Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Received on December 3, 2003; revised on March 16, 2004; accepted on March 18, 2004
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Abstract |
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Key words: cellobiohydrolase I (Cel7A) / isoforms / N-glycosylation / phosphodiester linkages / Trichoderma reesei
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Introduction |
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In an attempt to explain these nonconclusive data, effects of growth conditions on the glycosylation of Cel7A from T. reesei Rut-C30 were investigated (Stals et al., 2004). An array of hydrolytic activities, present in the extracellular media and optimally active at pH 56, alter the initially complex N- and O-glycosylation pattern of Cel7A. In minimal growth conditions, the medium acidifies, and fully glycosylated and phosphorylated proteins are found (Garcia et al., 2001; Harrison et al., 2002
; Maras et al., 1997
; Pakula et al., 2000
). Rich cultivations maintain the initial pH and postsecretorial enzymatic trimming of N-glycosylation by an endoglycosidase H (Endo H)type activity and of O-glycosylation by a mannosidase and a phosphatase is mainly responsible for variations in glycosylation patterns (Harrison et al., 1998
; Hui et al., 2001
, 2002
; Klarskov et al., 1997
).
High cellulase-producing mutants of T. reesei, obtained by several rounds of random mutagenesis and screening (Figure 1), are used in biotechnological applications. As such, unknown alterations in their protein glycosylation machinery may have occurred. The high-yielding Rut-C30 mutant strain was initially isolated on the basis of its catabolite repression resistance (Montenecourt and Eveleigh, 1979) and exhibits an elevated content of endoplasmatic reticulum (Ghosh et al., 1982
). This strain and RL-P37 were shown to be hyperproducers of cellulase (Sheir-Neiss and Montenecourt, 1984
). They were both isolated employing a 2-deoxyglucose selection system, an antimetabolite that has been shown to interfere with glycosylation (Hubbard and Ivatt, 1981
).
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Results |
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Complex patterns of isoforms were found in each case (Figure 2), contrasting with the single band observed for N-deglycosylated core protein (isoelectric point [IP] 3.6). Heterogeneous modifications at the three glycosylation sites probably account for the presence of compounds with more acidic IPs. Four bands are detected for core Cel7A isolated from the Rut-C30 strain, whereas an additional more acidic form can be discriminated for the QM6A and QM9414 strains. In each case the most abundant isoform coincides with deglycosylated protein.
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Further structural evidence is obtained by Q-Trap ESI-MS in the positive ion mode. The masses of the neutral oligosaccharides from Rut-C30 and RL-P37 Cel7A correspond to those of the reported structures (data in Table II). The mass of the main glycan m/z 1054.3 Da obtained from the other strains is consistent with Hex5GlcNAc, but also masses corresponding to Hex678GlcNAc present in minor amounts are detected (data in Table III).
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FACE analysis allows further differentiation of several compounds. The glycans isolated from Rut-C30 Cel7A (Figure 5A, lane 4) appear in the order d', c', b', c''. The electrophoretic patterns of the neutral and the charged fractions are strikingly similar (Figure 5A, lanes 3 and 4): c' and d' are the charged (mannophosphorylated) counterparts of c and d (apparent shift of two residues). The presence of a minor amount of c'' is due to acid hydrolysis of the phosphodiester linkage (use of trifluoracetic acid [TFA] in the fractionation step). -(1
2)-Mannosidase hydrolyzes only compound b' and an apparent loss of two mannose residues (lanes 4 and 8) is observed. Comparing lanes 3 and 4 (Figure 5B), molecular weight structures higher than Man5GlcNAc2 are expected for the charged fraction from QM9414. Because the presence of a phosphodiester bond equals a shift of two sugar residues (see previous discussion), the following structures can be proposed: ManPMan678GlcNAc2. The N-glycans of Cel7A from the wild-type and the two VTT strains give similar profiles as those from QM9414 (data not shown).
