Heterogeneity of homologously expressed Hypocrea jecorinaTorny Eriksson1,*, Ingeborg Stals2,*, Anna Colle´n1, Folke Tjerneld1, Marc Claeyssens2, Henrik Sta˚lbrand1 and Harry Brumer3 1 De
Trang 1Heterogeneity of homologously expressed Hypocrea jecorina
Torny Eriksson1,*, Ingeborg Stals2,*, Anna Colle´n1, Folke Tjerneld1, Marc Claeyssens2, Henrik Sta˚lbrand1 and Harry Brumer3
1
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden;2Laboratory for
Biochemistry, Department of Biochemistry, Physiology and Microbiology, Ghent University, Belgium;3Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, Sweden
The catalytic module of Hypocrea jecorina (previously
Trichoderma reesei) Cel7B was homologously expressed by
transformation of strain QM9414 Post-translational
modi-fications in purified Cel7B preparations were analysed by
enzymatic digestions, high performance chromatography,
mass spectrometry and site-directed mutagenesis Of the
five potential sites found in the wild-type enzyme, only
Asn56 and Asn182 were found to be N-glycosylated
GlcNAc2Man5was identified as the predominant N-glycan,
although lesser amounts of GlcNAc2Man7 and glycans
carrying a mannophosphodiester bond were also detected Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein However, partial deami-dation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations Keywords: protein glycosylation; O-glycan; N-glycan; Tricho-derma reesei; cellulase
The filamentous fungus Hypocrea jecorina (previously
Trichoderma reesei[1]) produces several extracellular
cellu-lases, which cooperate in the degradation of paracrystalline
cellulose The five known endoglucanases, including Cel7B,
generally act by hydrolysing the b(1fi4) glucan chains
internally [2,3], whereas the two cellobiohydrolases (Cel6A
and Cel7A) release cellobiose from the nonreducing and
reducing chain ends, respectively [4,5] b-Glucosidase
even-tually hydrolyses this cellobiose to glucose which is taken up
by the fungal hyphae All but one of the Hypocrea jecorina
cellulases share a similar modular structure comprised
of a catalytic module connected to a carbohydrate-binding
module (CBM) by a flexible linker peptide The 3D structures
of the catalytic modules of Cel7A (formerly
cellobiohydro-lase I, CBH I [6]) and Cel7B (formerly endoglucanase I,
EG I) both exhibit a similar overall fold but are different in
their active site topologies; the former has a tunnel-shaped
active site whereas the latter possesses an open cleft [7,8] This reflects their different specificity, i.e exo vs endo activity [8] The catalytic modules of fungal glycoside hydrolases are often glycosylated on asparagine residues in the consensus sequence Asn-Xaa-(Ser/Thr), where Xaa is not Pro [9] This post-translational modification is thought to affect protein secretion and enzyme stability [10,11] Of the structures studied so far, most fungal N-glycans contain the mamma-lian-type core structure (Man3GlcNAc2) [12] However, the occurrence of a single N-acetyl glucosamine on Cel7A from
H jecorinastrains ALKO2877 and QM9414 [13,14], indi-cates glycosylation may be processed differently in some cases The N-glycosylation of both Cel7A and Cel7B, isolated from different strains and grown under different conditions, has been studied by several groups, but dispar-ate and inconclusive results have been published [8,13–18] The Hypocrea jecorina Cel7B catalytic module (Swiss-Prot number P07981) possesses five potential N-glycosylation sites Single N-acetyl glucosamine (GlcNAc) residues have been observed by X-ray crystallography on Asn56 and Asn182 of Cel7B produced in the H jecorina strain QM9414 [8] In a later study, this enzyme was suggested to carry only one high mannose N-glycan, some forms of which carried mannophosphodiester linkages [16] O-Mannosylation was also indicated by this study, but the sites of attachment of this and the N-glycan were not determined Recently, Cel7B from the H jecorina strain Rut-C30 was shown to bear a single GlcNAc on Asn56, while Asn182 was occupied with higher-order glycans, primarily GlcNAc2Hex8[18]
In the present study, we describe the glycoform analysis
of the catalytic module of H jecorina Cel7B homologously expressed in a QM9414 decendent strain using a range of experimental techniques Detailed analysis using mass spectrometry and high-performance chromatography indi-cated that two of the five potential N-glycosylation sites of
Correspondence to H Brumer, Department of Biotechnology,
Royal Institute of Technology (KTH), AlbaNova
University Centre, S-106 91 Stockholm, Sweden.
