Báo cáo Y học: Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding pot

Eur. J. Biochem. 269, 977–988 (2002) Ó FEBS 2002

Tyrosine sulfation and N-glycosylation of human heparin cofactor II
from plasma and recombinant Chinese hamster ovary cells
and their effects on heparin binding
Christoph Bohme1, Manfred Nimtz2, Eckart Grabenhorst3, Harald S. Conradt3, Annemarie Strathmann1
ă
and Hermann Ragg1
1

Faculty of Technology, University of Bielefeld, Germany; 2Molecular Structure Research and 3Protein Glycosylation, Gesellschaft fuăr
Biotechnologische Forschung mbH, Braunschweig, Germany

The structure of post-translational modifications of human
heparin cofactor II isolated from human serum and from
recombinant Chinese hamster ovary cells and their effects on
heparin binding have been characterized. Oligosaccharide
chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood
are complex-type diantennary and triantennary chains in a
ratio of 6 : 1 with the galactose being > 90% sialylated with
a2 fi 6 linked N-acetylneuraminic acid. About 50% of the
triantennary structures contain one sLex motif. Proximal
a1 fi 6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in > 90%
of the diantennary and triantennary glycans, the latter

being slightly less sialylated with exclusively a2 fi 3-linked
N-acetylneuraminic acid units. Applying the ESI-MS/
MS-MS technique, we demonstrate that the tryptic peptides
comprising tyrosine residues in positions 60 and 73 were
almost completely sulfated irrespective of the protein’s origin. Treatment of transfected Chinese hamster ovary cells
with chlorate or tunicamycin resulted in the production of

heparin cofactor II molecules that eluted with higher ionic
strength from heparin–Sepharose, indicating that tyrosine
sulfation and N-linked glycans may affect the inhibitor’s
interaction with glycosaminoglycans.

Heparin cofactor II (HCII) is a single-chain glycoprotein
with a carbohydrate content of about 10% circulating in
blood [1]. The protein is a member of the serpin (serine
protease inhibitor) superfamily, most members of which
inhibit serine proteases by forming SDS stable complexes
with their target enzymes. HCII functions as an inhibitor of
cathepsin G, chymotrypsin and thrombin. After addition of
heparin or dermatan sulfate, the rate of HCII/thrombin
interaction is enhanced several orders of magnitude and the
inhibition rate approaches the value observed for thrombin
inhibition by antithrombin (AT) in the presence of heparin,
indicating that HCII may be an interesting anticoagulant
and antithrombotic agent. Increased plasma levels of HCII
have been found under a variety of pathophysiological
conditions, suggesting that the protein may be involved in
the acute phase response [2,3].

HCII has been biochemically characterized in some
detail. As deduced from the cDNA sequence, the mature
human protein consists of 480 amino acids and contains
three potential N-glycosylation sites at positions Asn30,
Asn169 and Asn368 [4,5]. In the mouse, two HCII variants
that appear to differ in number and/or structure of their
glycans circulate in blood [6]. In addition, two tyrosine
sulfation signals with the characteristic accumulation of

acidic amino acids are present close to the N-terminus of all
HCII sequences known [7,8]. Sulfation of these tyrosine
residues has been demonstrated for HCII secreted from a
human hepatoma cell line [9].
Composition and stoichiometry of the monosaccharide
units of the inhibitor molecule have been analyzed [1,10]
with mannose, galactose, N-acetylglucosamine, and sialic
acid being the main elements of HCII glycans, the structure
of the carbohydrate chains, however, is unknown. Glycosylation may have profound effects on a variety of biological
features of proteins [11]. This is also evident for human AT,
which exists in two isoforms (AT-a and AT-b). These
variants differ substantially in their affinity for heparin
(elution from heparin–Sepharose at about 0.8 and 1.2 M
NaCl, respectively) due to differential N-glycosylation
[12,13]. As a consequence, AT-b is predominantly associated
with endothelial and subendothelial cells [14,15], providing
the vessel wall with a strongly antithrombotic surface.
HCII isolated from human plasma displays a low affinity
for heparin as indicated by the low NaCl concentration
required for its dissociation from heparin–Sepharose (about
0.25 M NaCl). Various experiments, however, have shown
that HCII has a considerably higher intrinsic propensity for

Correspondence to H. Ragg, Faculty of Technology, University of
Bielefeld, Universitatsstr. 25, D-33501 Bielefeld, Germany.
ă
Fax: + 49 521106 6328, Tel.: + 49 521106 6321,
E-mail:
Abbreviations: AT, antithrombin; CHO, Chinese hamster ovary;
dhfr, dihydrofolate reductase; DMEM, Dulbecco’s modified Eagle’s

medium; GAG, glycosaminoglycan; HCII, heparin cofactor II;
HPAEC-PAD, high pH anion-exchange chromatography with pulsed
amperometric detection; MEM, minimal essential medium; MTX,
methotrexate; NeuAc, N-acetylneuraminic acid; PEG, poly(ethylene
glycol); PNGase F, polypeptide:N-glycosidase F; sLex, sialyl Lewis X.
(Received 26 September 2001, revised 6 December 2001, accepted 11
December 2001)

Keywords: heparin cofactor II; glycosylation; tyrosine
sulfation; heparin binding; serpins.


978 C. Bohme et al. (Eur. J. Biochem. 269)
ă

heparin binding, approaching that of AT-a. HCII mutants
devoid of an acidic region (positions 53–75), which resembles the C-terminal tail of hirudin, require > 0.7 M NaCl for
elution from heparin–Sepharose [16–18], suggesting that
this polyanionic domain may affect the heparin-binding
properties of HCII. Here, we report on the structure of posttranslational modifications of plasma-derived and recombinant human HCII from CHO cells and their effects on
heparin binding.