Q-Trap ESI-MS in the negative ion mode confirms the chromatographic and electrophoretic analyses. The presence of charged structures other than the fully characterized compound (c') in Rut-C30 Cel7A (Maras et al., 1997) is proven: Three major doubly charged ions at m/z values of 899.2, 980.2, and 1061.2 correspond to PHex8910GlcNAc2 (Figure 7A, upper spectrum, and Table II). Minor fractions with masses consistent with PHex67GlcNAc2 and PHex11121314GlcNAc2 are also evident. The distribution of charged N-glycans from QM9414 Cel7A is more complex (Figure 7B, upper spectrum, and Table III). The masses of the four major singly charged ions m/z 1110.3, 1272.3, 1434.2, and 1596.3 are consistent with phosphorylated structures, such as PHex5678GlcNAc (circled in Figure 7B). Moreover, six doubly charged components are detected at the m/z values 756.8, 837.9, 918.8, 999.9, 1080.9, and 1162.3. The latter could correspond to structures containing two phosphate residues: P2Hex789101112GlcNAc (boxed in Figure 7B).
Further analyses of charged glycans
Mild acid hydrolysis removes the terminal mannose in a phosphodiester linkage and subsequent alkaline phosphatase digestion is used to release the ensuing phosphate (Thieme and Ballou, 1971). The products formed in both steps were analyzed by FACE (Figure 5A and B) and HPAEC-PAD (Figure 6A and B); in combination with results of ESI-MS measurements (Figure 7A and B), structural deductions could be made.
Mild acid hydrolysis apparently does not affect the retention times of compounds on HPAEC-PAD (Figure 6A and B, chromatograms 2), but the liberation of mannose can clearly be demonstrated. With glycans derived from QM6A and QM9414 Cel7A, several products are formed, including one that corresponds to mannobiose. FACE analysis (Figure 5A and B, lanes 5) shows faster-migrating (higher-charged) compounds, probably indicating removal of mannosyl residues and/or formation of terminal phosphoryl groups. Remarkably, all glycans from the QM9414 strain seem to be converted to one compound, suggesting the formation of terminal phosphate by losses of several mannosyl residues.
Subsequent treatment with alkaline phosphatase results in the complete disappearance of charged compounds and in the formation of their uncharged counterparts: mainly GlcMan7GlcNAc and GlcMan8GlcNAc for the Rut-C30 strain (Figure 6A, chromatogram 3, and Figure 5A, lane 6) and Man5GlcNAc and Man6GlcNAc for the other strains (Figure 6B, chromatogram 3, and Figure 5B, lane 6).
Corroborative evidence is gathered from Q-Trap ESI-MS analyses (in the negative mode) of the acid-treated samples (lower spectra in Figure 7A and B). A loss of one mannosyl residue (162 Da) is expected after hydrolysis of a phosphodiester bond. For the charged Rut-C30 Cel7A glycans, one major doubly charged species (m/z 899.22) is detected as the hydrolysis product of compound c' (m/z 980.22) (Table II). Other glycans, detected in the original sample, possibly representing (ManP)GlcMan8GlcNAc2 at m/z 1061.22 and (ManP)Man7GlcNAc2 at m/z 899.22 may, on mild acid treatment, result in the formation of PGlcMan8GlcNAc2 (m/z 980.22) and PMan7GlcNAc2 (m/z 818.72).
Mild acid treatment of the charged QM9414 Cel7A glycans (Figure 7B) leads to the accumulation of one major singly charged m/z 1110.41 and two new doubly charged species: m/z 594.72 and 675.82. Hydrolysis of the major compound (ManP)Man5GlcNAc at m/z 1272.31 indeed yields PMan5GlcNAc at m/z 1110.41. The considerable amount of m/z (1434.2)1, corresponding to either (ManP)Man6GlcNAc or (Man2P)Man5GlcNAc, is probably also converted to the same component PMan5GlcNAc at m/z 1110.41, because only a small amount of PMan6GlcNAc m/z (1272.4)1 can be detected after acid hydrolysis. This is further proof for the occurrence of terminal mannobiosyl residues: (Man2P)Man56GlcNAc.