Fax: + 46 85537 8468, Tel.: + 46 85537 8367,
E-mail: harry@biotech.kth.se
Abbreviations: Endo H, Streptomyces plicatus endoglycosidase H;
CID MS/MS, collision-induced dissociation tandem mass
spectro-metry; HPAEC-PAD, high-performance anion-exchange
chromato-graphy with pulsed amperometric detection; PAG-IEF,
polyacrylamide gel isoelectric focusing.
Enzyme: endoglycosidase H (EC 3.2.1.96).
*Note: These authors contributed equally to this work.
Note: A website is available at http://www.biotech.kth.se/
woodbiotechnology/
(Received 18 November 2003, revised 16 January 2004,
accepted 6 February 2004)
Trang 2the enzyme were glycosylated with high-mannose structures,
predominantly GlcNAc2Man5 Additional heterogeneity
in the purified protein arises from a partially occupied
O-glycosylation site, as well as from partial deamidation
of asparagine
Materials and methods
Enzyme production
The gene sequence encoding the catalytic module (Glu1–
Thr371) of H jecorina Cel7B (Cel7Bcat) was expressed
under the control of the gpdA promotor from Aspergillus
nidulans as described previously [19] by transforming the
vector pAC1 into H jecorina (Trichoderma reesei) QM9414,
to yield strain QM9414-Cel7Bcat
H jecorina strain QM9414-Cel7BcatN182Q, expressing
Cel7Bcat(Asn182Gln) under the regulation of the A nidulans
gpdA promotor, was constructed as follows Site directed
mutagenesis was carried out using the PCR, according
to the QuickChange method (Stratagene, La Jolla, CA,
USA) using native Pfu polymerase and vector pAC1 as the
template The following oligonucleotide primer was used:
5¢-CGTCCAGACATGGAGGcaaGGtACCCTCAACAC
TAGC-3¢ Mismatches are indicated in lower case and the
introduced KpnI restriction site used for screening of
transformants is shown in bold Amplified and purified
plasmid preparations were screened using KpnI and two
positives from 10 were found The open reading frame of
the construct was sequenced prior to transformation
into H jecorina QM9414 (gift from M Penttila¨, VTT
Biotechnology, Espoo, Finland) to yield strain
QM9414-Cel7BcatN182Q Transformation and selection was
per-formed as described by Collen et al [19], based upon the
method described by Penttila¨ et al [20]
The strains H jecorina QM9414-Cel7Bcatand H jecorina
QM9414-Cel7BcatN182Qwere cultivated in minimal
med-ium with glucose as the sole carbon source according to
Colle´n et al [21], which is a modification of the medium
used by Nakari-Seta¨la¨ et al [22] and Penttila¨ et al [20] The
medium contained 30 gÆL)1 K2HPO4, 8 gÆL)1 KH2PO4,
4 gÆL)1(NH4)2SO4, 0.6 gÆL)1CaCl2, 0.6 gÆL)1MgSO4, 5
mgÆL)1FeSO47H2O, 1.6 mg L)1MnSO4H2O, 1.4 mgÆL)1
ZnSO47H2O, 2 mgÆL)1CoCl2and 4% (w/v) glucose The
pH was adjusted to 6.0 The fermentation was performed in
1 L baffled shake-flasks with 200 mL medium at 28C and
180 r.p.m The glucose concentration was monitored daily
as described previously [21] and was kept above 1% (w/v)
After 7 days of cultivation, the mycelia were removed and
the buffer was exchanged to 20 mM NH4OAc, pH 4.5
(Buffer A) by ultrafiltration The proteins were purified by
anion-exchange chromatography (Source Q; Amersham
Pharmacia Biotech) using a linear gradient generated by
mixing Buffer A with Buffer B (1MNH4OAc, pH 4.5) All
fractions containing significant activity toward
p-nitro-phenyl-b-cellobioside (measured as described in [23]) were
pooled and used in further analyses
Polyacrylamide gel isoelectric focusing (PAG-IEF)
PAG-IEF experiments were performed with a
PhastSys-temTM(Amersham Biosciences, Uppsala, Sweden) using a
dry precast homogeneous polyacrylamide gel (3.8 cm · 3.3 cm) The gel was rehydrated with 120 lL PharmalyteTM
pH 2.5–5 (Amersham Biosciences, Uppsala Sweden),
20 lL ServalytTMpH 3–7 (Serva Electrophoresis GmbH, Heidelberg, Germany) and 1860 lL bidistilled water for
2 h In a prefocusing step, the pH gradient was generated (75 Vh, 2000 V, 2.5 mA) and 1 lL samples (10 mg pro-teinÆmL)1) were subsequently applied at the cathode end Electrophoresis was started at low voltage (15 Vh) and run
to a final 450 Vh (2.5 mA, 2000 V) At the end of the run the locations of Cel7B activity were revealed by immersing the gel in 2 mM 4-methylumbelliferyl b-lactoside (NaOAc buffer, pH 5) Staining with Coomassie Blue R-350 was performed according to the manufacturer’s instructions (Pharmacia Biotech)
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)
A HPAEC-PAD system (DionexTM, Sunnyvale, CA, USA), equipped with an ED40 electrochemical detector, a GP40 gradient pump and a LC30 chromatography oven (40C) was used Chromatographic data were analysed using