MATERIALS AND METHODS
Outdated citrated human plasma was a gift of Centeon AG.
Bovine insulin, MTX, PEG, polybrene, sodium chlorate,
sulfate-deficient DMEM/Ham’s F12 1 : 1 mixture, and
tunicamycin were obtained from Sigma. Culture media and
fetal bovine serum were purchased from Life Technologies.
Heparin-HiTrap (5 mL), Mono Q HR5/5 and Superdex
200 HR 10/30 were from Pharmacia. Standard grade BSA

was supplied from Serva. A Sartocon Micro 30-kDa
MWCO cross flow module was from Sartorius. Human
transferrin was purchased from Bayer AG. PeptideN4-(N-acetyl-4-D-glucosaminyl)asparagine amidase F (from
Flavobacterium meningosepticum) from recombinant
Escherichia coli was bought from Roche Molecular Biochemicals. Vibrio cholerae sialidase was from Calbiochem.
Stable expression and amplification of HCII in CHO cells
Plasmid pWTBI2 was constructed by ligating a 1.6-kb
HindIII/EcoRI human HCII cDNA fragment into a
HindIII/EcoRI-cleaved expression vector of the pSV2
plasmid series [19]. The HCII cDNA spanned the protein
coding sequence (including the signal peptide) down to the
EcoRI site at position 1559 in the 3¢ untranslated region
(numbering as described previously [4]). The expression of
the thrombin inhibitor cDNA in this construct is regulated
by the SV40 early promoter and the polyadenylation signal
for SV40 early mRNAs.
Plasmid pWTBI2 and plasmid pSV2dhfr, which encodes
a mouse dihydrofolate reductase cDNA under the control
of the SV40 early promoter, were mixed at a ratio of 5 : 1
(w/w) and transfected into DHFR-deficient CHO cells
(routinely cultured in Ham’s F12 medium) by the polybrene
method, following established procedures [20]. After
2–3 weeks of selection in nucleoside-free a-MEM containing 10% dialyzed serum, 2 mM L-glutamine and 0.3 mM
L-proline, individual colonies were picked and expanded.
Cell lines expressing increased amounts of HCII were
isolated by augmenting the MTX concentration in increments (0.05, 0.25, 2.5, and 100 lM, respectively).
The amount of recombinant HCII secreted from MTXselected cell clones was determined by a sandwich ELISA
employing a monoclonal mouse anti-HCII capturing Ig and
a horseradish peroxidase-coupled monoclonal mouse antiHCII detection antibody. Assays were performed in 96-well
microtiter plates (Nunc) with HCII from human plasma as

standard. The enzyme-catalyzed oxidation of 3,3¢,5,5¢
tetramethylbenzidine was monitored at 405 nm with a
microtiter plate autoreader (BioTek Instruments). Cells
were counted using the trypan blue exclusion assay and the
specific HCII productivity (lg per 106 cells per day) was
calculated.

Ó FEBS 2002

Treatment of HCII expressing CHO cells with inhibitors
of N-glycosylation and tyrosine sulfation
Recombinant CHO cells producing human HCII were
grown close to confluency in DMEM/Ham’s F12 medium.
After washing twice with serum-free medium, cells were
incubated in serum-free medium in the presence of tunicamycin at the concentrations indicated in the figure legends.
After 4 h, fresh medium containing the same concentration
of the glycosylation inhibitor was added. Three days later,
the conditioned medium was harvested and inspected by
Western blotting for the presence of HCII as described
previously [21]. Treatment of cells with sodium chlorate
(20 mM) followed the same procedure, except that cells were
cultivated in sulfate-deficient DMEM/Ham’s F12 1 : 1
mixture during the presence of the sulfation inhibitor.
Purification of HCII from plasma and from recombinant
CHO cells
HCII from outdated citrated human plasma of a single
blood donor was isolated essentially as described previously,
including precipitation with barium chloride and poly(ethylene glycol) [22], chromatography on heparin–Sepharose and
on Mono Q [6]. Highly purified HCII suitable for
glycoanalysis was obtained through inclusion of a final gel

filtration step on Superdex 200 HR 10/30.
For mass production of recombinant HCII, cells from the
CHO clone Bi2/100/6/13/3/19 were trypsinized and cultured
in a Superspinner [23], consisting of a Duran flask (capacity
1 L) equipped with a magnetic membrane stirrer in order to
improve the oxygen supply by bubble-free aeration. The
device was placed on a stirring plate in a CO2 incubator
together with a small membrane pump, which supplied the
membrane stirrer with the incubator gas. The starting
volume (550 mL) was inoculated with 2.5 · 105 cellsỈmL)1
from five confluent T175-flasks, which had been propagated
in DMEM/Ham’s F12 medium with 2% fetal bovine
serum. To remove bovine HCII, the serum content was
successively reduced further in a combined repeated/fed
batch process. To this end, the cell suspension culture was
diluted to a cell density of 2.5 · 105 cellsỈmL)1, filled up to
the maximum working volume (1 L) with serum-free
DMEM/Ham’s F12 medium supplemented with 1 gỈL)1
BSA, 3 mM glutamine, 10 lgỈmL)1 bovine insulin,
10 lgỈmL)1 human transferrin and antibiotics, and grown
to the early stationary phase. After two further dilution
steps, the cells were finally cultivated for 5 days in serumfree medium supplemented with 0.5 gỈL)1 BSA, amino
acids, and glucose.
For isolation of recombinant HCII, the cells were
removed by centrifugation, and after addition of phenylmethanesulfonyl fluoride (10 lM), the supernatant was
concentrated about threefold in a cross-flow ultrafiltration
module and dialyzed against 10 mM Tris/HCl, 1 mM
EDTA, 10 lM phenylmethanesulfonyl fluoride, 5 mM
NaCl, pH 7.4. The following isolation procedures included
fractionation on heparin–Sepharose and chromatography

on Mono Q essentially as described above, except that the
Tris/EDTA buffer system was used for heparin–Sepharose
chromatography. For glyco-analysis, HCII was rechromatographed on heparin–Sepharose with a linear 0–1 M NaCl
gradient (40 mL) and on Superdex-200 HR 10/30. Purity