From structures putatively substituted with two phosphodiester bonds (boxed in Figure 7B), liberation of two hexose residues (324 Da) are expected after acid hydrolysis. Indeed, the major doubly charged N-glycan (ManP)2Man6GlcNAc at m/z 837.92 yields a new product P2Man6GlcNAc with m/z 675.8). Again, the higher-molecular-weight structures P2Hex9101112GlcNAc at m/z 918.82, 999.92, 1080.92, and 1162.32all seem to be hydrolyzed mainly to the same P2Man56GlcNAc core structure, suggesting that the diester bond originally carried extra mannose residues.
Repartition of N-glycans over the glycosylation sites
The previously characterized (Maras et al., 1997) and presently proposed glycan structures from both the Rut-C30 and QM9414 strains (as represented in Tables II and III) can be assigned to the three Asn sites of core Cel7A (Table IV). In the case of core Cel7A from the Rut-C30 strain (Figure 3A), the mass of the major glycoform (51,078 Da) corresponds to core protein (45,968 Da, as calculated from the amino acid composition) substituted with compound c at the three N-glycosylation sites (3 x 1703 Da; total 51,079 Da). The presence at one site of a glycan carrying a phosphodiester bond (compound c') is indicated by the 51,317-Da fraction. The 51,561 and 51,804 Da species correspond to core protein carrying two and three mannophosphoryl groups, respectively. The predominant glycoforms (49,627, 50,045, 50,445, and 50,849 Da) for QM9414 Cel7A (Figure 3B) represent core proteins substituted with a combination of GlcNAc2Man5 (1217 Da) and GlcNAc2Man7P structures (1621 Da) at the three N-glycosylation sites.
The random repartition of neutral and charged oligosacharides gives rise to four IPs as observed for Rut-C30 Cel7A (Figure 2, lane 1). The IP of proteins substituted with three uncharged N-glycans coincides with that of N-deglycosylated protein. Mannophosphodiester groups substituted at one, two, or three glycosylation sites account for the more acidic Cel7A forms. The presence of a fifth IP for QM9414 Cel7A (Figure 2, lanes 3 and 5) is probably the result of the occurrence of glycans with two mannophosphodiester linkages. In a separate experiment, the different Cel7A bands were isolated and identified by ESI-MS (spectra not shown, Table IV).
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Discussion |
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All data obtained with the uncharged N-glycans of Cel7A from both the high producing Rut-C30 and RL-P37 strains point to 1-3 glucosylated structures (GlcMan78GlcNAc2) (Maras et al., 1997
) (Table II). For Cel7A isolated from the other strains, this fraction consists of Man56GlcNAc2 (Table III).
Several charged structures other than the fully characterized compound (ManP)GlcMan7GlcNAc2 (Maras et al., 1997) consist of the mannophosphodiester counterparts of the uncharged species in Rut-C30 Cel7A (Table II). The composition of the charged N-glycan species of proteins isolated from the QM9123 lineage is even more complex (Table III). At least seven major negatively charged compounds are detected, corresponding to structures carrying either one or two phosphodiester bonds. As observed with yeast cell wall mannoproteins (Jigami and Odani, 1999
), alternative substitutions on the
1-3 branch of Man6GlcNAc are proposed. Moreover, the phosphodiester bonds can be substituted by oligomannosyl residues. The presence of such a residue was proven by mild acid hydrolysis and subsequent chromatrographic, electrophoretic, and mass spectrometric analyses.
The characterized N-glycans can be assigned to the three glycosylation sites of Cel7A core, and this accounts for the different glyco-/isoforms detected by MS and electrophoresis (Table IV). For the Rut-C30 strain, the major glycoform corresponds to core protein substituted with three neutral glycans (GlcMan7GlcNAc2), but fractions carrying the charged compound ManPGlcMan7GlcNAc2 on one, two, or three Asn sites are also detected. This random distribution explains the detection of four IPs for core Cel7A. For the other strains, substitution of three Man5-GlcNAc2 structures results in a major glycoform, and the presence of mono- and biphosphorylated glycans at the three different sites accounts for the other more acidic isoforms.
