Glycosylation of acetylxylan esterase from Trichoderma reesei

Mathew J. Harrison3, Indira M. Wathugala3, Maija Tenkanen1,4, Nicolle H. Packer2,5 and K.M. Helena Nevalainen3

3Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; 4VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044, VTT Finland; and 5Proteome Systems Ltd, Locked Bag 2073, North Ryde, NSW 1670, Australia

Received on November 1, 2001; revised on January 17, 2002; accepted on January 24, 2002.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The nature of the N- and O- linked glycosylation of acetylxylan esterase (AXE) of the Trichoderma reesei strain Rut-C30 has been characterized using different enzymatic, chromatographic, and mass spectrometric techniques. The combined data showed that the AXE N-glycan is phosphorylated and highly mannosylated. The predominant N-glycans on the single glycosylation site on AXE can be represented as GlcNAc2Man(1–6)P. The linker–substrate binding domain peptide separated from the core by papain digestion is heavily O-glycosylated and consists of mannose, galactose, and possibly glucose as monosaccharide and disaccharide substituents. In addition to glycosylation, sulfation was observed in the linker region. Both N- and O- linked glycans show remarkable heterogeneity. Three isoforms of AXE, separated by 2D SDS–PAGE, are described with pI values of 5.0, 5.3, and 5.9. The three isoforms can be explained by posttranslational modification of the enzyme by glycans, phosphate, and sulfate. Advancing the knowledge on the nature of the glycans produced by T. reesei is elementary for its use as a host for the expression of heterologous glycoproteins of industrial and pharmaceutical importance.

Key words: acetylxylan esterase/glycosylation/hemicellulase/isoforms/Trichoderma reesei


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Xylans are the major hemicelluloses in the cell walls of angiosperms and gymnosperms, modified by acetylation and methylation. Acetylation has been shown to increase the solubility, digestibility, biodegradability, and enzymatic hydrolysis of plant cell wall material (Matsuo and Mizuno, 1974Go; Morris and Bacon, 1977Go; Wood and McCrae, 1986Go; Grohmann et al., 1989Go; Poutanen et al., 1987Go; Poutanen and Puls, 1988Go). Several esterases acting on different acetylated side chains have been purified from various microorganisms (reviewed in Christov and Prior, 1993Go). Acetylxylan esterase (AXE) secreted by the filamentous fungus Trichoderma reesei is capable of liberating acetic acid from polymeric acetyl xylan (acetylated hemicellulose) constituents in plants (Tenkanen, 1998Go). The gene encoding this enzyme has been isolated from T. reesei Rut-C30 by Margolles-Clark et al. (1996)Go. T. reesei AXE occurs in several isomeric forms with similar apparent molecular mass but differing pI values (Poutanen et al., 1990Go; Poutanen and Sundberg, 1988Go; Sundberg and Poutanen, 1991Go). The apparent molecular mass of all isoforms obtained by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is approximately 34 kDa, which is somewhat higher than that of the expected theoretical mass of amino acids only (27.45 kDa) (Poutanen et al., 1990Go; Poutanen and Sundberg, 1988Go; Sundberg and Poutanen, 1991Go). The amino acid sequence deduced from the axe1 gene of T. reesei Rut-C30 shows that the enzyme comprises a globular catalytic domain and cellulose (substrate) binding domain (SBD), spatially separated by a proline and hyroxy-amino acid–rich linker region. This linker region is analogous in structure to the highly O-glycosylated linker regions found between the catalytic domains and SBDs of cellulases of T. reesei and Aspergillus awamori (Van Tilbeurgh et al., 1986Go; Neustroev et al., 1993Go).

Even though most fungal hydrolases are glycoproteins of considerable biotechnical interest, limited information is available on the sites, type, and composition of glycosylation on these enzymes. In general, the fungal N-linked glycan core has shown to be identical to the mammalian N-linked core (Man3GlcNAc2). However, the occurrence of single N-acetyl-glucosamine reported on the main cellobiohydrolase I (CBHI) of T. reesei ALKO2877 and QM9414 (Harrison et al., 1998Go; Klarskov et al., 1997Go) suggests that strains of T. reesei N-glycosylate CBHI differently. Structural characterization and studies into the effects of N-linked carbohydrate chains of different enzymes, such as {alpha}-galactosidase (Savel'ev et al., 1997Go) and cellobiohydrolase I from T. reesei (Maras et al., 1997Go), glucoamylase from A. awamori X 100/D27 (Eriksen et al., 1998Go), and {alpha}-amylase from A. awamori (Chen et al., 1994Go) have been carried out. The importance of N-linked glycosylation for secretion or stability of extracellular enzymes from filamentous fungi appears to differ between fungi (Neustroev et al., 1993Go; Eriksen et al., 1998Go; Chen et al., 1994Go).