DIONEX PEAKNET software (release 5.1) Monosaccharide mixtures resulting from total acid hydrolysis were analysed
on a CarboPac PA-10 column using isocratic elution (16 mM NaOH, 1 mLÆmin)1) Enzymatically released N-glycans were separated on a CarboPac PA-100 column; neutral oligosaccharides were first resolved using a 0–60 mM
NaOAc gradient in 100 mM NaOH for 35 min (1 mLÆmin)1) A 60–500 mMNaOAc gradient in 100 mM
NaOH was subsequently applied to elute carbohydrates carrying negatively charged substituents
Mass spectrometry Mass spectrometric analysis was carried out on a Q-TofTM
II mass spectrometer fitted with a nano Z spray source (Waters Corporation, Micromass MS Technologies, Man-chester, UK), essentially as described previously [24] Endoglycosidase H digestion
Enzymatic N-deglycosylation was performed by adding 0.02 U Endo H (Sigma-Aldrich, Bornem, Belgium) per microgram of Cel7Bcator Cel7BcatN182Qin 10 mMNaOAc buffer, pH 4.5, for 12 h at 37C N-Deglycosylated proteins were subsequently precipitated with three volumes
of ethanol and were redissolved in bidistilled water prior to PAG-IEF analysis For carbohydrate analysis, the super-natant was desalted [25] on a Carbograph column (Alltech Associates Inc., Lokeren, Belgium) After extensive washing with bidistilled water, N-glycans were eluted with 2 mL 25% (v/v) CH3CN(aq)containing 0.05% (v/v) trifluoroacetic acid Following evaporation of the solvent, the N-glycans were redissolved in bidistilled water for further analysis Alkaline phosphatase treatment
Enzymatic dephosphorylation of released N-glycans was attempted on both untreated and mild acid-hydrolysed samples (0.01 HCl, 100C, 30 min) as follows One unit
Trang 3of calf intestine alkaline phosphatase (Roche Diagnostics,
Vilvoorde, Belgium) dissolved in 20 lL 100 mMTris/HCl,
pH 8.8 containing 10 mM ZnCl2, was added to 20 lL
oligosaccharides (25 lgÆmL)1) Reactions were allowed to
proceed overnight at room temperature prior to product
analysis by HPAEC-PAD
a-Mannosidase treatment
Jack bean mannosidase (1 unit; Sigma-Aldrich, Bornem,
Belgium) was added to oligosaccharide mixtures (20 lL,
25 lgÆmL)1) obtained from Cel7Bcator the reference protein
RNAse B in 20 mMNaOAc buffer, pH 5, containing 2 mM
ZnCl2 The products of the overnight reaction at room
temperature were analysed by HPAEC-PAD
Total acid hydrolysis
Oligosaccharide samples (25 lgÆmL)1) were hydrolysed in
4Mtrifluoroacetic acid After heating at 100C for 4 h in
Teflon capped tubes, the acid was removed by evaporation
and the sugars were identified by HPAEC-PAD
Protease digestions
Prior to protease digestion, proteins were denatured and
reduced by incubating 0.8 mgÆmL)1 Cel7Bcat or 0.05
mg mL)1Cel7BcatN182Qin 0.1MNH4HCO3, containing
6M urea and 5 mM dithiothreitol, for 30 min at 60C
Iodoacetamide (25 mMfinal concentration) was added and
the samples were incubated in the dark for 30 min at 25C
Subsequent dialysis was performed against 1Murea either
in 10 mMNH4HCO3(for trypsin digestions) or in 10 mM
sodium phosphate buffer, pH 7.5 (for V8 protease
diges-tions) Modified trypsin (Promega, Madison, WI, USA)
was added in a protease/cellulase ratio of 1/20 (w/w; 37C,
12 h) V8 protease digestions were performed by incubating
Staphylococcus aureus V8 protease (V8 endoproteinase
Glu-C; Sigma, St Louis, MO, USA) in a 1/20 (w/w)
protease/cellulase ratio (37C, 12 h)
Results
Protein expression and purification
The gene sequence encoding Glu1–Thr371 of Hypocrea
jecorina Cel7B (Cel7Bcat, Fig 1) was homologously
expressed under the regulation of the gpdA promotor of
Aspergillus nidulans [19] After cultivation (7 days) the
endoglucanase activity was 0.63 nkatÆmL)1, corresponding
to an extracellular expression level of 27 mgÆL)1 The pH
was 5.5 at the start of the cultivation and decreased to
approximately 3 at days six and seven of growth
Purifica-tion by anion-exchange chromatography yielded several
endoglucanase-active peaks, which suggested the presence
of protein isoforms (data not shown) These combined
fractions were used in further experiments to ensure that all
produced isoforms of the protein were analysed SDS/
PAGE analysis indicated a major protein band with an
apparent molecular mass of 44 ± 1 kDa (data not shown),
which is higher than that calculated for Cel7Bcat(39.1 kDa)
The results from the cultivation of H jecorina expressing
Cel7BcatN182Qwere similar to that of Cel7Bcat, except that the detected endoglucanase activity in the cultivation broth was lower (0.18 nkatÆmL)1)