Our study thus clearly shows that the synthesis of phosphodiester bonds is not atypical for Trichoderma sp., and the proposed structures (Table III) resemble the mannophosphorylated species observed in yeast cell walls (Jigami and Odani, 1999; Karson et al., 1977
). Yeast mannosidase can only hydrolyze one
-(1
2)-mannosyl linkage (middle arm) (Herscovics, 1999
), whereas the fungal enzyme can splice all
-(1
2)-mannosyl residues (Maras et al., 2000
). This explains the presence of extra
(1
2)-mannobiosyl linkages in the yeast structure. The glucose end-capped
1-3 branch, as observed for Cel7A from the Rut-C30 and RL-P37 strains, hinders a possible second phosphomannosyl transfer. Therefore glycans carrying two mannophosphoryl groups are detected only with the wild-type QM6A and strains derived from QM9123. Up to now, no mannophosphoryl transferase could be demonstrated in Trichoderma, but the activity and specificity has been studied thoroughly in yeast (Karson and Ballou, 1978
).
The ratio of uncharged versus singly and doubly phosphorylated N-glycans is probably determined by the localisation and the activity of -(1
2)-mannosidase and a phosphomannosyltransferase; both enzymes compete for the Man
1-2Man residues (Karson and Ballou, 1978
; Maras et al., 2000
). Because the majority of the N-glycan population in T. reesei Cel7A consists of Man5GlcNAc2 and only a minor amount becomes doubly phosphorylated, the
-(1
2)-mannosidase apparently trims the N-glycans efficiently. T. reesei
-(1
2)-mannosidase has no endoplasmic reticulum (ER) retention signal and its precise localisation is unknown (Callewaert et al., 2001
), whereas the putative phosphomannosyl transferase seems to reside in the Golgi (Pakula et al., 2000
). A lower turnover of
-(1
2)-mannosidase for one of the four
-(1
2)-linkages in the Man9GlcNAc2 intermediate may, however, favour a mannophosphoryl transfer.
Deglucosylation by glucosidase I and II (Helenius, 1994) of Glc3Man9GlcNAc2 on newly synthesized glycoproteins and reglucosylation by UDP-Glc:glycoprotein glucosyltransferase (Parodi, 1999
) participate in the ER folding pathway that involves the chaperones calnexin and calreticulin. The reactions are, however, transient, and the oligosaccharides are finally deglucosylated and further processed after the protein has acquired its tertiary and quaternary structure (Hebert et al., 1995
).
Although it is rare to find monoglucosylated oligosaccharides in mature glycoproteins, such glycans were found on the cell surface of Leishmani sp. (Funk et al., 1997). The authors concluded that this structure was present when organisms lack glucosidase II or have a low level of glucosidase II activity. Storage proteins more often carry monoglucosylated glycans, as detected in arylphorin of silkworms (Kim et al., 2003
), hen albumin (Ohta et al., 1991
), and the major glycoprotein present in the egg jelly coat of starfish (De Waard et al., 1987
). Glc1Man9GlcNAc2 also makes up some 2040% of the total glycans in
-mannosidase extracted from jack bean storage tissue (Kimura et al., 1999
). All these data suggest a major role for the glycan Glc1Man9GlcNAc2.