O-linked glycans of glucoamylase from A. awamori (Neustroev et al., 1993Go), Aspergillus niger (Gunnarsson et al., 1984Go) and CBHI of T. reesei (Harrison et al., 1998Go) include di- and trisaccharides containing terminal glucose, mannose, and galactose. In CBHI from T. reesei, a glucose residue has been found to be directly linked to the polypeptide chain (Gum and Brown, 1976Go). Analysis of stability changes following decreased glycosylation of A. awamori linker region (Neustroev et al., 1993Go) suggests that O-linked sugars essentially contribute to the stabilization of glucoamylase. Furthermore, the linker glycopeptide seems to stabilize the binding domain against reversible thermal and chemical denaturation (Williamson et al., 1992Go). With respect to the diversity of sugar residues present in the glycoproteins, filamentous fungi bear greater similarity to mammalian cells than yeast, which typically hypermannosylate. Apart from O-and N-linked glycosylation, further structural diversification may occur by covalent attachment of phosphate, sulfate, acetyl, or methyl groups to the sugar (reviewed in Lis and Sharon, 1993Go).

Previous partial characterization of AXE of T. reesei RutC-30 has shown that the enzyme is modified by both N- and O-linked sugars comprising up to 12–15% by weight of the molecule (Margolles-Clark et al., 1996Go; Sundberg and Poutanen, 1991Go). However, the exact nature of the glycosylation of the acetyl xylan esterase of T. reesei has not been resolved. Characterization of the N- and O-linked carbohydrates of AXE presented in this article will contribute to the understanding of the effect of posttranslational modifications on fungal hydrolytic enzymes. Composition, positional information of O-linked sugars on the peptide, nature of oligosaccharide heterogeneity, and modifications of glycosylation of the purified glycoprotein AXE from T. reesei Rut-C 30 will be discussed. In a wider perspective, in the production of heterologous glycoproteins in filamentous fungi, especially those of therapeutic importance, a good knowledge of the nature of the glycans produced by the host is elementary.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Preparation of core and linker-SBD peptides from native AXE by papain digestion
AXE of T. reesei was digested into the catalytic core and linker-SBD glycopeptides by limited proteolysis with high concentrations of papain, a cysteine protease with broad substrate specificity (10:1 substrate:papain by weight); peptides were analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). From the calculated mass of the known amino acid sequence it is likely that the peak at about 8.4 kDa (Figure 1A) is the linker-SBD glycopeptide with the core observed as both singly and doubly charged forms (about 16 kDa and 32 kDa). Papain was also observed and correlated with its predicted mass of approximately 24 kDa (Figure 1A). The mass of the linker-SBD peptide (Figure 1B) was seen to exist as isoforms in the range of 7.9–8.8 kDa.



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Fig. 1. MALDI-TOF MS spectra of papain-digested AXE. The top spectrum (A) shows the full-scan spectrum for a typical digest of AXE with papain. The AXE domains globular core and linker-SBD glycopeptide are indicated by a (hypothetical) graphic of AXE. Papain cleavage site and mass arising from papain are also shown. The lower spectrum (B) is a magnification of the linker-SBD region as indicated by the boxed area in the upper spectrum. Masses of AXE regions are summarized in Table I. The mass differences of 80Da and 162 Da corresponding to sulfate and hexose additions, respectively, are also shown.

