Glycobiology vol. 12 no. 4 pp. 291–298, 2002
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
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
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, 1974; Morris
and Bacon, 1977; Wood and McCrae, 1986; Grohmann et al.,
1989; Poutanen et al., 1987; Poutanen and Puls, 1988). Several
esterases acting on different acetylated side chains have been
purified from various microorganisms (reviewed in Christov
and Prior, 1993). Acetylxylan esterase (AXE) secreted by the
1Present
2To
address: KCL, P.O. Box 70, FIN-02151, Espoo, Finland
whom correspondence should be addressed
© 2002 Oxford University Press
filamentous fungus Trichoderma reesei is capable of liberating
acetic acid from polymeric acetyl xylan (acetylated hemicellulose) constituents in plants (Tenkanen, 1998). The gene
encoding this enzyme has been isolated from T. reesei Rut-C30
by Margolles-Clark et al. (1996). T. reesei AXE occurs in
several isomeric forms with similar apparent molecular mass
but differing pI values (Poutanen et al., 1990; Poutanen and
Sundberg, 1988; Sundberg and Poutanen, 1991). 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., 1990; Poutanen and Sundberg, 1988; Sundberg and Poutanen, 1991). 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., 1986; Neustroev et al., 1993).
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-acetylglucosamine reported on the main cellobiohydrolase I (CBHI)
of T. reesei ALKO2877 and QM9414 (Harrison et al., 1998;
Klarskov et al., 1997) 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 α-galactosidase (Savel’ev et al., 1997) and
cellobiohydrolase I from T. reesei (Maras et al., 1997),
glucoamylase from A. awamori X 100/D27 (Eriksen et al.,
1998), and α-amylase from A. awamori (Chen et al., 1994)
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., 1993; Eriksen et al., 1998; Chen et al., 1994).
O-linked glycans of glucoamylase from A. awamori (Neustroev
et al., 1993), Aspergillus niger (Gunnarsson et al., 1984) and
CBHI of T. reesei (Harrison et al., 1998) 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, 1976). Analysis of stability changes following
decreased glycosylation of A. awamori linker region
(Neustroev et al., 1993) suggests that O-linked sugars
essentially contribute to the stabilization of glucoamylase.
Furthermore, the linker glycopeptide seems to stabilize the
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M.J. Harrison et al.
binding domain against reversible thermal and chemical
denaturation (Williamson et al., 1992). 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, 1993).
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., 1996; Sundberg and Poutanen, 1991).
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.
Preparation of core and linker-SBD peptides from native AXE
by papain digestion
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 linkerSBD 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.
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.
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
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.
Results
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Glycosylation of T. reesei acetylxylan esterase
on the observed mass intensities, the mixed fraction was used
as such for qualitative experiments and the subsequent results
interpreted accordingly.
Table I. Electrospray mass spectral data for intact and papain-digested AXE
from T. reesei
Assignment
Observed (Da)
Theoretical (Da)
MS of linker-SBD peptide
Intact AXE protein
∼31000–33000
27451.2
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.
Core peptide
+ HexNAc2Hex26
31832.5
31829.2
+ HexNAc2Hex27
31997.4
31991.3
32158.3
32153.4
+ HexNAc2Hex28
Linker-SBD peptide (Gly216 – Leu271)
∼7900–9000
5814.263
+ 13 Hex
7921.75
7921.129
+ 13 × Hex, 1 × SO3
8001.56
8001.194
+ 14 × Hex
8083.91
8083.272
+ 14 × Hex, 1 × SO3
8163.64
8163.336
+ 15 × Hex
8245.16
8245.414
+ 15 × Hex, 1 × SO3
8325.41
8325.478
+ 16 × Hex
8408.87
8407.557
+ 16 × Hex, 1 × SO3
8487.73
8487.621
+ 17 × Hex
8569.21
8569.699
+ 17 × Hex, 1 × SO3
8650.27
8649.763
+ 18 × Hex, 1 × SO3
8811.26
8811.906
Site glycosylation of linker-SBD peptide
Edman degradation using the GlycoSite sequenator (Gooley
and Williams, 1997), 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.
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).
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.
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).
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M.J. Harrison et al.
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.
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.
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) showed no evidence of phosphoamino acids (data not
shown).
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, 2000).
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] H 2PO4Na+.
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.
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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
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.
Glycosylation of T. reesei acetylxylan esterase
peptide also produced two isoforms of approximate pI 4.4 and
5.4 (Figure 8B).
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
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., 1998). 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., 1997; Gunnarsson et al., 1984; Takayanagi et al.,
1992) 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., 1997;
Harrison et al., 1998).
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., 1990), 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.,
1997; Harrison et al., 1998). 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, 1998). Sulfated carbohydrates
have also been shown to play specific roles in well-defined
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M.J. Harrison et al.
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., 1996). 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-yetunidentified biologically significant function.
Materials and methods
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-molecularweight 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).
Proteolytic digestion by papain
Papain digestion of AXE has been previously reported by
Margolles-Clark et al. (1996). 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 2× reaction buffer (Bio-Rad; 100 mM sodium phosphate,
pH 7.5), and 2.5 µl of denaturing solution (2% SDS, 1 M β-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
296
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
Sephasil™ C8 reversed-phase column (100 mm × 2.1 mm) for
protease digests, or graphitized carbon cartridge (HyperCarb
10 mm × 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., 1998). 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 (HPAECPAD). 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-deoxyD-glucose as internal standard. Released monosaccharides
Glycosylation of T. reesei acetylxylan esterase
were quantitated by chromatography on a Dionex HPAEC-PAD
system fitted with a Dionex CarboPac PA10 column (250 mm
× 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 × 10 min with water, then incubated 2 × 30 min
with 0.05% 2,7 naphthaline-disulfonic acid, then washed 2 ×
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 × 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 × 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). 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 ionexchange column (250 mm × 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, 1997).
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
This work was supported by a Macquarie University Research
Grant.
Abbreviations
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, highperformance 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-Nglycosidase F; SBD, substrate binding domain; SDS–PAGE,
sodium dodecyl sulfate–polyacrylamide gel electrophoresis;
TFA, trifluoracetic acid.
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