Association of urokinase-type plasminogen
activator (uPA) to cells via binding to its specific cellular receptor
(uPAR) augments the potential of these cells to support plasminogen
activation, a process that has been implicated in the degradation of
extracellular matrix proteins during cell migration and tissue
remodeling. The uPA receptor is a glycolipid-anchored membrane protein
belonging to the Ly-6/uPAR superfamily and is the only multidomain
member identified so far. We have now purified the three individual
domains of a recombinant soluble uPAR variant, expressed in Chinese
hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four
are utilized, as the tryptic peptide derived from domain III containing
Asn233 was quantitatively recovered without
carbohydrate. The remaining four attachment sites were shown to exhibit
site-specific microheterogeneity of the asparagine-linked carbohydrate.
The glycosylation on Asn52 (domain I) and
Asn172 (domain II) is dominated by the smaller biantennary
complex-type oligosaccharides, while Asn162 (domain II) and
Asn200 (domain III) predominantly carry tri- and
tetraantennary complex-type oligosaccharides. The carbohydrate moiety
on Asn52 in uPAR domain I could be selectively removed by
N-glycanase treatment under nondenaturing conditions. This
susceptibility was abrogated when uPAR participitated in a bimolecular
complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of
carbohydrate on domain I exhibited altered binding kinetics toward uPA
(a 4-6-fold increase in Kd) as assessed by real
time biomolecular interaction analysis.
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INTRODUCTION |
The proteolytic potential of the plasminogen activation system is
critically involved in vascular fibrinolysis (1) and tissue remodeling
under pathological conditions such as cancer invasion and wound healing
(2-4). Both plasminogen and its principal activators,
tissue-type plasminogen activator and urokinase-type plasminogen
activator (uPA)1 are mosaic
glycoproteins composed of a COOH-terminal chymotrypsin-like serine
protease domain and a modular, non-catalytic NH2-terminal region, which exerts the specific cofactor activities of these proteins, exemplified by the cellular binding of uPA and
fibrin-specific binding of tissue-type plasminogen activator. The
modular composition of the amino-terminal fragment (ATF) of uPA
includes an epidermal growth factor-like module and a kringle module,
the former of which is responsible for the specific cell binding of uPA
(5). The glycan structure of these proteins have been analyzed in great detail (6-9) and in certain cases particular glycoforms of these proteins were even found to exhibit different biological properties (10-13).
Binding of uPA to cells through the high-affinity association
(Kd of 0.1-1 nM) between the epidermal
growth factor-like module of uPA and its membrane receptor facilitates
cell associated plasmin generation catalyzed by uPA (14), a mechanism
implicated in cancer invasion (3). The human uPA receptor (uPAR) is
encoded as a single polypeptide chain of 313 amino acids including 5 potential attachment sites for N-linked carbohydrate (15).
During post-translational processing an approximately 30-residue signal
peptide is excised from the COOH terminus of uPAR with the concomitant
addition of a glycolipid membrane anchor (16). Like plasminogen and its activators, uPAR also has a multidomain protein architecture being constructed from three homologous Ly6/uPAR-like (49) domains (17, 18,
49). These domains plausibly adopt an overall folding topology similar
to those of CD59 (19, 20) and the nonglycosylated snake venom
-neurotoxins (21).
A pronounced heterogeneity in the glycosylation pattern of uPAR
expressed in various cell lines of neoplastic origin has been observed
(22). It has, furthermore, been reported that cytokine treatment
(transforming growth factor
1, epidermal growth factor, basic
fibroblast growth factor, and phorbol 12-myristate 13-acetate) of
various cell lines (U937, A549, and HeLa) leads to a 5-20-fold increase in the number of surface exposed receptor molecules as well as
a roughly proportional decrease in their ligand binding affinity
(23-26). Although the molecular mechanism underlying this phenomenon
remains to be elucidated, differences in the glycosylation pattern
might be involved, since phorbol 12-myristate 13-acetate induced
differentiation of U937 cells is accompanied by an increase in the size
heterogeneity of uPAR, presumably reflecting an altered processing of
its glycan moieties (22, 27, 28). In accordance with this speculation
is the finding that abolishment of the N-linked glycosylation of Asn52 in uPAR domain I by site-directed
mutagenesis (Asn52
Gln) leads to an approximately
5-fold reduction in its binding affinity, when expressed in murine LB-6
cells (29). The present paper reports the first structural
determination of the glycosylation pattern of uPAR and demonstrates in
a purified system the direct influence of the carbohydrate moiety of
uPAR domain I on real time receptor-ligand binding kinetics using
surface plasmon resonance analysis.