 
The digestion occurred rapidly and was approximately 90% complete after 2 h. Liquid chromatography MS (LC-MS) of this digestion resolved two distinct species at 8.4 and 9.8 min (Figure 2A), which gave molecular masses consistent with identity of the first peak as the linker-SBD glycopeptide and the latter peak as a mixture of the linker-SBD glycopeptide and the catalytic core peptide. Also present in this fraction were masses consistent with the intact mass of the protease papain (~24 kDa), and the intact glycosylated mass of AXE (~31–33 kDa) (Figure 2B) Fractions were collected from these chromatographic peaks to give a linker-SBD fraction and an impure core fraction. Subsequent rechromatography of the core fraction over longer and more shallow gradients or over size-exclusion columns did not sufficiently resolve the core peptide (approximately 23 kDa) from papain or the intact AXE protein to allow the purification of core fraction. As the proportion of the core peptide exceeded that of the intact AXE by a factor of approximately 10:1 based on the observed mass intensities, the mixed fraction was used as such for qualitative experiments and the subsequent results interpreted accordingly.



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Fig. 2. Reversed-phase LC-ESI-MS of a typical papain digest after 2 h. (A) A total ion chromatogram; the proposed elution positions of the linker glycopeptide and catalytic core peptide are indicated. Mass data from spectra derived from this chromatogram was used to calculate the mass data presented in Table I. (B) A combined spectrum of the 7.5–12.5 min region of the chromatogram in (A); the identities and charge states of peaks are indicated.

 
MS of linker-SBD peptide
Rechromatography of the purified linker peptide on the reversed phase column over a more shallow high-performance liquid chromatography (HPLC) gradient, and subsequent analysis of peak fractions by MALDI-TOF MS showed that the linker-SBD peptide could be fractionated further into two peaks (Figure 3A). Mass isoforms, which corresponded to differences in the number of attached hexoses (i.e., multiples of 162.05 Da), were observed for the linker-SBD peptide (Figure 3B) in the first peak. The same mass pattern was obtained from the second peak in which the linker-SBD peptide also exhibited a second mass isoform series which was 80 Da greater than each of the 162 Da isoforms (Figure 3C). The mass difference of +80 Da suggested the possibility of a population of phosphorylated (+79.966 Da monoisotopic) or sulfated (+79.957 Da monoisotopic) isoforms interposed with the distribution of 162 Da (hexose) modifications. Table I summarizes the mass data and assigned compositions of the peptides produced by papain digestion of AXE.



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Fig. 3. Rechromatography of the purified AXE linker-SBD peptide by HPLC (A) and analysis of the peak fractions by MALDI-TOF MS. (B) A MALDI -TOF spectrum of the earlier-eluting peak. (C) A MALDI-TOF spectrum of the later-eluting peak. Each of the peaks in (C) is 80 Da greater in mass than the peaks in (B). As in Figure 1B the 162 Da mass difference series shown is due to hexose heterogeneity.

 

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Table I. Electrospray mass spectral data for intact and papain-digested AXE from T. reesei
 
Site glycosylation of linker-SBD peptide
Edman degradation using the GlycoSite sequenator (Gooley and Williams, 1997Go), which identifies glycoamino acids in a protein sequence, was applied for the characterization of the purified linker-SBD peptide (Gly215–Thr252) of AXE, which comprises 11 (6 Thr, 5 Ser) possible O-glycosylation sites. The region was sequenced and glycosylation sites assigned (Figure 4). The region Arg244–Thr252, made up of two additional threonines and serines, could not be reliably assigned due to poor repetitive yield and accumulated carryover from the cluster of glyco-amino acids interspersed with proline in the previous sequence. Of the region sequenced, all four threonines and all three serines were fully glycosylated with predominantly a disaccharide but also some monosaccharide. There was no evidence of unglycosylated serines or threonines. It was difficult to quantitate the microheterogeneity at each site because of the high degree of intercycle carryover contamination in sequencing through these highly glycosylated regions.



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Fig. 4. Glycosylation sites of AXE, as determined by Edman degradation. The AXE linker peptide could not be fully sequenced because of excessive lag induced by the proline- and glycoamino acid–rich region at the start of this sequence. The part of the sequence that was sequenced is underlined. Within this sequenced part, all threonines and serines were fully glycosylated by at least one hexose or two hexoses (diamonds).

 
Characterization of glycans
Monosaccharide analyses of the core protein was calculated as 3.2 moles of glucosamine (the hydrolysis product of N-acetylglucosamine), 0.9 moles of galactose, 2.9 moles of glucose, and 23.2 moles of mannose per mole of protein (Figure 5A). The purified linker-SBD contained 0.9 moles galactose, 4.5 moles glucose, and 16.3 moles mannose per mole of peptide (Figure 5B). No glucosamine was detected in the linker-SBD.