Ó FEBS 2002

Heparin cofactor II: N-glycans and sulfation (Eur. J. Biochem. 269) 979

was assessed by reducing SDS/PAGE on 10% gels run in a
Tris/glycine buffer system, according to the manufacturer’s
protocol (Novex), and subsequent visualization of proteins
by Coomassie blue staining. The homogeneity of HCII from
plasma and from recombinant CHO cells was checked by
N-terminal sequence analysis using an Applied Biosystems
ProciseTM instrument.
Reduction, carboxamidomethylation and tryptic
digestion of HCII from plasma and from recombinant
CHO cells
One to two nanomoles of the purified protein were reduced,
carboxamidomethylated and digested with trypsin;
RP-HPLC separation of resulting peptides was performed
as described previously [24].
Separation of N-glycans by Mono Q anion-exchange
chromatography
Oligosaccharides were liberated quantitatively by treatment
of the glycoprotein preparations (2 mgỈmL)1) with 15 U of
recombinant PNGase F in 50 mM sodium phosphate buffer
(pH 7.2) for 8 h at 37 °C.

In order to separate native N-glycans according to
charge, desalted oligosaccharides were dried using a SpeedVac and redissolved in 0.5 mL of MilliQ water. The
N-glycans were applied to a Mono Q HR 5/5 column at
room temperature and eluted at 1 mLỈmin)1 with a mixture
of water (solvent A) and 0.5 M NaCl (solvent B). Chromatographic conditions were: a 5-min isocratic run with
100% A, followed by a linear gradient to 10% B for 15 min,
an isocratic run using 10% B for 10 min and a final linear
gradient to 100% B over 1 min. Oligosaccharides were
detected by their ultraviolet absorption at 206 nm. Fractions of 0.5 mL were collected.
Enzymatic and chemical removal of sialic acid
Vibrio cholera neuraminidase (2.5 lL; 1 mL)1) was
added to the samples containing 0.5 nmol of total oligosaccharides in 25 lL of sodium acetate, pH 5.0, 5 mM
CaCl2 and 0.02% NaN3 (w/v). The reaction mixture was
incubated for 2 h at 37 °C. For the chemical removal of
NeuAc, N-glycans were incubated in 100 lL of 0.2%
trifluoroacetic acid for 1 h at 82 °C.
High-pH anion-exchange chromatography with pulsed
amperometric detection
Purified native and desialylated oligosaccharides were
analyzed by high-pH anion-exchange (HPAE) chromatography using a Dionex BioLC system (Dionex, Sunnyvale,
CA, USA) equipped with a CarboPac PA1 column
(0.4 · 25 cm) in combination with a pulsed amperometric
detector (PAD) [25,26]. Detector potentials (E) and
pulse durations (T) were: E1: + 50 mV, T1: 480 ms;
E2: + 500 mV, T2: 120 ms; E3: ) 500 mV, T3: 60 ms,
and the output range was 500–1500 nA. The oligosaccharides were then injected into the CarboPac PA1 column that
was equilibrated with 100% solvent C. Elution (flow rate of
1 mLỈmin)1) was performed by applying a linear gradient
(0–20%) of solvent D over a period of 40 min followed by a

linear increase from 20 to 100% solvent B over 5 min
Solvent C was 0.1 M NaOH in bidistilled H2O, solvent D
consisted of 0.6 M NaOAc in solvent C.
Reduction and permethylation of oligosaccharides
The enzymatically liberated N-glycans were reduced and
permethylated as described previously [27].
MALDI-TOF MS
The reduced and carboxamidomethylated tryptic peptides
of the HCII preparations were subjected to positive ion
matrix-assisted laser desorption/ionization (MALDI) mass
spectrometry, using a Bruker REFLEX time-of-flight
(TOF) instrument equipped with delayed-extraction and
reflectron systems and a N2 laser (337 nm) operating with
3-ns pulse width and 107)108 WỈcm)2 irradiance at the
surface of 0.2 mm2 spots. In addition to the positive mode
standard procedure for the detection of sulfated peptides
using the reflectron for enhanced resolution, the peptide
mixture was also analyzed in the positive and negative
linear ion mode. One-microliter samples containing equal
volumes of peptide solution (10 pmolỈlL)1) and the
ultraviolet-absorbing matrix [19 mg a-cyano-4-hydroxycinnamic acid in 400 lL acetonitrile and 600 lL 0.1% (v/v)
trifluoroacetic acid in H2O] were spotted onto the stainless
steel target and dried at room temperature. Determination
of the molecular masses of reduced and permethylated
oligosaccharides was carried out similarly in the positive
ion mode using the reflectron. One-microliter samples
containing equal volumes of reduced and permethylated
oligosaccharide solution ( 10 pmolỈlL)1) and a-cyano4-hydroxycinnamic acid matrix were mixed and spotted on
the target. Sodium chloride was added to the matrix to a
final concentration of 5 lM in order to guarantee the

exclusive generation of sodium adducts of the carbohydrate molecular ions.
Tandem electrospray ionization (ESI) mass spectrometry
The peptide samples were dissolved in a 1 : 1 mixture of
MeOH/H2O (the reduced and permethylated oligosaccharide samples in 9 : 1 MeOH/H2O) to a concentration of
 3 pmolỈlL)1, and gold-coated nanospray glass capillaries
(Protana, Odense, Denmark) were filled with 3 lL of this
solution. The tip of the capillary was placed orthogonally in
front of the entrance hole of a QTOF II mass spectrometer
(Micromass, Manchester, England) equipped with a nanospray ion source, and a voltage of  800 V was applied. For
collision-induced dissociation experiments, parent ions were
selectively transmitted from the quadrupol mass analyzer
into the collision cell. Argon was used as the collision gas
and the kinetic energy was set from )15 to )60 eV for
optimal fragmentation. The resulting daughter ions then
were separated by an orthogonal TOF mass analyzer.