PAG-IEF analysis of intact and Endo H digested Cel7Bcat
After PAG-IEF over a narrow pH gradient, at least five enzymatically active isoforms were observed Analysis of a sample treated with endoglycosidase H (Endo H), which cleaves the core GlcNAcb(1fi4)GlcNAc bond in high mannose-type N-glycans, yielded one predominant and one minor isoform (Fig 2)
ESI-MS analyses of intact and Endo H-digested Cel7Bcat
X-ray crystallography has previously revealed that the N-terminal residue in Cel7B is pyroglutamate and that the protein contains eight disulphide bonds [8] TOF MS analysis of tryptic digests confirmed the presence of the N-terminal pyroglutamate in the protein produced in this study (Table 1) After correction for these post-translational modifications, the calculated molecular mass of Cel7Bcat
is 39133.8 Da Figure 3A shows the reconstructed zero-charge spectrum of purified Cel7Bcat, in which a range of peaks is observed The mass of the major component corresponds well with the calculated molecular mass for Cel7Bcat substituted with Man5GlcNAc2 on two Asn residues (calculated molecular mass, 41 567.6 Da; observed molecular mass 41 566.6 Da) The observation of species of increasing mass spaced by 162 Da reflects the presence of glycoforms with an increasing number of hexose (probably mannose) units Phosphorylation is indicated by the pres-ence of +80 Da species interspersed within the hexose ladder This was further confirmed by carbohydrate analysis (see below) The peak at 40 552.4 Da may result from
a glycoform on which one of the high-mannose glycans has been trimmed to a single GlcNAc MS analysis of
Fig 1 Primary amino acid sequence of H jecorina Cel7B cat Labels T1–T16 and S1–S11 denote predicted peptides from trypsin or
S aureus V8 protease digestion, respectively Predicted N-glycosyla-tion sites are shown in bold italic type, with Asn underlined Q, Gln-derived pyroglutamate; Cys residues are highlighted in bold.
Trang 4Endo H-treated Cel7Bcat shows a major peak with a
molecular mass of 39 538.0 Da (Fig 3B), which corresponds
to the Cel7Bcatpolypeptide plus two N-acetyl glucosamine
residues (calculated molecular mass, 39 540.2 Da) The
additional peak observed at 39 700.6 Da (39 538+
162 Da) is probably due to O-linked glycosylation of the protein (see below) The results are summarized in Table 2 Identification of N-glycosylation sites
A series of detailed TOF MS and CID MS/MS experiments were performed to identify the N-glycosylation sites of wild-type Cel7Bcat After deglycosylation by Endo H, and prior
to digestion with either trypsin or V8 protease, the protein was denatured, the disulphide bonds reduced and the free Cys residues converted to carboxyamidomethyl derivatives The predicted cleavage sites for trypsin and V8 protease under the conditions used for each digestion are indicated in Fig 1 Peptides were infused directly into the MS without prior separation, and the observed m/z values were matched against those calculated for various protonated forms of the peptides (Table 1) Matching values were found for the T8, S7 and T12 peptides, thus indicating absence of glycosyla-tion at Asn142 and Asn259 Due to the presence of Pro in the second position of the Asn-Xaa-(Ser/Thr) consensus sequence, glycosylation at Asn344 in the T15 peptide is not expected [9] Indeed, no evidence for glycosylation at this site was observed in the TOF MS data No signals were observed for the remaining two potential tryptic glycopep-tides, T5 (which contains Asn56) and T9 (which contains both Asn182 and Asn186) However, peaks arising from these two peptides, each with an appendant GlcNAc residue, were observed (Table 1), thus indicating that these two peptides are N-glycosylated in the intact glycoprotein The identities of peptides containing all five predicted N-glycosylation sites were further confirmed by CID MS/MS experiments Fragmentation of ions correspond-ing to peptides T5+GlcNAc (Table 1, m/z 1159.2, [M+3H]3+) and T9+GlcNAc (Table 1, m/z 986.4,
Table 1 Selected proteolytic fragments of Cel7Bcore Peptides represent sequential fragment numbering from the N-terminus, T, tryptic peptides (cleavage after K and R except when preceeding P); S, S aureus V8 proteolytic fragments (cleavage after E except when preceeding E or P) Potential N-linked glycosylation sites are shown in bold; C, carboxyamidomethyl cysteine; Q, Gln-derived pyroglutamic acid All masses are monoisotopic Ions indicated in bold were selected for CID MS/MS experiments N.O., not observed.