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Fig. 5. Monosaccharide composition of AXE by HPAEC. (A) The core peptide. Sample of the core peptide used for this result contained some intact AXE protein. (B) Purified linker-SBD peptide. The elution positions of monosaccharide standards is superimposed on both chromatograms.

 
Ion chromatography was used as an auxiliary technique to discriminate between phosphorylation and/or sulfation of the core and linker peptides summarized in Table I. As ion chromatography is a fairly insensitive technique, it was necessary to use a large amount (>=1 nmol) of the desalted linker peptide and AXE native protein. Hydrolysis of the native AXE protein produced both sulfate and phosphate (Figure 6A), whereas the linker-SBD peptide produced only sulfate (Figure 6B). It is therefore reasonable to conclude that the linker contains sulfation. By comparison of the peak heights of sulfated and unsulfated forms in HPLC and MS, sulfation occurs on close to 50% of peptides. By subtraction, the phosphate is inferred to be located on the AXE core peptide. Phosphoamino acid analysis performed according to the method reported by Yan et al. (1996)Go showed no evidence of phosphoamino acids (data not shown).



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Fig. 6. Ion chromatography of the acid hydrolysates of AXE. (A) Intact AXE protein, showing strong peaks for both sulfate and phosphate ions. (B) The AXE linker-SBD glycopeptide, showing only a sulfate peak.

 
Treatment of AXE with the N-glycan-specific endoglycosidase peptide-N-glycosidase F (PNGaseF) resulted in a discernible difference in apparent molecular weight on 1D SDS–PAGE (data not shown), implying the presence of N-glycosylation. The released N-glycans were desalted and their mass determined by graphitized carbon LC-MS (Harrison and Packer, 2000Go). The mass data revealed a range of singly charged species from approximately 600–1500 Da, at 162-Da intervals (Figure 7). This mass series corresponds with a high degree of accuracy to the mass of a N-linked, high-mannose composition series HexNAc2Hex[1–6] H2PO4Na+.



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Fig. 7. Graphitized carbon LC-ESI-MS of PNGaseF-released N-linked oligosaccharides of AXE. A single combined spectrum of the sugar-containing region eluting between 37.9 and 38.5 min is shown, together with the inferred identities of major peaks. Note that spectra obtained at high cone voltages produced greater signal/noise, consistent with positive ion ESI-MS of phosphorylated sugars; a side effect of this high voltage is some fragmentation. At lower cone voltage, only the series from (c)–(f) is seen.

 
Gel separation of charged isoforms
Two-dimensional PAGE of AXE in this study consistently produced three distinct isoforms, differing in pI only (Figure 8A). When 2D PAGE of a typical papain digest of AXE was performed, only two pI isoforms of approximately pI 4.5 and 4.9 were observed for the core peptide, and the linker-SBD peptide also produced two isoforms of approximate pI 4.4 and 5.4 (Figure 8B).



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Fig. 8. 2D PAGE of AXE and AXE digests. (A) Native AXE, showing pI isoforms of pH 4.90, 5.25, and 5.98 of equal apparent mass. (B) A 4-h (complete) papain digest of AXE. The inferred identity of spots is indicated on a magnified area of the gel pH range.

 
The observation of two pI isoforms of each of the core and linker-SBD peptides on 2D gels together implies that each of these peptide regions is heterogeneous for at least one charged modification (Figure 8B). One can speculate that the two clear spots of each papain cleavage product on 2D PAGE may be explained by the absence or presence of a single sulfation of O-linked sugars on the linker-SBD peptide and the absence or presence of a single phosphorylation on the core peptide N-glycans. The mixture of these possibilities on the whole native protein would agree with the observation of the three charged isoforms on the 2D PAGE gel (Figure 8A).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Filamentous fungi are an important source of industrially applied glycosylhydrolases. Though glycosylation is a recurrent feature of this family of proteins, there is little known about the structures and biological functions of such glycosylation or of glycosylation in filamentous fungi in general. In this study, we describe the characterization of the posttranslational modifications of one such glycosylhydrolase, AXE, from T. reesei strain Rut-C30.

