Heterogeneity of homologously expressed
Hypocrea jecorina
(
Trichoderma reesei
) Cel7B catalytic module
Torny Eriksson
1,
*, Ingeborg Stals
2,
*, Anna Colle
´n
1
, Folke Tjerneld
1
, Marc Claeyssens
2
, Henrik Sta
˚lbrand
1
and Harry Brumer
3
1
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden;
2
Laboratory for
Biochemistry, Department of Biochemistry, Physiology and Microbiology, Ghent University, Belgium;
3
Department 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.
GlcNAc
2
Man
5
was identified as the predominant N-glycan,
although lesser amounts of GlcNAc
2
Man
7
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 (Man
3
GlcNAc
2
) [12]. However, the
occurrence of a single N-acetyl glucosamine on Cel7A from
H. jecorina strains 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 GlcNAc
2
Hex
8
[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:EndoH,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)
Eur. J. Biochem. 271, 1266–1276 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04031.x
the enzyme were glycosylated with high-mannose structures,
predominantly GlcNAc
2
Man
5
. 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 (Cel7B
cat
) 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-Cel7B
cat
.
H. jecorina strain QM9414-Cel7B
cat
N182Q, expressing
Cel7B
cat
(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 KpnIandtwo
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-
Cel7B
cat
N182Q. 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-Cel7B
cat
and H. jecorina
QM9414-Cel7B
cat
N182Q were cultivated in minimal med-
ium with glucose as the sole carbon source according to
Colle
´net 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
K
2
HPO
4
,8gÆL
)1
KH
2
PO
4
,
4gÆL
)1
(NH
4
)
2
SO
4
,0.6gÆL
)1
CaCl
2
,0.6gÆL
)1
MgSO
4
,5
mgÆL
)1
FeSO
4
7H
2
O,1.6mgL
)1
MnSO
4
H
2
O, 1.4 mgÆL
)1
ZnSO
4
7H
2
O, 2 mgÆL
)1
CoCl
2
and 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 28 Cand
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 m
M
NH
4
OAc, 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 (1
M
NH
4
OAc, 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 lLPharmalyte
TM
pH 2.5–5 (Amersham Biosciences, Uppsala Sweden),
20 lLServalyt
TM pH 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 lLsamples(10mgpro-
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 m
M
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 (40 C)
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 m
M
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 m
M
NaOAc gradient in 100 m
M
NaOH for 35 min
(1 mLÆmin
)1
). A 60–500 m
M
NaOAc gradient in 100 m
M
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 Cel7B
cat
or Cel7B
cat
N182Q in 10 m
M
NaOAc
buffer, pH 4.5, for 12 h at 37 C. 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) CH
3
CN
(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
M
HCl, 100 C, 30 min) as follows. One unit
FEBS 2004 Heterogeneity of H. jecorina (T. reesei) Cel7B (Eur. J. Biochem. 271) 1267
of calf intestine alkaline phosphatase (Roche Diagnostics,
Vilvoorde, Belgium) dissolved in 20 lL100m
M
Tris/HCl,
pH 8.8 containing 10 m
M
ZnCl
2
, 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 Cel7B
cat
or the reference protein
RNAse B in 20 m
M
NaOAc buffer, pH 5, containing 2 m
M
ZnCl
2
. The products of the overnight reaction at room
temperaturewereanalysedbyHPAEC-PAD.
Total acid hydrolysis
Oligosaccharide samples (25 lgÆmL
)1
) were hydrolysed in
4
M
trifluoroacetic acid. After heating at 100 Cfor4hin
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
Cel7B
cat
or 0.05
mg mL
)1
Cel7B
cat
N182Q in 0.1
M
NH
4
HCO
3
, containing
6
M
urea and 5 m
M
dithiothreitol, for 30 min at 60 C.
Iodoacetamide (25 m
M
final concentration) was added and
the samples were incubated in the dark for 30 min at 25 C.
Subsequent dialysis was performed against 1
M
urea either
in 10 m
M
NH
4
HCO
3
(for trypsin digestions) or in 10 m
M
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; 37 C,
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 (37 C, 12 h).