Our study shows the presence of partially processed glucosylated oligosaccharides (Glc1Man78GlcNAc2) on secreted cellulases from the filamentous fungi T. reesei (Maras et al., 1997). Remarkably, only glycoproteins isolated from the high-producing Rut-C30 and RL-P37 mutants carry these structures. The wild-type fungus and other strains originating from a different mutation branch (Figure 1) seem to follow the normal ER glycosylation pathway and show fully trimmed Man56GlcNAc2 structures. An inefficient ER-glucosidase II in the hyperproducing Rutgers strains may explain the presence of monoglucosylated N-glycans. Both mutants are derived from a common parent using a 2-deoxyglucose selection procedure. With Neurospora crassa, the resistance to 2-deoxyglucose is caused by several mutations involved in glucose transport or glucose metabolism (Allen et al., 1989
). By selecting T. reesei strains that are less sensitive to catabolite repression of cellulase expression, they could have selected for glycosylation mutants. Interestingly, they secrete approximately three to five times more total cellulase activity than the QM9414 or the wild-type strain QM6A (Sheir-Neiss and Montenecourt, 1984
). Moreover, morphological differences have been observed for the Rut-C30 strain (swollen ER and wide internal cisternal space filled with amorphous material) (Ghosh et al., 1982
), suggesting enhanced intracellular protein synthesis. This was attributed to a mutation(s) inactivating a regulatory function controlling protein synthesis or secretion (Ghosh et al., 1984
). Retention of monoglucosylated glycoproteins in the ER until saturation of lectin-like chaperones may explain the high ER content and can be related to the high secretion capacity observed.
These observations and the results obtained in our comparative study merit further investigation into the effects of the different selection procedures that have led to the hyperproducing T. reesei strains and their secretory pathways.
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Materials and methods |
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Purification of Cel7A and preparation of the catalytic core domain was identical as described in the companion paper (Stals et al., 2004).
Preparation of N-glycans
Enzymatic deglycosylation
To 1 mg Cel7A, denatured in 1% sodium dodecyl sulfate, 0.5 M mercaptoethanol and 0.1 M ethylenediamine tetra-acetic acid, 1 U PNGase F or Endo H is added after neutralization of sodium dodecyl sulfate with TX-100. The digestion proceeds for 17 h at 37°C in 200 mM sodium phosphate, pH 8.5 (Jackson, 1994) or 100 mM sodium acetate, pH 5; the resulting solution is desalted/treated by carbon chromatography as described shortly.
Clean-up and fractionation of charged and neutral oligosaccharides
The clean-up of the N-glycans is conducted according to a procedure described by Packer et al. (1998). The carbograph solid phase extraction column (with 150 mg nonporous graphitized carbon) can adsorb up to 40 mg oligosaccharides (Alltech Associates, Inc., Lokeren, Belgium) and is regenerated with 80% CH3CN0.1%TFA and rinsed extensively with water. After deglycosylation, the reaction mixture is loaded on the solid phase extraction column and subsequently rinsed with water to elute monosaccharides, salt, detergents, and buffer components. Oligosaccharides are subsequently eluted with 2 ml 25% CH3CN0.05% TFA. After evaporation, the residue is dissolved in 0.5 ml water. To fractionate uncharged from charged components, 2 ml 25% CH3CN is added to elute neutral sugars, whereafter the charged (phosphorylated, sulfated, or sialylated) components are eluted by the addition of 2 ml 25% CH3CN0.05% TFA. This procedure, as tested with a standard mixture (Man, Glu, cellotetraose, mannose-1-phosphate, and mannose-6-phosphate) on HPAEC-PAD, was found to be very efficient with yields of nearly 100%. After evaporation, the N-glycans (
60 pmol) were dissolved in 200 µl water. The water used was always of Milli-Q purity.
Analysis of N-glycans
HPAEC-PAD
An integrated HPAEC-PAD system comprising a GP40 gradient pump, a LC30 chromatography oven, and an ED40 electrochemical detector (equipped with a gold working electrode) was used (Dionex NV, Wommelgem, Belgium). Samples of 20 µl were injected using a Gilson auto-injector. Chromatographic data are analysed using Dionex Peaknet software.