Papain digestion of AXE produced two peptides, amino acids pyrGlu1–Gly215, which constitute the catalytic core domain, and amino acids Gly216–Leu271, constituting the linker and SBDs. The linker-SBD peptide was found to be O-glycosylated with hexose residues and modified by sulfate. Rather than simply releasing the O-glycans and obtaining average structural information, the density of O-glycosylation on the linker peptide was determined using a modified Edman amino acid sequencing technique. This approach showed not only the sites that were occupied but also that each theonine and serine was heterogeneously glycosylated with at least one or two hexoses. Although evidence of the attachment site of sulfation is unknown, we presume that it is most likely attached to one of the O-glycans present in the linker region by extrapolation to the CBHI glycopeptide linker. This peptide is sulfated in the T. reesei strain ALKO2877 but possesses no free hydroxy-amino acids or tyrosine within the linker to accommodate a sulfate group (Harrison et al., 1998Go). All of the oligosaccharide masses of N-linked glycans liberated by PNGaseF treatment corresponded to the oligosaccharide composition HexNAc2Hex3–7PNa, which describes a typical N-linked core of HexNAc2Hex3, plus up to four additional hexoses and phosphate.

Monosaccharide analyses of the whole intact AXE protein revealed the presence of mannose, galactose, glucose, and N-acetlyglucosamine. Analysis of the linker-SBD peptide in isolation revealed only mannose, galactose, and glucose in a ratio supporting the view that N-acetylglucosamine derives from the sole N-glycan attached to the core peptide. It was not possible to derive a monosaccharide composition for the core peptide, due to the coelution of the core peptide and native AXE over reversed-phase C8 and gel-filtration columns. The observed average ratio of sugars for the linker-SBD peptide in monosaccharide analysis was 16.9:1 (moles of sugar per mole of peptide, excluding 4.4 moles of glucose) and is consistent with the observation that the most intense isoform of the linker peptide possessed 16 hexoses. It is difficult to verify the true presence of glucose in hydrolysis-based monosaccharide analyses becaus glucose is often viewed as a contaminant. Others (Maras et al., 1997Go; Gunnarsson et al., 1984Go; Takayanagi et al., 1992Go) have confirmed the presence of N- and O-linked glucose on Aspergillus and Trichoderma glycoproteins by several techniques, including nuclear magnetic resonance, and given the high amount of glucose observed in this report, we believe that glucose is likely to be also present on AXE. The presence of mannose, galactose, and probably glucose on the AXE linker is in agreement with that observed by us for another glycosylhydrolase, T. reesei CBHI (Nevalainen et al., 1997Go; Harrison et al., 1998Go).

A previous report on the characterization of the AXE glycoprotein observed two protein activities with pIs of 6.8 and 7.0 by gel electrophoresis (Poutanen et al., 1990Go), compared to the three more acidic spots of pI 4.90, 5.25, and 5.98 observed in this report. Discrepancy between our results may be due to the nature of carrier ampholytes used in the previous report or difference in cultivation conditions, which may have an effect on the number of AXE forms detected. Given that the theoretical pI value of the published sequence of AXE obtained from SWISS-PROT (www.expasy.ch/sprot) is 5.56, and that the presence of sulfation and/or sulfation decrease the pI, it is probable that the most basic spot of 5.98 indeed represents the native AXE protein sequence without further modification. The three pI isomers of AXE observed in this report on 2D gels are consistent with our identification of two heterogeneous, charged modifications. Assuming that the overall contribution of phosphorylation and sulfation to the (decrease in the) pI of the full AXE protein is approximately equal within the effective pH resolution limits of the 2D gels used, and that the presence or absence of N-glycan phosphorylation occurs independently of the presence or absence of linker sulfation, it would be expected that AXE would resolve into three spots. From these, the center spot, comprising a mixture of protein molecules modified either by phosphorylation or sulfation, would be the most intense.

Two-dimensional PAGE of a complete papain digestion of AXE produced a pI isoform doublet of approximately 25 kDa apparent molecular mass, corresponding in mass to the catalytic core peptide; a second pI doublet of about 13 kDa apparent molecular mass, corresponding in mass to the linker peptide; and a fifth spot of about 22 kDa, corresponding to the apparent molecular mass of the protease papain. The observation of two spots for each of the linker-SBD and core peptides is consistent with the identification of partial sulfation of the linker and phosphorylation of the N-linked glycan on the core peptide. The large separation of the linker peptide spots in the charge dimension is likely due to the very large effect of the presence/absence of sulfation on such a small peptide because of its accordingly low buffering capacity.