RESULTS
Expression of HCII from CHO cells
Human HCII cDNA was expressed under the control of the
SV40 early promoter in DHFR-deficient CHO DUKX-B1


980 C. Bohme et al. (Eur. J. Biochem. 269)
ă

cells, which had been cotransfected with plasmid pSV2dhfr.
More than 20 individual clones growing in selection
medium were examined, but initially no cell lines producing
sufficient HCII detectable by the ELISA technique were
identified. Therefore, individual clones were isolated at

random, exposed to 0.05 lM MTX, expanded, and checked
for the presence of human HCII mRNA by RT-PCR.
Positive cell lines were picked and after several rounds of
selection with increasing MTX concentrations, cell lines
expressing up to 17 lg HCII per 106 cells per day were
isolated.
Production, purification and characterization
of recombinant and plasma HCII
The high producer clone Bi2/100/6/13/3/19, selected in
medium containing 100 lM MTX was cultivated in
suspension in a 1-L superspinner by successively reducing
the serum concentration. During the production phase,
the serum-free cultivated cells accumulated HCII to a
concentration of about 11 lgỈmL)1 within a 5-day period.
The recombinant inhibitor and HCII from plasma
were purified to apparent homogeneity as assessed by
SDS/PAGE (not shown) and subjected to sequence
analysis. The N-terminus of the inhibitor consisted of a
single sequence (GSKGPLDQLEKGGE), irrespective of
whether HCII had been isolated from plasma or from
recombinant CHO cells. Thus, these results are in agreement
with the accurate and efficient cleavage of the 19-amino-acid
signal peptide in CHO cells [5,28].
Characterization of the tryptic peptides
of serum-derived and recombinant HCII
Natural and recombinant HCII were characterized further
by peptide mapping. After reduction and carboxamidomethylation, the proteins were digested with trypsin and the
resulting peptide mixtures were subjected to RP-HPLC
mapping. The elution profiles of both protein preparations
were almost identical (data not shown). This result was

further corroborated by MALDI-TOF-MS analysis of the
tryptic peptide mixtures (compare Fig. 1 and Table 1).
Most signals could be assigned, resulting in a sequence
coverage of almost 70%. The pattern of peptides obtained
from both proteins was very similar, indicating an identical
polypeptide backbone of the natural and the recombinant
HCII. Position 218, for which an amino-acid polymorphism
has been reported (Lys or Arg [4,5]), is populated with a
lysine residue in the plasma derived protein. We note that
higher amounts of peptides containing oxidized methionine
were detected in the recombinant polypeptide preparation
(see legend to Table 1).
Identification of sulfated tyrosine peptides
and glycosylation site occupancy in trypsin digests
of serum-derived and recombinant HCII
Human HCII from HepG2 cells has been found to be
sulfated at two tyrosine residues [9]. In our experiments,
however, solely the unmodified forms of the corresponding
tryptic peptides T43–65 and T66–101 were detected when
the cleavage products from both protein preparations were
analyzed by the standard MALDI-TOF-MS techniques.

Ó FEBS 2002

Even after desialylation by mild acid hydrolysis, no MALDI
signals corresponding to N-glycopeptides were identified in
the pertinent spectra (Fig. 1). The tryptic peptides of both
protein preparations were therefore additionally subjected
to ESI-MS analysis. Using this ionization technique, the
tyrosine containing peptides T43–65 and T66–101 were

found predominantly in their post-translationally modified
forms (Fig. 2). These findings can be explained by the
pronounced instability of tyrosine sulfate under the conditions routinely applied for positive ion MALDI-TOF-MS
of peptides.
A complete series of sequence-specific fragment ions
was only found with the smaller sulfopeptide upon
ESI-MS/MS, whereas the larger one yielded an intense
signal generated by elimination of SO3 and a relatively
weak fragment due to peptide cleavage at the proline
residue. The daughter ion spectra of the sulfated and
desulfated
peptide
ENTVTNDWIPEGEEDDDYLDLEK(43–65) obtained after increasing the collision energy
were found to be almost identical (data not shown),
confirming the amino-acid sequence of the corresponding
peptide from the recombinant and the serum-derived HCII.
As no modified fragment ions were detected after collisioninduced dissociation from the sulfated peptide, the position
of sulfation could not be deduced from this spectrum. Such
behaviour can be explained by the spontaneous elimination
of SO3 from any possible peptide fragment generated.
Therefore, the determination of the sequence position of
sulfated tyrosine residues appears to be impossible by
classical mass spectrometric peptide sequencing techniques.
However, in the case of the peptides under consideration
here, the position of sulfation is unambiguous, as both
peptides contain only a single tyrosine residue, and
sulfation at Ser/Thr residues is improbable, in view of the
characteristic consensus sequence for tyrosine sulfation
[29,30] present in both peptides.
In addition to the identification of tyrosine sulfate

residues detected with the ESI technique, we were also
able to identify the dominant glycoforms of all three
potential glycopeptides [NLSMPLLPADFHK(30–42),
DFVNASSK(166–173), SMTNR(T) (365–370)] after desialylation by mild acid hydrolysis (Table 1). As the corresponding unmodified peptides were neither detectable by
MALDI nor by ESI-MS, we deduce from these results that
all three potential HCII N-glycosylation sites are completely
glycosylated. This conclusion is also supported by the SDS/
PAGE pattern of HCII from CHO cells treated with limited
concentrations of tunicamycin (Fig. 3).
In contrast to the serum-derived protein, which predominantly contained a diantennary carbohydrate structure
attached to each glycosylation site, the monofucosylated
(plus 146 Da) derivative was observed for the glycopeptides
of the recombinant protein. The expected linkage position
of this fucose unit to the proximal GlcNAc residue could be
confirmed by MS/MS of the respective molecular ions by
the identification of a fragment ion generated by the
cleavage of the chitobiose bond of the N-glycan derived
from serum HCII. Generally, intense carbohydrate specific
fragments were detected (fragment ions generated by
elimination of monosaccharide residues from the molecular
ion, as well as intense pure carbohydrate fragments), but
only very weak peptide sequence specific fragment ions
(data not shown).