Peptide Residues Sequence
Calculated Observed Calculated Observed Calculated Observed
DEAT C GK
T5 +
GlcNAc
40–68 WMHDANYNS C TVNGGV NTT L C P
DEAT C GK + GlcNAc (203.079)
LYLSQMDENGGANQYNTAGANY GSGY C DAQ C PVQTWR
[M+4H]4+
1616.48 1293.37
[M+5H]5+
1293.59
T9 +
GlcNAc
40–68 NGT L NTS HQGF CC NEMDILEGNSR
+ GlcNAc (203.079)
SGNAGP C SSTEG NPS NILANNPNT HVVFSNIR
1524.96 [M+4H] 4+
1524.99 1220.17
[M+5H] 5+
1220.21+ 1016.98
[M+6H] 6+
1017.00
a
[M+4H]4+ion also observed at m/z 869.62.
Fig 2 PAG-IEF of Cel7B cat (PyrGlu1–Thr371) expressed in H
jeco-rina QM9414-Cel7B cat over the pH range 2.5–7 Lane 1, markers
(amyloglucosidase, pI 3.50; methyl red dye, pI 3.75; soybean trypsin
inhibitor, pI 4.55; b-lactoglobulin A, pI 5.20; bovine carbonic
anhyd-rase, pI 5.85) Lane 2, purified Cel7B cat ; lane 3: purified Cel7B cat after
Endo H treatment.
Trang 5[M+3H]3+) produced the peptide sequence tags
NTTLCPDEATC and NGTLNTSHQGFCCNEMDL
LEGNSR, respectively Isobaric Leu/Ile is denoted by
L, while C denotes carboxylamidomethyl Cys In both
cases, a neutral loss of 203 Da, the mass of GlcNAc, is
observed from the [M + H]+ion in deconvoluted,
single-charge spectra In contrast, the T8 peptide is too large to
generate useful CID MS/MS information In this case,
fragmentation of the much smaller S7 peptide ion
(Table 1, m/z 628.8, [M + 2H]2+), which also contained
the potential glycosylation site Asn142, produced a
confirmatory peptide sequence tag SQMD
CID MS/MS of the nonglycosylated T12 peptide ion (Table 1, m/z 1292.2, [M + 2H]2+) gave rise to two series
of daughter ions, which correspond to the sequences LTQFNTDNGSPSGNLVSITR and LTQFNTDDGSPS GNLVSITR (Fig 4) The data indicate that deamidation of Asn259 has occurred to produce an aspartic acid residue at this location (bold) The resulting 1 Da increase in the peptide mass results in the production of an [M + 2H]2+ ion for the Asn259Asp variant (m/z 1292.7) which was not resolved from the parent peptide ion in the quadrupole stage
of the MS; simultaneous CID of both ions generates the overlapping series of daughter ions
Fig 3 Reconstructed zero-charge spectra of Cel7B cat (A) and Endo H-treated Cel7B cat (B).
Table 2 Potential glycan structures correlated with observed glycoprotein masses.
Spectrum
Observed mass (Da)
Proposed occupation of glycosylation sites
Trang 6Fig 4 CID MS/MS analysis of the double-charged Cel7B cat T12 peptide ion at m/z 1292.2 (A) Complete MaxEnt3 spectrum (B) Expansion of spectrum A (C) Assignment of the second series of daughter ions with aspartic acid as residue 259.