Sulfation on a linker peptide and presence of phosphorylated N-glycans has been reported for T. reesei CBHI (Nevalainen et al., 1997Go; Harrison et al., 1998Go). In the absence of further information concerning the biological/enzymatic differences between the differently modified enzymes in the different strains of T. reesei, the biological significance of these modifications is unclear. However, both modifications (phosphorylated N-glycans and sulfated O-glycans) have been implicated to some extent in organisms such as Leishmania and Dictyostelium in protein targeting and secretion (Haynes, 1998Go). Sulfated carbohydrates have also been shown to play specific roles in well-defined biological processes, such as control of the circulatory half-life of lutenizing hormone, symbiotic interactions between leguminous plants and nitrogen-fixing bacteria, and homing of lymphocytes to lymph nodes (reviewed in Hooper et al., 1996Go). Therefore, it is possible that the addition of a sulfate moiety turns a relatively common structure into a unique carbohydrate with the potential to be recognized by a specific receptor molecule. We have shown that the linker region of the filamentous fungal glycosylhydrolase AXE is heavily and heterogeneously O-glycosylated and possesses an unusual charged modification, sulfate. It is not unreasonable to presume that these perform some as-yet-unidentified biologically significant function.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Enzymes and reagents
Immobilized pH gradient (IPG) strips were from Pharmacia (Uppsala, Sweden). Tributyl phosphine (97%) and 3-[(3-cholamidopropyl) dimethyl-amino]-1-propanesulfonate (CHAPS), were from Fluka (Buchs, Switzerland). Tris, SDS, urea, glycine, acrylamide, Ready Gels, carrier ampholytes, colloidal Coomassie Blue G-250, and Coomassie and low-molecular-weight protein markers were from Bio-Rad (Hercules, CA). Glycerol was from BDH (Poole, UK). Acetonitrile and trifluoroacetic acid (TFA) were from Hewlett Packard (Böblingen, Germany). Water was MilliQ grade (Millipore), papain was from Boehringer Mannheim GmbH (Germany). AXE from T. reesei Rut-C30 was purified as reported in Sundberg and Poutanen (1991)Go.

Proteolytic digestion by papain
Papain digestion of AXE has been previously reported by Margolles-Clark et al. (1996)Go. A slightly different protocol was used in the present study. Two hundred micrograms of AXE in 200 µl of 100 mM ammonium acetate, pH 8.0, was incubated with 8 µl of papain (1.4 mg/ml in 0.2 M phosphate buffer, 5 mM L-cysteine, 2 mM ethylenediamine tetra-acetic acid, pH 7.0) at 37°C for 120 min. A previous analytical scale experiment performed over a time-course of 30, 60, 120, and 240 min revealed that digestion of AXE into core and linker-SBD peptides was essentially complete at 120 min. Digested material was analyzed by MALDI-TOF and LC-MS. Analysis of core and linker peptides from this stock were performed by LC-MS (with a 1:20 split of the postcolumn flow to the MS), or by HPLC using the same LC method as described.

PNGaseF deglycosylation of AXE
PNGaseF digests followed the PNGase-F (Bio-Rad) denaturing protocol advised by the manufacturer. Twenty-five microliters of a solution of 0.6 mg/ml AXE (15 µg) in 100 mM ammonium bicarbonate, pH 8.0, was denatured by boiling for 5 min with 25 µl of 2x reaction buffer (Bio-Rad; 100 mM sodium phosphate, pH 7.5), and 2.5 µl of denaturing solution (2% SDS, 1 M ß-mercaptoethanol). The solution was cooled on ice prior to the addition of 2.5 µl of NP-40 (15%) and 4 µl of PNGaseF (2.5 U/ml in 20 mM Tris, pH 7.5, 50 mM NaCl, 1 mM ethylenediamine tetra-acetic acid). The digest was performed for 14 h at 37°C. The digest was verified by a shift in the apparent molecular weight of the native versus the PNGaseF-treated form on SDS–PAGE and the released N-linked oligosaccharides were subsequently analysed by graphitized carbon LC-MS as described.