Monosaccharide analysis
The enzymatically released N-glycans (20 µl, 6 pmol) are hydrolysed in Teflon-capped tubes (1 ml 4 M TFA, 100°C, 4 h). The acid is subsequently removed by evaporation, and the resulting monosaccharides are analyzed as their oxyanions at alkaline pH on a CarboPac PA-10 pellicular anion-exchange column. Isocratic elution (1 ml/min) uses 16 mM NaOH solution; in between injections the column was washed with 200 mM NaOH. In a test mixture, monosaccharides were separated in the following order: Fuc, GalNAc, GlcNAc, Gal, Glc, Man.
Oligosaccharide analysis. Neutral and charged N-glycans.
(20 µl, 6 pmol) were separated at 1 ml/min on a CarboPac PA-100 column (40°C) using the following gradient program: a 060 mM sodium acetate gradient in 100 mM sodium hydroxide for 35 min (neutral oligosaccharides) and a 60500 mM sodium acetate gradient for another 35 min (charged oligosaccharides)
Fluorescent labeling and electrophoresis of N-glycans.
Derivatization of the N-glycans with the fluorophore 8-amine-1,3,6-naphtalene-trisulfonic acid was performed according to the method of Jackson (1994). The derivatized oligosaccharides were precipitated with ice-cold acetone and kept frozen as a stock solution in water. Before loading samples (5 µl, 12 pmol) on a FACE oligosaccharide profiling gel, they were mixed with a 50% glycerol solution containing bromofenol. Fractionation of derivatized PNGase F/Endo H released glycans is obtained on a 25%/30% gel (AA:Bis, 1:37.5). On each gel, a derivatized dextran ladder and the high-mannose oligosaccharides of RNase B (12 pmol) are loaded as a reference. Gels are run on ice with a Protean III apparatus (Bio-Rad, Nazareth Eke, Belgium) with ice-cold running buffer for 15 min at 120 mV and 90 min at 200 mV. Photographic images can be taken directly through the assembly glass plates after irradiation by 520 nm light on a photoluminager (Boehringer Mannheim, Brussels, Belgium)
ESI-MS
Mass spectra were acquired on a Applied Biosystems Q-Trap, equipped with a nanospray source (needle voltage:±1000 V). The samples were dissolved in a 50% MeOH solution. Fractionated neutral and charged oligosaccharides are measured in the positive mode and negative mode, respectively. The mass range of the system is m/z 501700. Thus the charged glycans from the Rut-C30 strain cannot be measured as singly charged ions.
Enzymatic treatments
Alkaline phosphatase treatment
It is reported that mannosylphosphate moieties can be removed by mild acid hydrolysis, followed by alkaline phosphatase digestion (Thieme and Ballou, 1971).
To the mixture of charged oligosaccharides before and after mild acid treatment (0.02 N HCl, 100°C, 30 min), 1 U calf intestine alkaline phosphatase (Sigma-Aldrich, Bornem, Belgium) in 20 µl 100 mM TrisHCl, pH 8.8, containing 10 mM ZnCl2 was added. The reaction was allowed to proceed overnight and followed both by FACE and HPAEC-PAD as described. Reaction mixtures were dried before loading with glycerol on a 25% polyacrylamide gel. For PAD detection, the buffer concentration cannot exceed 10 mM, the reaction mixtures are therefore diluted with water before injection.
-Mannosidase treatment
N-glycans (of Cel7A and RNase B) are treated with jack bean -(1
2,3,6)-mannosidase (Sigma-Aldrich) and T. reesei
-(1
2)-mannosidase (Maras et al., 2000
). One unit of mannosidase is added to the oligosaccharides dissolved in 20 mM sodium acetate buffer, pH 5 (containing 2 mM ZnCl2 in the case of
-specific mannosidase). The reaction is allowed to proceed overnight at room temperature. Hydrolysis is evaluated both by FACE and HPAEC-PAD, as described.