Trang 7Analysis of the Cel7BcatN182Q mutant
MS/MS analysis of the T9 peptide produced from
Cel7Bcat failed to provide information about the site of
N-glycan attachment (either Asn182 or Asn186), as the
GlcNAc moiety is readily lost during CID To resolve
this ambiguity, a glycosylation site mutant was
construc-ted in which Asn182 was replaced by glutamine This
mutant was expressed, purified and analysed in the same
way as the wild-type Cel7Bcatprotein Figure 5 shows the
reconstructed zero-charge spectra of the Cel7BcatN182Q
protein before and after Endo H treatment Accounting
for an N-terminal pyroglutamate and eight cystine
bridges, the calculated average molecular mass for the
Cel7BcatN182Qpolypeptide chain is 39 148.9 Da The
dif-ference of 1216.6 Da between this value and that observed
for the major isoform of the intact Cel7BcatN182Qprotein
(40 365.5 Da) corresponds to a modification of the
polypeptide chain with a single GlcNAc2Man5 N-glycan
(calculated molecular mass, 1217.1 Da) As with the
wild-type Cel7Bcat, a range of isoforms was observed by
MS; those separated by 162 Da reflect variable
mannosyla-tion of the N-glycan or O-glycosylamannosyla-tion, while those
separated from this series by 80 Da indicate glycan
phos-phorylation
Endo H treatment significantly reduced the number of
glycoforms of the Cel7BcatN182Qobserved by MS The
observed mass of the major species corresponds well with
that expected for the Cel7B N182Qpolypeptide bearing
one asparagine-linked GlcNAc residue (calculated mole-cular mass, 39 352.0; observed molemole-cular mass, 39 351.0) Similar to wild-type Cel7Bcat (Fig 3B), the zero-charge spectrum of Endo H-treated Cel7BcatN182Qexhibits a minor peak 162 Da larger than the main glycoform, possibly reflecting an O-linked hexose modification
To confirm that Asn182 had indeed been mutated to Gln and that the remaining glycosylation site was identical
to that of the wild-type protein, Endo H-treated Cel7BcatN182Qwas similarly subjected to trypsin digestion and TOF MS analysis As in Cel7Bcat(Table 1), peptide T5
of Cel7BcatN182Qwas observed bearing a single GlcNAc residue (Table 3), and thus carried the N-glycan The T9 peptide ionized as a triple-charged ion, m/z 923.4 (Table 3), and yielded the complete sequence QGTLNTSHQGF CCNEMDLLEGNSR upon CID MS/MS analysis, which verified the mutation and the absence of N-glycosylation The remaining peptides bearing potential N-glycosylation sites (T8, T12 and T15) were only observed as their unmodified forms (Table 3)
Characterization of Endo H-released N-glycans Total acid hydrolysis indicates that N-glycans released
by Endo H treatment contain only mannose and N-acetyl glucosamine in a ratio of 6 : 1 Oligosaccharide analysis
by HPAEC-PAD showed the presence of GlcNAcMan5, GlcNAcMan7 and, eluting at high salt concentrations, small amounts of negatively charged glycans (Fig 6B)
Fig 5 Reconstructed zero-charge spectra of Cel7B cat N182Q mutant (A) and Endo H treated Cel7B cat N182Q mutant (B).
Trang 8a-Mannosidase hydrolyses only the neutral oligosaccharides
to mannose and GlcNAcMan (Fig 6C) Acid treatment of
the oligosaccharides (0.01M HCl, 100C) leads to the
formation of mannose (Fig 6D) The concomittant
forma-tion of a terminal phosphate could not be demonstrated
with HPAEC-PAD, but PAGE analysis of fluorescent
labelled oligosaccharides clearly indicates a shift in
elec-trophoretic mobility (data not shown) Alkaline
phospha-tase digestion alone did not change the HPAEC-PAD
elution profile Conversion of charged N-glycans to their
uncharged counterparts (GlcNAcMan5and GlcNAcMan7)
could only be achieved by combined mild acid hydrolysis
and alkaline phosphatase treatment (Fig 6E), thus
indica-ting the presence of a phosphodiester linkage between sugar
residues [26]
O-glycosylation
The presence of O-glycosylation at serine or threonine
residues was indicated by the observation of a second
minor species with a mass 162 Da greater than the
major glycoform in both Endo H-digested Cel7Bcat and
Cel7BcatN182Q(Figs 3B and 5B) Analysis of the TOF MS
data obtained for the tryptic digest of wild-type enzyme
indicates that, in addition to peaks arising from the
unmodi-fed T6 peptide (Fig 1) at m/z 1384.64 ([M + 3H]3+) and
m/z 1038.72 ([M + 4H]4+), peaks corresponding to T6 +
Hex (T6 +162 Da) are present at m/z 1438.68 ([M +3H]3+)
and m/z 1079.26 ([M + 4H]4+) CID MS/MS analysis of the
m/z 1384.64 ion yielded near-complete sequence coverage
for the T6 peptide: LEXXDYAASGVTTSGSSLTMNQY
MPSSSGGYSSVSPR CID MS/MS analysis of the
puta-tive glycopeptide ion at m/z 1438.68 generated less complete
sequence tag information, and failed to pinpoint the site of
glycosylation due to facile cleavage of the
carbohydrate-peptide linkage (spectrum not shown)
Discussion