Ó FEBS 2002

Heparin cofactor II: N-glycans and sulfation (Eur. J. Biochem. 269) 981

Fig. 1. MALDI-TOF-MS tryptic peptide fingerprints of natural (A) and recombinant (B) HCII. Amino-acid sequence, calculated, and experimentally detected masses of the peptide fragments are summarized in Table 1. Both protein preparations yielded a very similar peptide pattern,

suggesting an identical polypeptide backbone. The oxidation rate of the methionine residues, however, was markedly higher for the recombinant
protein, as can be clearly seen, e.g. for the peptides T311-343 [m/z 3672.1) or T420-449 [m/z 3206.2], each containing a single methionine residue. We
failed to detect any of the N-glycopeptides using the MALDI technique, even after desialylation. The dominant glycoforms of all three glycopeptides, however, were readily observed employing the ESI technique. Peptides T43-65 and T66-101, which had been reported to be sulfated [9],
were observed only in the desulfated form due to elimination of SO3. By ESI-MS (see Fig. 2) it could be unequivocally demonstrated that serum
HCII as well as the recombinant species are predominantly sulfated. *, incompletely cleaved peptide.

Detailed characterization of the N-glycans from serum
and CHO cell-derived HCII
For a more detailed investigation of the N-glycan structures,
we performed additional mass spectrometric analyses of the
oligosaccharides liberated by PNGase F. After reduction
and permethylation of the total glycan mixtures, the
MALDI-TOF spectra depicted in Fig. 4 revealed the
presence of a mainly diantennary disialylated structure
and approximately 15% of triantennary trisialylated chains
in both proteins (compare legend to Fig. 4), confirming the
results obtained from the ESI-MS analysis of the tryptic
peptides described above. The CHO cell-derived N-glycans
were almost completely fucosylated at the proximal
GlcNAc residue, whereas only about 10% of the serum
protein showed fucosylation of the diantennary and

triantennary N-glycans. Approximately 60% of the triantennary structures isolated from the serum HCII contained
a fucose residue. In order to determine the linkage position
of the fucose to the triantennary structure, ESI-MS/MS
analysis was performed on the triply charged molecular ion
[M + 3Na]3+ after isolation of the trisialylated oligosaccharide fraction by anion exchange chromatography on a
Mono Q column [31]. From the resulting daughter ion
spectrum (Fig. 5), we conclude that the fucose unit was not
linked to the proximal GlcNAc residue, which is characteristic for the CHO cell-derived material, but was rather

attached to a peripheral GlcNAc residue as is indicated
by the weak fragment at m/z 1021.6 [NeuNAc-Hex(dHex)HexNAc + Na] and its more intense secondary
fragment at m/z 646.3 [HO-Hex-(dHex)HexNAc + Na]
(see fragmentation scheme and legend of Fig. 5). This


Ó FEBS 2002

982 C. Bohme et al. (Eur. J. Biochem. 269)
ă

Table 1. Amino-acid sequence and tryptic peptides of human HCII. The peptides indicated below were detected by MALDI-TOF- and/or ESI-MS
(see Figs 1 and 2, respectively) in tryptic digests of human HCII isolated from serum or recombinant CHO cells, respectively, indicating their
identical polypeptide backbones. In the serum-derived protein  10–20% of the methionine residues were oxidized, whereas in the recombinant
polypeptide about 50% of these residues occurred in the oxidized form. Peptides smaller than 650 Da were not detected (ND). With the inclusion of
the glycopeptides detected only by ESI-MS, a sequence coverage of more than 80% was achieved. N-glycosylation consensus sequences are
underlined, sulfated tyrosine residues are typed in bold. w, m, s ¼ weak, middle or strong signal, respectively. GP, glycopeptide. glyc, glycan.
Amino-acid sequence

Residue nos

[M + H]+

Calculated

GSK
GPLDQLEK
GGETAQSADPQWEQLNNK
NLSMPLLPADFHK
ENTVTNDWIPEGEEDDDYLDLEK

IFSEDDDYIDIVDSLSVSPTDSDVSAGNILQLFHGK
SR
IQR
LNILNAK
FAFNLYR
VLK
DQVNTFDNIFIAPVGISTAMGMISLGLK
GETHEQVHSILHFK
DFVNASSK
YEITTIHNLFR
K
LTHR
LFR
K
NFGYTLR
SVNDLYIQK
QFPILLDFK
TK
VR
EYYFAEAQIADFSDPAFISK
TNNHIMK
LTK
GLIK
DALENIDPATQMMILNCIYFK
GSWVNKFPVEMTHNHNFR
LNER
EVVK
VSMMQTK
GNFLAANDQELDCDILQLEYVGGISMLIVVPHK
MSGMK

TLEAQLTPR
VVER
WQK
SMTNR(T)
TR
EVLLPK
FK
TR
NYNLVESLK
LMGIR
MLFDK
NGNMAGISDQR
IAIDLFK
HQGTITVNEEGTQATTVTTVGFMPLSTQVR
FTVDRPFLFLIYEHRTSCLLFMGR
VANPSR
S

1–3
4–11
12–29
30–42
43–65
66–101
102–103
104–106
107–113
114–120
121–123
124–151

