephadex carbonate columns as described previously (Gemmill and Trimble, 1996).
Jack bean [alpha]-mannosidase and coffee bean [alpha]-galactosidase digestions were performed on 10 nmol of oligosaccharide in 100 µl PCR tubes for 3 days at 37°C in 50 µl of 20 mM sodium acetate containing 2 mM CaCl2 at pH 4.5 and 6.5, respectively. The reactions were stopped by the addition of 450 µl of methanol and centrifuged to remove precipitated protein. The supernatants were evaporated to dryness under vacuum and resuspended in 100 µl 90% methanol. The samples were again centrifuged to remove insoluble protein and the supernatants evaporated under vacuum, resuspended in 50 µl water, and analyzed directly by HPAEC-PAD as described below. The unusual glycosidase buffer described here was used to allow analysis of small amounts of analytes without risking possible loss due to the ion exchange columns normally used to remove protein and buffer salts. The enzymes were at least 80% as active with their respective PNP-substrates under these conditions when compared with standard buffering conditions, and they retained at least 50% of their original activity after 3 days at 37°C.
HPAEC-PAD oligosaccharide chromatography was performed on a Dionex system using one of two methods. Analytical determinations were performed using a 4 × 250 mm PA1 column at 1 ml/min using 20 mM NaOH as the eluant. Preparative chromatography was performed using a 10 × 250 mm PA-100 column with the same conditions, except at a flow rate of 2 ml/min. In both cases PAD settings were E1 = 0.05, E2 = 0.7, E3 = -0.15; T1 = 4, T2 = 3, T3 = 6 with attenuation set at either 1000 or 3000 nA full scale, depending on sample load.
Samples were permethylated by the NaOH method (Ciucanu and Karek, 1984) and hydrolyzed in 4M CF3CO2H for 4 h at 100°C. The resulting partially methylated monosaccharides were separated by TLC and visualized as described previously (Mukerjea et al., 1996).
Samples were prepared as described previously (Ziegler et al., 1994) and examined on a 500 MHz Bruker Avance spectrometer at 300 K using acetone as an internal reference ([delta], ppm = 2.225). The 1D proton and 2D DQF COSY experiments were performed as described previously (Ziegler et al., 1998). The HMQC experiment, which detects coupling between protons and the carbons they are bonded to, was performed using a JCH of 145 Hz. The HMBC experiment, which detects coupling between the C1-H protons and the carbons attached by a glycosidic linkage, was performed using a JCH of 145 Hz and a 3JCH of 14.8 Hz. The carbon dimensions in both the HMBC and HMQC experiments were referenced to the spectrometer frequency and not to an internal chemical shift marker.
Bio-Gel P-4 chromatography methods have been described previously (Ziegler et al., 1994). Neutral hexose quantitation was by a modification (Byrd et al., 1982) of the phenol-sulfuric acid method (Dubois et al., 1956).
We thank Dr. John Cavanagh, Ms. Lynn McNaughton, and Dr. Susan Baxter of the Wadsworth Structural NMR Facility for help with the NMR data acquisition and Ms. Cathy Lubowski for preparing a portion of the S.cerevisiae Man[alpha]1,2Man-ol. We are indebted to Dr. Charles Hauer and Mr. Robert Stack of the Wadsworth Biological Mass Spectrometry Laboratory for providing the MALDI/TOF mass spectra. We gratefully acknowledge the Wadsworth Center photography unit for help with the illustrations and Ms. Tracy Godfrey for final manuscript preparation. This work was supported, in part, by USPHS Grant GM-23900 to R.B.T.
ER, endoplasmic reticulum; Endo H, endo-ß-N-acetylglucosaminidase H (E.C. 3.2.1.96); PNGase F, peptide-N4-asparagine amidohydrolase F (E.C. 3.2.2.52); HPAEC, high pH anion exchange chromatography; PAD, Pulsed amperometric detection; TLC, thin-layer chromatography; MALDI/TOF, matrix assisted laser desorption ionization time of flight; MS, mass spectrometry; Man-ol, mannitol; HexN-ol, HexN-1Hexitol; Galf, galactofuranose; DQF, double quantum filtered; HMBC, heteronuclear multiple bond connectivity; HMQC, heteronuclear multiple quantum coherence.
3To whom correspondence should be addressed at: Wadsworth Center, C-547, N.Y.S. Department of Health, P.O. Box 509, Albany, NY 12201-0509
Pool
Mr (Da)
Amount obtained (µMol)
Isomera
Proposed structure
2A
344.1
18
2A
Gal[alpha]1,2Man-ol
2B
343.7
12
2B
Man[alpha]1,2Man-ol
3
505.8
10
3A
3BGal[alpha]1,2Man[alpha]1,2Man-ol
Man[alpha]1,2Man[alpha]1,2Man-ol
4A
668.2
65
4A
Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol
4B
668.6
26
4B
Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol
5A
5B831.6
829.64.8
2.45AB
Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,2Man-ol
Pool
Ringa
Sugar
(ppm) (Hz)
(ppm)
C1-H (J1,2)b
C2-H
C3-H
C4-H
C5-H
2A
Ga
Gal[alpha]1,2->
5.11 (4.0)
3.83
3.89
4.02
4.13
2B
Mt
Man[alpha]1,2->
5.02 (4.7)
4.01
3.88
3.81
3A
Mx
->2Man[alpha]1,2->
5.36 (4.9)
4.02
3.92
3.83
Ga
Gal[alpha]1,2->
5.19 (3.8)
3.83
3.93
4.03
4.13
3B
Mx
->2Man[alpha]1,2->
5.27 (4.9)
4.03
3.71
Mt
Man[alpha]1,2->
5.07 (4.8)
4.09
3.86
4A
Md
->2,3Man[alpha]1,2->
5.33 (4.8)
4.16
4.11
3.98
3.87
Ga
Gal[alpha]1,2->
5.25 (4.1)
3.81
3.93
3.99
4.11
Gb
Gal[alpha]1,3->
5.28 (2.9)
3.87
4B
Md
->2,3Man[alpha]1,2->
5.24 (4.9)
4.21
4.11
3.95
3.88
Mt
Man[alpha]1,2->
5.21 (4.7)
4.04
3.87
3.85
3.87
Gb
Gal[alpha]1,3->
5.28 (3.5)
3.87
5AB
Md
->2,3Man[alpha]1,2->
5.43 (4.9)
4.23
4.09
3.97
3.87
Mx
->2Man[alpha]1,2->
5.26 (4.9)
3.99
3.98
3.75
3.83
Ga
Gal[alpha]1,2->
5.25 (4.0)
3.82
3.93
4.02
4.10
Gb
Gal[alpha]1,3->
5.31 (3.1)
3.85
3.89
4.02
4.14
Discussion
Materials and methods
Cell growth, glycoprotein preparation, and N-deglycosylation
O-Glycan preparation
Glycosidase reactions
HPAEC-PAD oligosaccharide chromatography
Permethylation analysis
NMR methods
Other methods
Acknowledgments
Abbreviations
References
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