Understanding the factors that contribute to the glycoform heterogeneity of proteins expressed in fungal sources is a fundamental concern in a wide range of applications from basic biochemical characterization to crystallographic stud-ies and medicinal applications [12] Previous studstud-ies on the catalytic module of Cel7B from H jecorina have generated conflicting results about the extent and localization of glycosylation of this protein [8,16,18] The present detailed structural study of Cel7Bcat expressed in an H jecorina QM9414 derivative strain aims to elucidate discrepancies found in previous publications
N-glycan heterogeneity in Cel7Bcat
PAG-IEF analysis clearly shows that the purified wild-type Cel7Bcat consists of several isoforms and that this heterogeneity is considerably reduced after enzymatic N-deglycosylation (Fig 2) Comparative MS analysis of the intact protein before and after Endo H treatment confirms that it consists of a mixture of glycoforms, which vary in the extent of both mannosylation and phosphory-lation Moreover, the data on the intact protein alone are sufficient to show that the protein carries two N-linked high-mannose glycans
The results of detailed carbohydrate analysis (Fig 6) and peptide MS experiments (Fig 3A) define the glycoform structure and the sites of glycan attachment in the present enzyme preparation TOF MS and CID MS/MS analyses
of Endo H-treated Cel7Bcatand Cel7BcatN182Qproteolytic digests unequivocally show that Asn56 and Asn182 of the Cel7B catalytic module are glycosylated These two residues are located on exposed loops of the protein (Protein Data Bank code 1EG1 [8]), as is often observed for N-glycosy-lation sites [27] The multiplicity of glycoforms in the intact
Table 3 Selected proteolytic fragments of Cel7BcoreN182Q Peptides represent sequential fragment numbering from the N-terminus; T, tryptic peptides (cleavage after K and R except when preceeding P) Potential N-linked glycosylation sites are shown in bold; C, carboxyamidomethyl cysteine; Q, Gln-derived pyroglutamic acid All masses are monoisotopic Ions indicated in bold were selected for CID MS/MS experiments N.O., not observed.
Peptide Residues Sequence
Calculated Observed Calculated Observed Calculated Observed
DEAT C GK
T5 +
GlcNAc
40–68 WMHDANYNS C TVNGGV NTT L C P
DEAT C GK + GlcNAc (203.079)
LYLSQMDENGGANQYNTAGANY GSGY C DAQ C PVQTWR
[M+4H] 4+
1616.46 1293.37
[M+5H] 5+
1293.56
T9 +
GlcNAc
40–68 QGTL NTS HQGF CC NEMDILEGNSR
+ GlcNAc (203.079)
SGNAGP C SSTEG NPS NILANNPNT HVVFSNIR
1524.96 [M+4H]4+
1524.97 1220.17
[M+5H]5
1220.18 1016.98+
1016.96 [M+6H]6+
a [M+4H] 4+ ion also observed at m/z 869.60.
Trang 9protein (Fig 3A and Table 2) can be explained by the
presence of Man5GlcNAc2 and Man7GlcNAc2, their
derived phosphodiesters and/or one extra O-linked
man-nose residue The predominant Man5GlcNAc2 N-glycan
reflects normal processing in the endoplasmatic reticulum
by a(1fi3) glucosidases and further trimming by a
non-specific a(1fi2) mannosidase present in H jecorina [28,29]
As suggested previously, the presence of minor amounts of
GlcNAc2Man6)7could be due to reduced catalytic activity
of the latter enzyme Previous studies on both Cel7A and
Cel7B isolated from strain Rut-C30 report glucosylated
N-glycans (GlcMan7GlcNAc2) [15,18] Thus, although the
glycosylation sites are invariant, the structures of the
attached glycans can be markedly different between strains
of different mutational lineage [30,31]
N-glycan phosphorylation has previously been
demon-strated in proteins from both strain QM9414 and RUT-C30
(e.g [15,16,32]) In the present study, the presence of Man-Pi
-Man phosphodiester structures in Cel7B is confirmed by
mild acid hydrolysis (release of Man) and conversion of the
free glycans to uncharged counterparts by subsequent
phosphatase treatment (Fig 6)
The observed differences in N-glycan structures in this
study compared to the other studies of Hypocrea jecorina
Cel7B [8,16,18] may further be explained, in part, by
differences in cultivation and/or purification conditions
Previous studies have shown N-glycan structures on Cel7B
with only a single GlcNAc [16,18], which may indicate
that H jecorina produces one or more enzymes respon-sible for processing glycan structures during cultivation Indeed, recent results indicate that H jecorina produces an array of extracellular glycan processing enzymes, including
an enzyme with Endo H-like activity [32a] Most interest-ingly, these activities exhibit widely different pH profiles, which implies that the spectrum of enzyme activities changes with pH variations in the medium during fermentation Thus, the incongruous results could be explained by postsecretorial effects due to the growth conditions used in the different studies The minimal medium used here results in a low pH at the final culture stages, where the glycan hydrolases become inactive As such, the high-mannose N-glycans decorated with phos-phodiester modifications represent the initial complexity of secreted fungal proteins Reports of Cel7B carrying single GlcNAc residues undoubtly result from cultivations in rich medium, which buffers the pH near the optima for the extracellular hydrolases [32a]