Results
Protein expression and purification
The gene sequence encoding Glu1–Thr371 of Hypocrea
jecorina Cel7B (Cel7B
cat
, 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 Cel7B
cat
(39.1 kDa).
The results from the cultivation of H. jecorina expressing
Cel7B
cat
N182Q were similar to that of Cel7B
cat
,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 Cel7B
cat
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 Cel7B
cat
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 Cel7B
cat
is 39133.8 Da. Figure 3A shows the reconstructed zero-
charge spectrum of purified Cel7B
cat
, in which a range of
peaks is observed. The mass of the major component
corresponds well with the calculated molecular mass for
Cel7B
cat
substituted with Man
5
GlcNAc
2
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.
1268 T. Eriksson et al.(Eur. J. Biochem. 271)FEBS 2004
Endo H-treated Cel7B
cat
shows a major peak with a
molecular mass of 39 538.0 Da (Fig. 3B), which corresponds
to the Cel7B
cat
polypeptide 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 Cel7B
cat
. 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/zvalues 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/z1159.2,
[M+3H]
3+
) and T9+GlcNAc (Table 1, m/z986.4,
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
[M+H]
+
[M+2H]
2+
[M+3H]
3+
Calculated Observed Calculated Observed Calculated Observed
T1 1–13 QQPGTSTPEVHPK 1388.68 1388.65 694.84 694.84 463.57 N.O.
T5 40–68 WMHDANYNSCTVNGGVNTTLCP
DEATCGK
3272.35 N.O. 1636.68 N.O. 1091.46 N.O.
T5 +
GlcNAc
40–68 WMHDANYNSCTVNGGVNTTLCP
DEATCGK + GlcNAc (203.079)
3475.43 N.O. 1738.22 1738.29 1159.15 1159.17
a
T8 123–181 LNGQELSFDVDLSALPCGENGS
LYLSQMDENGGANQYNTAGANY
GSGYCDAQCPVQTWR
6466.94 N.O. 1616.46
[M+4H]
4+
1616.48 1293.37
[M+5H]
5+
1293.59
S7 142–152 NGSLYLSQMDE 1256.55 1256.61 628.78 628.81 419.52 N.O.
T9 182–205 NGTLNTSHQGFCCNEMDILEGNSR 2754.17 N.O. 1377.59 N.O. 918.73 N.O.
T9 +
GlcNAc
40–68 NGTLNTSHQGFCCNEMDILEGNSR
+ GlcNAc (203.079)
2957.25 N.O. 1479.13 1479.16 986.42 986.43
T12 248–271 TFTIITQFNTDNGSPSGNLVSITR 2583.31 N.O. 1292.16 1292.18 861.77 861.79
T15 308–363 ALSSGMVLVFSIWNDNSQYMNWLD
SGNAGPCSSTEGNPSNILANNPNT
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/z869.62.
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.
FEBS 2004 Heterogeneity of H. jecorina (T. reesei) Cel7B (Eur. J. Biochem. 271) 1269
[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/z628.8, [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/z1292.2, [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/z1292.7) 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
Asn56 Asn182 Thr/Ser
Fig. 3A 40 552 GlcNAc Man
5
GlcNAc
2
41 566 Man
5
GlcNAc
2
Man
5
GlcNAc
2
41 729 Man
5
GlcNAc
2
Man
5
GlcNAc
2
Hex
41 890 Man
5
GlcNAc
2
Man
7
GlcNAc
2
41 970 Man
5
GlcNAc
2
(ManP)Man
6
GlcNAc
2
42 133 Man
5
GlcNAc
2
(ManP)Man
6
GlcNAc
2
Hex
42 295 Man
7
GlcNAc
2
(ManP)Man
6
GlcNAc
2
42 374 (ManP)Man
6
GlcNAc
2
(ManP)Man
6
GlcNAc
2
42 457 Man
7
GlcNAc
2
(ManP)Man
6
GlcNAc
2
Hex
Fig. 3B 39 537 GlcNAc GlcNAc
39 700 GlcNAc GlcNAc Hex
1270 T. Eriksson et al.(Eur. J. Biochem. 271)FEBS 2004