HPLC electrospray ionization TOF-MS (LC-MS)
Enzyme digests were routinely analyzed or preparatively purified by liquid chromatography on a SMART HPLC (Pharmacia). The instrument was fitted with either a SephasilTM C8 reversed-phase column (100 mm x 2.1 mm) for protease digests, or graphitized carbon cartridge (HyperCarb 10 mm x 4 mm, Shandon, Cheshire, UK) for oligosaccharide separations. For peptide separation, a linear gradient of 0.1% formic acid to 90% acetonitrile, 0.1% formic acid at a flow rate of 100 µl/min was routinely used, with separations proceeding over the time shown for each result. Typically, this was 15 min, though the resolution of sulfated/unsulfated forms was performed over 90 min. Separation of carbohydrates was performed over graphitized carbon from 0.1% formic acid to 25% acetonitrile, 50% butanol-saturated water, 0.1% formic acid at a flow rate of 100 µl/ min as a modification of chromatography previously reported (Packer et al., 1998Go). In both cases, postcolumn flow was directed to an electrospray ionization TOF mass spectrometer (Micromass, UK), except for preparative purifications of the core and linker peptides by HPLC in the absence of a mass spectrometer, in which case postcolumn flow was fraction-collected and the identity of fractions confirmed by direct injection into the mass spectrometer. Spectra were routinely acquired in the positive ion mode using two alternating scan functions, which differed only with respect to the voltage on the cone. Each of the "low" cone-voltage (30 V) and "high" cone-voltage scans (80 V) were acquired into separate files at a rate of 1 scan/s over the m/z ranges given in the results. Typically, these were m/z 50 to m/z 3000 for protein analyses, m/z 50 to m/z 1000 for O-linked carbohydrates, and m/z 50 to m/z 2500 for N-linked carbohydrates. Spectra in this report are presented without background subtraction or smoothing.

MALDI-TOF MS
MALDI-TOF MS spectra of papain digests of AXE were acquired on a Voyager DE-STR (Perseptive BioSystems, Framingham, MA) delayed-extraction, time-of-flight/reflectron instrument and/or a TofSpec2E (Micromass) delayed-extraction, time-of-flight/reflectron instrument. Samples were prepared on stainless steel or gold 96-well target plates by the dried-droplet method and allowed to air-dry at room temperature. Generally, this involved 0.5 µl of analyte with 0.5 µl of freshly prepared matrix solution; generally 10 mg/ml sinapinic acid in 50% acetonitrile, 0.1% TFA. Spectra were externally calibrated to reference spectra of myoglobulin and/or trypsin. Spectra are presented without background subtraction or smoothing.

Monosaccharide analysis
Monosaccharide compositional analyses were performed by both TFA or HCl acid hydrolysis and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Lyophilized protein samples of between 300–750 pmol in screw cap Eppendorf tubes were resuspended in 50 µl of either 2 M TFA or 4 M HCl and hydrolyzed at 100°C for 4 h. Hydrolyzed samples were then dried in a Savant Speed-Vac and resuspended in 50 µl of water containing 0.5 mol 2-deoxy-D-glucose as internal standard. Released monosaccharides were quantitated by chromatography on a Dionex HPAEC-PAD system fitted with a Dionex CarboPac PA10 column (250 mm x 4 mm). Separations were performed isocratically with 18% NaOH at 1 ml/min over 20 min, with between run washes of 0.4 M NaOH for 20 min. Amounts are presented from concurrently performed TFA (for glucose, mannose, and galactose) and HCl (for glucosamine and galactosamine) hydrolyses.

2D PAGE
IPG strips (7 cm; pH 3–10 or pH 4–7 linear) were rehydrated with 5 µl of Orange G and 120 µl of sample solution (8 M urea, 4% CHAPS, 2 mM tributylphosphine, 40 mM Tris-base, 0.5% ampholytes, pH 9.5) for 6–7 h in covered rehydration trays (Bio-Rad). Reduced samples (2% dithiothreitol) in sample buffer, 1 µl bromophenol blue, 100°C, 5 min) of 30–50 µl in volume were loaded in cups and isoelectric focusing performed at 20°C with a Pharmacia Multiphor II. Focusing was performed for 6 h at 100 V, 5 h at 300 V, 2 h at 600 V, 1 h at 1000 V, and 3 h at 3000 V, for a total of approximately 13600 Vh. After isoelectric focusing, the strips were stored at –80°C until required for the second dimension separation. Focused IPG strips were equilibrated in 10-ml plastic Falcon tubes with approximately 5 ml of 6 M urea, 20% glycerol, 2% tributylphosphine, 0.375% Tris and 2.5% acrylamide, pH 8.8, with gentle rocking for 20 min.