O-glycan heterogeneity in Cel7Bcat
In addition to the observed N-glycan heterogeneity, Cel7Bcat has one partially occupied O-glycosylation site located in peptide T6 Asn69–Arg109 Whereas N-glyco-sylation has been observed only in the catalytic module of fungal cellulases, O-linked glycosylation has been shown
to be a typical feature of the linker peptide Heterogene-ous phosphorylation or sulfation of mono- di- or tri-hex-ose (predominantly manntri-hex-ose) units on serine or threonine residues has been reported [14,17,32] The function of this O-glycosylation is not fully understood but it has been suggested that it contributes to enzyme stability [11] and helps define the conformation of the linker [33] In contrast, the O-glycosylation of core modules of H jeco-rinacellulases has not been widely documented To our knowledge, the present work is only the second report of this phenomenon; the first is the observation of O-linked a-mannosyl units on multiple serine and threonine residues in Cel6A (formerly cellobiohydrolase II, CBHII) [34] CID MS/MS analysis did not allow the determin-ation of the exact site of hexose attachment within the T6 peptide Analysis of the Cel7Bcatstructure (PDB code 1EG1) shows that this peptide is rich in surface-exposed serine and threonine residues found in tandem or triple repeats Although further chemical analysis will be required, it is tempting to speculate that the sequence 97-SSS-99, which is at the tip of a loop region, may be a likely O-glycosylation site
Heterogeneity in Cel7Bcatdue to protein deamidation The spontaneous deamidation of asparagines to yield aspartic acid residues is well documented for both proteins and peptides [35] In particular, the sequence Asn–Gly has been shown to be especially susceptible [36] Our results indicate that partial deamidation at Asn259–Gly260 has occurred in Cel7Bcat Deamidation has recently been observed in the protein hydrophobin HFBI expressed in
H jecorina[37], and it is likely that this process contributes
to a further increase in the heterogeneity of H jecorina proteins in general
Fig 6 HPAEC-PAD analysis of N-glycans released from Cel7B cat
(A) Reference N-glycans (B) N-glycans released from Cel7B.
(C) N-glycans released from Cel7B treated with Jack bean
a-man-nosidase (D) N-glycans released from Cel7B treated with mild acid.
(E) N-glycans released from Cel7B treated with mild acid followed by
alkaline phosphatase.
Trang 10The three types of protein heterogeneity in Cel7Bcat
observed by MS analysis all contribute to the complex
PAG-IEF patterns observed with purified samples of the enzyme
Native Cel7Bcatexhibits a pattern resulting from at least five
different forms of the enzyme Because Endo H treatment
reduces the number of isoforms to two, the three bands
observed with more acidic isoelectric points probably
correspond to phosphorylated forms (substitution of
charged N-glycans on one or both glycosylation sites,
Table 2) Heterogenous mannosylation of neutral N-glycans
at one or both glycosylation sites is only expected to cause a
minor shift in the pI value The major component resulting
from Endo H treatment is Cel7Bcat bearing two single
GlcNAc substitutions (calculated pI 4.44), as observed by
MS The minor, more acidic isoform could either result from
the deamidation of Asn259 (calculated pI 4.39) or from the
presence of a species carrying charged O-glycosylation
Indeed, a species corresponding to the Cel7Bcatpolypeptide
bearing a hexosephosphate (or sulfate) moiety was observed
by MS analysis of the intact protein from some preparations
(not shown) However, the very low abundance of this peak
and the lack of a confirmatory observation in peptide digests
makes such an assignment uncertain
In conclusion, the heterogeity observed in the Cel7B
preparation by electrophoresis and mass spectrometry can
be attributed to N-glycosylation Moreover, the results
indicate both O-glycosylation and deamidation in the
catalytic domain are further complicating factors
Acknowledgements
I S and M C thank the Ghent University Research Council for
support (B/03197/04 IV 1) H.S thanks the Swedish Research Council
(Vetenskapsra˚det) and the Carl Trygger Foundation (CTS) for funding.
The purchase of mass spectrometry equipment at the Royal Institute of
Technology was funded by the Wallenberg Consortium North.
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