152–165
166–173
174–184
185
186–189
190–192
193
194–200
201–209
210–218
219–220
221–222
223–242
243–249
250–252
253–256
257–277
278–295
296–299
300–303
304–310
311–343
344–348
349–357
358–361
362–364
365–370
370–371
372–377
378–379

380–382
383–391
392–396
397–401
402–412
413–419
420–449
450–473
474–479
480

ND
899.5w
1973.1m
GP1a
2739.4mb
3912.1mb
ND
ND
785.5w
930.6s
ND
2952.7w
1660.9m
GP2a
1405.9m
ND
ND
ND
ND

870.5s
1079.8wd
1120.8w
ND
ND
2312.3w
857.5m
ND
ND
2500.4wc
2201.3me
ND
ND
823.5w
3672.1m
ND
1028.7m
ND
ND
GP3a
ND
ND
ND
ND
1079.8wd
ND
ND
1162.7w
819.6 m
3206.2s

3017.8mc,e
ND
ND

291.2
899.5
1972.9
1482.8 + glyc
2739.2
3911.9
262.1
414.3
785.5
930.5
58.3
2952.5
1660.8
867.4 + glyc
1406.7
147.1
526.3
435.3
175.1
870.4
1079.6
1120.6
248.2
274.2
2312.1
857.4

361.2
430.3
2500.2
2201.1
530.3
474.3
823.4
3671.8
552.2
1028.6
502.3
461.2
608.3 + glyc
276.2
698.4
294.2
389.2
1079.6
588.3
653.3
1162.5
819.5
3206.5
3017.5
643.3
105.0

a
Glycopeptide; using ESI-MS, the most abundant glycoforms could be detected (diantennary complex type with two NeuAc residues
(serum protein) or diantennary complex type with proximal fucose and two NeuAc units (recombinant protein). b Peptide containing a

sulfated tyrosine. Due to the easy elimination of SO3, MALDI-MS allowed the detection of only the desulfated molecular ion. With
ESI-MS, however, the predominant presence of a sulfopeptide was unequivocally detected (see text and Fig. 2). c Peptide bearing a
carboxamidomethylated cysteine residue. d The presence of two peptides with identical molecular masses but different amino-acid sequences
could be unequivocally shown by ESI-MS/MS. e Peptide with one missed cleavage site.


Ó FEBS 2002

Heparin cofactor II: N-glycans and sulfation (Eur. J. Biochem. 269) 983

interpretation was further corroborated by the detection of
the intense fragment at m/z 316.2 [GlcNAc-ol + Na]
instead of the fragment characteristic for proximally
fucosylated N-glycans at m/z 490 [dHex-HexNAc-ol +
Na]. Even the linkage position of the fucose residue at the
N-acetyllactosamine antennae could be assigned from the
daughter ion spectra, due to the well-known preferential
elimination of the 3-linked substituent of the GlcNAc
residue of the N-glycan antenna [32]. A weak, but reproducible signal at m/z 440.3 was detected, which is generated
by the elimination of fucose from the fragment ion at m/z
646.3. Therefore, fucose must be linked to O-3 of a GlcNAc
residue, indicating the presence of a sLex unit rather than a
sLea motif in the N-linked oligosaccharides from serumderived HCII.
HPAEC-PAD mapping of the desialylated oligosaccharides enabled the quantitation of the basic oligosaccharide chains present in both glycoprotein preparations
and demonstrated again the very similar sialylation
degree of both proteins. Table 2 summarizes the glycosylation characteristics of recombinant and serum HCII

based on mass spectrometry results and HPAEC-PAD
mapping using oligosaccharides of known structure as
standard.

Heparin binding properties of HCII treated
with inhibitors of post-translational modifications
In order to investigate the influence of post-translational
modifications on heparin binding, recombinant CHO cells
were treated with inhibitors of tyrosine sulfation and
N-glycosylation under conditions that allowed partial
inhibition of these modifications. After dialysis, the conditioned medium was fractionated on heparin–Sepharose with
a linear NaCl gradient (Fig. 6). HCII produced in the
presence of 20 mM sodium chlorate dissociated in a bimodal
manner from the affinity matrix. The first peak is observed
at  280 mM NaCl, a concentration characteristic for HCII
synthesized in the absence of the sulfation inhibitor.
A second peak is present at  430 mM NaCl, and a
considerable amount of HCII eluted at still higher ionic
strength, a property not associated with HCII from cells

Fig. 2. Mass region of the triply charged molecular ions of the two tyrosine sulfated tryptic peptides recorded by ESI-MS of (A) serum-derived HCII
and (B) the recombinant protein from CHO cells. The detected molecular ions of these peptides (43-ENTVTNDWIPEGEEDDDYLDLEK-65 and
66-IFSEDDDYIDIVDSLSVSPTDSDVSAGNILQLFHGK-101) are compatible with the presence of one tyrosine O4-sulfate ester in each peptide
(calculated m/z of monoisotopic masses for the triply charged monosulfated molecular ions [M + 3H]3+: 940.4 and 1331.3, corresponding to a
molecular mass of 2818.1 and 3990.8 Da, respectively). Arrows indicate the expected positions for the molecular ions of the triply charged
unsulfated peptide species. Approximately 10% of the smaller peptide were present in its unmodified form in both protein preparations. In contrast,
the larger peptide from the natural protein was completely sulfated, while about 20–30% of this fragment from the recombinant protein did not
contain this modification.


984 C. Bohme et al. (Eur. J. Biochem. 269)
ă

Fig. 3. Electrophoretic resolution of recombinant HCII from CHO cells

incubated with various concentrations of tunicamycin. After concentration ( fivefold), the medium was fractionated by SDS/PAGE and
examined by Western blotting for the presence of HCII. Lanes 1–5,
HCII from cells treated with the indicated concentrations of tunicamycin; lane 6, recombinant HCII from an independent experiment.
For comparison, PNGase F-treated HCII from CHO cells (lane 7) and
purified HCII from recombinant CHO cells (lane 8) were included.

grown in normal medium. A shift towards elution at higher
salt concentrations, albeit less pronounced, was also
observed for the totally unglycosylated  64 kDa HCII
form, secreted from CHO cells grown in the presence of
1 lgỈmL)1 tunicamycin. Similar results were obtained with
HCII from HepG2 cells cultivated in the presence of
tunicamycin (not shown).