Analysis of glycoproteins
ESI-MS and PAG-IEF data are gathered as described in the companion paper (Stals et al., 2004).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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Callewaert, N., Laroy, W., Cadirgi, H., Geysens, S., Saelens, X., Min Jou, W., and Contreras, R. (2001) Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris. FEBS Lett., 503, 173178.[CrossRef][ISI][Medline]
De Waard, P., Kamerling, J.P., Van Halbeek, H., Vliegenthart, J.F.G., and Broertjes, J.J.S. (1987) Characterization of N-linked gluco-oligomannose type of carbohydrate chains of glycoproteins from the ovary of the starfish Asterias rubens. Eur. J. Biochem., 168, 679685.[Abstract]
Funk, V.A, Thomas-Oates, J.E., Kielland, S.L., Bates, P.A., and Olafson, R.W. (1997) A unique, terminally glucosylated oligosaccharide is a common feature on Leishmania cell surfaces. Mol. Biochem. Parasitol., 84, 3348.[CrossRef][ISI][Medline]
Garcia, R., Cremata, J.A., Quintero, O., Montesino, R., Benkestock, K., and Ståhlberg, J. (2001) Characterization of protein glycoforms with N-linked neutral and phosphorylated oligosaccharides: studies on the glycosylation of endoglucanase 1 (Cel7B) from Trichoderma reesei. Biotechn. Appl. Biochem., 33, 141152.[CrossRef][ISI]
Ghosh, A., Alrabiai, S., Ghosh, B.K., Triminovazquez, H., Eveleigh, D.E., and Montenecourt, B.S. (1982) Increased endoplasmic reticulum content of a mutant of Trichoderma reesei (Rut-C30) in relation to cellulase synthesis. Enzyme Microb. Technol., 4, 110113.[CrossRef][ISI]
Ghosh, A., Ghosh, B.K., Triminovazquez, H., Eveleigh, D.E., and Montenecourt, B.S. (1984) Cellulase secretion from a hypercellulolytic mutant of Trichoderma reesei Rut-C30. Arch. Microbiol., 140, 126133.[ISI]
Harrison, M.J., Nouwens, A.S., Jardine, D.R., Zachara, N.E., Gooley, A.A., Nevalainen, H., and Packer, N.H. (1998) Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei. Eur. J. Biochem., 256, 119127.[Abstract]
Harrison, M.J., Wathugala, I.M., Tenkanen, M., Packer, N.H., and Nevalainen, K.M.H. (2002) Glycosylation of acetylxylan esterase from Trichoderma reesei. Glycobiology, 12, 291298.
Hebert, D.N., Foellmer, B., and Helenius, A. (1995) Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell, 81, 425433.[ISI][Medline]
Helenius, A. (1994) How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol. Biol. Cell., 5, 253265.[ISI][Medline]
Herscovics, A. (1999) Processing glycosidases of Saccharomyces cerevisiae. Biochim. Biophys. ActaGen. Subj., 1426, 275285.[CrossRef]
Hubbard, S.C. and Ivatt, R.J. (1981) Synthesis and processing of asparagine-linked oligosaccharides. Ann. Rev. Biochem., 50, 555583.[CrossRef][ISI][Medline]
Hui, J.P.M., Lanthier, P., White, T.C., McHugh, S.G., Yaguchi, M., Roy, R., and Thibault, P. (2001) Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry. J. Chrom. B, 752, 349368.[ISI]
Hui, J.P.M., White, T.C., and Thibault, P. (2002) Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei. Glycobiology, 12, 837849.
Jackson, P. (1994) High-resolution polyacrylamide-gel electrophoresis of fluorophore-labeled reducing saccharides. Guide Techn. Glycobiol., 230, 250265.[ISI]
Jigami, Y. and Odani, T. (1999) Mannosylphosphate transfer to yeast mannan. Biochim. Biophys. ActaGen. Subj., 1426, 335345.[CrossRef]
Karson, E.M. and Ballou, C.E. (1978) Biosynthesis of yeast mannanproperties of a mannosylphosphate transferase in Saccharomyces cerevisiae. J. Biol. Chem., 253, 64846492.[Abstract]
Karson, E.M., Ballou, D.L., and Ballou, C.E. (1977) Mannosylphosphate transferase in Saccharomyces cerevisiae mannoprotein biosynthesis. J. Supramol. Struct., 17, 17.