Second-dimension gels were 1.5-mm-thick prepoured 10–20% Ready Gels (Bio-Rad), run using either the Mini Protean II or the Protean II Xi Multicell, both also from Bio-Rad. Either Coomasie low-molecular-weight or silver low-molecular-weight proteins (Bio-Rad) were used as molecular weight markers. Cathode running buffer was 192 mM glycine, 0.1% SDS, adjusted to pH 8.3 with Tris base. Equilibrated strips were embedded on top of the SDS–PAGE gels using molten 0.5% agarose in cathode buffer, and the gels were run at a constant current of 3 mA per gel for 1 h and then 12 mA per gel for 2 h or until the dye front ran off the bottom of the gel.

Gels were stained using either silver diamine or Coomassie colloidal G-250 (stained at least for 24 h in 0.1% colloidal Coomassie in 30% methanol) stains. Silver diamine staining was carried out as follows: SDS–PAGE gels were first placed in a fixative, 40% methanol, 10% acetic acid for 30 min, and then into a second fixer (45 g anhydrous sodium acetate in 30% methanol, 0.5% glutraldehyde) for 30 min. After fixing, the gels were washed for 3 x 10 min with water, then incubated 2 x 30 min with 0.05% 2,7 naphthaline-disulfonic acid, then washed 2 x 10 min with water, followed by a 30-min incubation in a solution of silver (1.5% ammonia, 0.08% NaOH, carefully mixed with 0.6% silver nitrate). The gels were then washed for 3 x 4 min with water and developed in 0.01% citric acid, 0.1% formaldehyde for 5 min. Development was stopped with 5% acetic acid for 10 min with subsequent washing 2 x 10 min with water. Stained gels were immediately imaged on a Molecular Dynamics SI densitometer before further use or analysis.

Ion chromatography
Separation of sulfate from phosphate was determined as described by Harrison and Packer (2000)Go. Approximately 1 nmol of protein was dried on a Savant Speed-Vac and resuspended in 50 µl of 4 M HCl. Samples were then hydrolyzed in screw-capped Eppendorf tubes at 100°C for 4 h. Hydrolyzed samples were dried and twice resuspended in 50 µl of water and redried to reduce the levels of residual HCl present in the sample. Dry, hydrolyzed samples and nonhydrolyzed controls were resuspended in water and analyzed for free phosphate or sulfate by ion chromatography. Ion chromatography was performed on a Dionex HPLC using a Dionex IonPacAS11 analytical ion-exchange column (250 mm x 4 mm) with a Dionex DX500 LC pump and a postcolumn conductivity detector with an in-line AMMS anion suppressor with neutralization using 0.5 N H2SO4. Separation was achieved over a shallow, concave gradient from 5% to 30% NaOH over 15 min at a constant flow rate of 1 ml/min. The column was reequilibrated with 5% NaOH, 95% water for 20 min between analyses. Sodium phosphate, sodium sulfate, sodium chloride, and sodium acetate were used as standards.

Solid-phase Edman degradation
Identification of glycosylated amino acids and N-terminal sequencing was performed by solid-phase protein sequencing on a Hewlett-Packard protein sequenator as previously described (Gooley and Williams, 1997Go).

General methods
Amino acid analysis and phosphoamino acid analysis were performed by the Australian Proteome Analysis Facility. Protein concentration of the AXE stock sample was measured colorimetrically using a Bio-Rad DC Protein Assay kit according to the manufacturer’s instructions.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by a Macquarie University Research Grant.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
AXE, acetylxylan esterase; CBHI, cellobiohydrolase I; CHAPS, 3-[(3-cholamidopropyl) dimethyl-amino]-1-propanesulfonate; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; HPLC, high-performance liquid chromatography; IPG, immobilized pH gradient; LC-MS, liquid chromatography mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry; PNGase F, peptide-N-glycosidase F; SBD, substrate binding domain; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFA, trifluoracetic acid.


    Footnotes
 
1 Present address: KCL, P.O. Box 70, FIN-02151, Espoo, Finland Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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