DISCUSSION
In this investigation we have analyzed the structure of posttranslational modifications of human HCII from circulating blood and genetically modified CHO cells and their
effects on heparin binding. All three potential N-glycosylation sites were found to be populated with complex type
carbohydrate chains. Interestingly, sLex motifs were detected in triantennary oligosaccharides of plasma HCII,
whereas only trace amounts of this structural motif were
present in the diantennary glycan fraction. It remains to be
determined which structural features discriminate N-glycans for addition of this modification, and which types of
a1,3/4-fucosyltransferases [33] are able to decode the
involved signals. The presence of sLex structures in HCII
is an intriguing finding in the light of reports which indicate
that several proteins associated with the acute phase
response in humans contain altered glycostructures
[34,35] and that HCII levels in plasma are increased under
inflammatory conditions [2,3]. Therefore, it could be
possible that the N-glycan structures of HCII are also
changed under certain (patho)-physiological situations. We

have detected triantennary oligosaccharides containing
the Lex motif also in a1-microglobulin, hemopexin, and
in a1-antitrypsin, whereas human serum AT contains this

Ó FEBS 2002

structural motif almost exclusively in 5% of the diantennary oligosaccharides (H. S. Conradt & M. Nimtz,
unpublished observations). It remains to be established
whether the amounts of sLex containing oligosaccharides
synthesized by the liver are accomplished by a concomitant
increase in the levels of the a2 fi 3 sialyltransferase and
a1 fi 3 fucosyltransferase VI.
sLex units linked to cell-bound molecules on the surface
of leukocytes have been found to interact with selectins
exposed on endothelial cells [36]. It may be envisaged that
sLex bearing HCII molecules could interfere with these
processes resulting in decreased plasma levels of the
inhibitor. On the other hand, interaction of HCII
molecules with receptors recognizing sLex-carrying structures on vessel-lining cells could lower the risk of
thrombotic events. Blood from a single donor was used
in this work for the analysis of post-translational modifications. As glycan structures between individuals may
differ, further investigations may determine whether
qualitative or quantitative changes in the carbohydrate
pattern of HCII correlate with specific (patho)-physiological situations.
There were several features specific for human HCII
expressed in CHO cells; compared to plasma HCII, only
a1 fi 6 fucosylation of the recombinant HCII was observed
and sLex structures were not detected (the trace amounts of
difucosylated oligosaccharides contain an additional fucose
residue a1 fi 2 linked to galactose, thus constituting the

Lewis H motif (for review see [37]). The NeuAc units are
linked in a CHO cell-characteristic manner by a2 fi 3
bonds, consistent with the lack of a2 fi 6 sialyltransferase
activity in CHO cells [38,39].
Tyrosine sulfate has been implicated in several biological
roles like leukocyte adhesion and haemostasis [40]. HCII
contains two adjacent sequences (positions 53–62 and
69–75, respectively) with similarity to the consensus signals
characteristic for tyrosine sulfation. The presence of
tyrosine O4-sulfate esters in this domain, which resembles
the acidic C-terminal tail of hirudin, has previously been
reported for HCII from HepG2 cells [9]. We were not able
to detect this modification when routine positive ion
MALDI-TOF conditions were used. This issue has recently
been addressed [41]; tyrosine sulfated peptides readily
eliminate SO3 and therefore solely the desulfated peptide
form was detected. Even when applying negative ion
MALDI in the linear mode, as has been recommended
[41], we could detect only very small amounts of the
sulfated molecular ion signal compared to the nonsulfated
peptide signal. Therefore, the detection and quantitation of
polypeptide modification by sulfate provides a major
challenge to mass spectrometric analysis. In contrast to
the situation with peptide phosphorylation, which can be
detected in the positive as well as in the negative ion mode,
it is difficult or even impossible to detect tyrosine sulfation
by MALDI-TOF-MS. With the electrospray technique
applying very soft nozzle/skimmer conditions, degradation
of the peptide sulfates was minimized or almost avoided.
During MS/MS experiments on both sulfated peptides, we

observed a very facile elimination of SO3 even under very
mild conditions where peptide bonds remained completely
intact.
The ESI-MS results presented here clearly show that
serum HCII as well as its recombinant counterpart


Ó FEBS 2002

Heparin cofactor II: N-glycans and sulfation (Eur. J. Biochem. 269) 985

Fig. 4. MALDI-TOF mass spectra of the reduced and permethylated total N-glycans enzymatically liberated from human HCII isolated (A) from
human serum or (B) produced by genetically engineered CHO cells. The following complex type carbohydrate structures were assigned to the detected
molecular ions [M + Na]+: 2622, diantennary monosialylated with one fucose residue; 2796, diantennary monosialylated with two fucose residues;
2809, diantennary disialylated; 2983, diantennary disialylated with one fucose residue; 3432, triantennary disialylated with one fucose residue; 3619,
triantennary trisialylated; 3793, triantennary trisialylated with one fucose residue; 4242, tetraantennary trisialylated with one fucose residue
(monoisotopic masses). The major difference between the oligosaccharides from the natural protein compared to its recombinant counterpart is the
almost complete proximal fucosylation and a slightly lower degree of sialylation of the recombinant material. The triantennnary trisialylated
N-glycan with one fucose residue (marked by an arrow) is the only major molecular ion observed in both glycoproteins. ESI-MS/MS (compare
Fig. 5), however, showed that the fucose residue in the triantennary structure from the serum protein is not linked to the proximal GlcNAc, but
peripherally to an N-acetyllactosamine antenna, thus constituting a Lex motif. F ¼ fragment; * ¼ artefacts due to the insertion of CH2O or CO2.

expressed by CHO cells are almost quantitatively sulfated at
the two tyrosine residues at positions 60 and 73, respectively.
The nearly complete tyrosine sulfation of the recombinant
HCII molecules was unexpected, as several reports showed
incomplete sulfate ester modification of recombinant proteins expressed in CHO cell lines [42,43]. Such individual
differences indicate that the efficiency of tyrosine sulfation
may depend on additional signals [44] and/or accessability
to the modifying enzyme.