Kim, S., Hwang, K., Dwek, R.A., Rudd, P.A., Ahn, Y., Kim, E., Cheong, C., Kim, S., Park, N., and Lee, S. (2003) Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein. Glycobiology, 13, 147157.
Kimura, Y., Hess, D., and Sturm, A. (1999) The N-glycans of jack bean alpha-mannosidasestructure, topology and function. Eur. J. Biochem., 264, 168175.
Klarskov, K., Piens, K., Stahlberg, J., Hoj, P.B., Van Beeumen, J., and Claeyssens, M. (1997) Cellobiohydrolase I from Trichoderma reesei: identification of an active-site nucleophile and additional information on sequence including the glycosylation pattern of the core protein. Carbohydr. Res., 304, 143154.[CrossRef][ISI][Medline]
Maras, M., De Bruyn, A., Schraml, J., Herdewijn, P., Claeyssens, M., Fiers, W., and Contreras, R. (1997) Structural characterisation of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei Rut-C30. Eur. J. Biochem., 245, 617625.[Abstract]
Maras, M., Callewaert, N., Piens, K., Claeyssens, M., Martinet, W., Dewaele, S., Contreras, H., Dewerte, I., Penttila, M., and Contreras, R. (2000) Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase. J. Biotechnol., 77, 255263.[CrossRef][ISI][Medline]
Montenecourt, B.S. and Eveleigh, D.E. (1979) Selective screening for the isolation of high yielding cellulase mutants of T. reesei. Adv. Chem. Ser., 181, 289301.
Ohta, M., Hamako, J., Yamamoto, S., Hatta, H., Kim, M., Yamamoto, T., Oka, S., Mizuochi, T., and Matsuura, F. (1991) Structures of asparagine-linked oligosaccharides from hen egg-yolk antibodyoccurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein. Glycoconj. J., 8, 400413.[ISI][Medline]
Packer, N.H., Lawson, M.A., Jardine, D.R., and Redmond, J.W. (1998) A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J., 15, 737747.[CrossRef][ISI][Medline]
Pakula, T.M., Uusitalo, J., Saloheimo, M., Salonen, K., Aarts, R.J., and Penttila, M. (2000) Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein. Microbiology, 146, 223232.
Parodi, A.J. (1999) Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells. Biochim. Biophys. ActaGen. Subj., 1426, 287295.[CrossRef]
Salovuori, I., Makarow, M., Rauvala, H., Knowles, J., and Kaariainen, L. (1987) Low-molecular-weight high-mannose type glycans in a secreted protein of the filamentous fungus Trichoderma reesei. Bio/Technology, 5, 152156.[ISI]
Sheir-Neiss, G. and Montenecourt, B.S. (1984) Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations. Appl. Microb. Biotechn., 20, 4653.[ISI]
Stals, I., Sandra, K., Geysens, S., Contreras, R., Van Beeumen, J., and Claeyssens, M. (2003) Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the N- and O- glycosylation pattern of Cel7A. Glycobiology, this issue.
Thieme, T.R. and Ballou, C.E. (1971) Nature of the phosphodiester linkage of the phosphomannan from the yeast Kloeckera brevis. Biochemistry, 10, 41214129.[ISI][Medline]
Tomme, P., McCrae, S., Wood, T.M., and Claeyssens, M. (1988) Chromatographic separation of cellulolytic enzymes. Meth. Enzymol., 160, 187193.[ISI]
van Tilbeurgh, H., Tomme, P., Claeyssens, M., Bhikhabhai, R., and Pettersson, G. (1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reeseiseparation of functional domains. FEBS Lett., 204, 223227.[CrossRef][ISI]
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