HCII from CHO cells incubated with inhibitors of
post-translational modifications eluted at higher ionic
strength from heparin–Sepharose than inhibitor molecules
isolated from untreated cells. In the case of tunicamycin,
this may be the consequence of reduced sterical hindrance
of heparin binding due to the missing N-glycans, similar
to the situation observed with AT [45,46]. This may
especially apply to the carbohydrate chain linked to
Asn169, a position in proximity to a site involved in
heparin binding by HCII [47,48]. Inhibition of tyrosine
sulfation had an even more profound effect on the
interaction between HCII and heparin. The observation

that higher NaCl concentrations are required for the
dissociation of the unsulfated inhibitor from the heparin
affinity matrix indicates that the N-terminal acidic domain
may affect the GAG binding domain, although it can not
be excluded that this effect is due to the reduction in the
protein’s overall negative charge.
In summary, we present evidence for a very strong
similarity of HCII from human serum and its recombinant
counterpart from CHO cells with respect to tyrosine
sulfation and N-glycosylation. The remarkable identity of
oligosaccharide antennarity and the extent of sialylation
observed in both preparations represents an example of the
importance of polypeptide structure governing protein
N-glycosylation, as it is known that CHO cells have a high
capacity to synthesize N-glycans of high tetraantennarity
with considerable N-acetyllactosamine repeats, which
however, were not detected in our recombinant glycoprotein. We demonstrate that tyrosine sulfation and N-glycans individually affect heparin binding of the inhibitor.

These findings may be exploited to generate HCII variants
with increased heparin affinity.


Ó FEBS 2002

986 C. Bohme et al. (Eur. J. Biochem. 269)
ă

Fig. 5. ESI daughter ion spectrum of the triply charged parent ion [M + 3Na]3+ of a reduced and permethylated triantennary trisialylated N-glycan
with one fucose residue isolated from serum HCII. The detected fragment ions, in particular the signal at m/z 646.3 [HO-Hex-(dHex-HexNAc) + Na], as well as the accompanying signal at m/z 440.3, which is generated by the elimination of the fucose residue from the former ion,
suggest a linkage to O-3 of the GlcNAc residue and therefore a Lex structure. The signal at m/z 1021.6 [NeuAc-Hex-(dHex-HexNAc) + Na] again
clearly indicates the presence of a peripheral fucose as shown in the fragmentation scheme. This is confirmed by the detection of a signal at m/z
316.2, which is typical for an unfucosylated proximal GlcNAc-ol residue. The presence of very small amounts of an isomeric carbohydrate structure
including a proximal fucose is indicated by the weak signal at m/z 490.3 characteristic for this structural feature. It should be noted that CHO cells
predominantly produce proximally fucosylated structures and very small amounts of structures with an additional fucose linked a1 fi 2 to the
galactose residue of an acetyllactosamine antenna (LeH-motif).

Table 2. N-glycan structures of serum-derived and recombinant HCII. After enzymatic liberation of the N-glycans from both protein preparations,
the carbohydrates were subjected to HPAEC-PAD after enzymatic desialylation. The major oligosaccharide from serum HCII was identified as a
diantennary type II N-acetyllactosamine oligosaccharide (Gal2GlcNAc2Man3GlcNAc2), whereas the major glycan of the CHO cell-derived HCII
contained the same structure with an additional proximally a1 fi 6 linked fucose. ND, not detected.
% Oligosaccharides in
Structure

Serum HCII

CHO cell HCII

Diantennary

Diantennary + prox. fucose
Diantennary + Lewis fucose
Triantennary
Triantennary + prox. fucose
Triantennary + periph. fucose (Lex)
a2 fi 6 or a2 fi 3 sialylation of terminal
Galb1–4GlcNAc

84
ND
2
7.5
ND
7

8.7
82
ND
1.5
7.6
ND

96

90

HCII may provide a valuable tool to study the fidelity of
post-translational modifications in recombinant cell lines
engineered with new transferases, e.g. fucosyltransferases or
sulfotransferases for the production of polypeptides which

are modified in novel ways and which may be used for the
study of the functional significance of post-translational
protein modifications.


Ó FEBS 2002

Heparin cofactor II: N-glycans and sulfation (Eur. J. Biochem. 269) 987

ACKNOWLEDGEMENTS
We thank Dr H. Karges, Centeon AG, Marburg, for human plasma
samples. We are grateful to Ulrike Beutling, Sabrina Herrmann,
Susanne Pohl, and Andrea Tiepold for their excellent technical
assistance.

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Fig. 6. Effect of inhibitors of post-translational modifications on heparin
binding. Recombinant CHO cells expressing human HCII were
exposed to 20 mM sodium chlorate or 1 lgỈmL)1 tunicamycin,
respectively. After dialysis, the medium was fractionated on heparin–
Sepharose with a linear NaCl gradient (0–1 M). Column eluates were
analyzed by Western blotting as described in the text. (A) HCII from
chlorate-treated cells. (B) HCII from tunicamycin-treated cells.
(C) HCII from untreated control cells. Lanes 1–10, column fractions
eluting at the salt concentrations indicated. The arrows indicate the
positions of fully glycosylated (78 kDa) and completely unglycosylated
(64 kDa) HCII, respectively.

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