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EXPERIMENTAL PROCEDURES |
Chemicals and Reagents--
Guanidine hydrochloride was of
ARISTAR grade from British Drug House (Poole, United Kingdom),
(4-amidinophenyl)-methanesulfonyl fluoride (APMSF) was from Boehringer
Mannheim (Germany),
-cyano-4-hydroxycinnamic acid was from Sigma,
and 2,5-dihydroxybenzoic acid (as a stock solution of 10 g/liter) was
from Hewlett-Packard (Palo Alto, CA). A 17-mer peptide antagonist of
the uPA-uPAR interaction, originally identified by bacteriophage
display technology (30) denoted AE78 (AEPMPHSLNFSQYLWYT) and a randomly
scrambled, non-binding version thereof denoted AE79
(AEWSNLMQPYYPSTHFL), were synthesized and characterized as described
previously (31). Water was drawn from a Milli-Q system with an
Organex-Q cartridge.
Purified Proteins--
A soluble recombinant variant of uPAR
(residues 1-277) was expressed in Chinese hamster ovary (CHO
dhfr
) cells, which secrete this protein to the culture
medium due to the truncation of the COOH-terminal signal sequence
responsible for the glycolipid membrane attachment of uPAR (16). Cells
were grown in the presence of 10 nM methotrexate and
secrete approximately 0.5 mg of uPAR per liter of harvest fluid (32).
The uPAR protein was purified by immunoaffinity chromatography using a
monoclonal anti-uPAR antibody (18). Active two-chain uPA (EC 3.4.21.31) was from Serono (Aubonne, Switzerland) and recombinant pro-uPA expressed in Escherichia coli was a kind gift from Dr. D. Saunders (Grünenthal, Germany). The ATF of uPA was kindly
provided by Drs. A. Mazar and J. Henkin (Abbott Laboratories, IL),
whereas the growth factor-like domain (GFD) of uPA was produced by
Glu-C digestion of two-chain uPA as described previously (33).
Porcine pepsin A (EC 3.4.23.1) was from Sigma, lot 120H8095. Modified
porcine trypsin (EC 3.4.21.4) of sequencing grade was obtained from
Promega (Madison, WI). N-tosyl-L-phenylalanine chloromethyl ketone-treated
-chymotrypsin (EC 3.4.21.1) was purchased from Worthington (Freehold, NJ). Purified lectin AAA from
Aleuria aurantia that specifically binds to
(1-6)-linked fucose in complex-type N-linked carbohydrates and the
following glycosidases were all purchased from Boehringer Mannheim
(Germany): neuraminidase (EC 3.2.1.18) from Clostridium
perfringens (0.1 units/µg), recombinant N-glycanase
(EC 3.2.2.18) from Flavobacterium meningosepticum expressed
in E. coli (25 units/µg), and a mixture of endoglycosidase
F1 and F2 (EC 3.2.1.96) isolated from F. meningosepticum.
Isolation of the Individual Domains of uPAR--
The
NH2-terminal domain I of uPAR (residues 1-87) was
liberated from domain II + III by cleavage after Tyr87 in
the linker region using limited proteolysis with chymotrypsin and
subsequently purified by gel filtration on a SuperdexTM 75 HR10/30 column (Pharmacia, Uppsala, Sweden) essentially as described
(18). Cleavage in the linker region between domain II + III was
accomplished by pepsin treatment, a procedure recently devised to
generate domain I + II from intact uPAR (34). In the present
experiment, lyophilized domain II + III was dissolved in 0.2 M acetic acid and treated for 2 h at 37 °C with
pepsin at an enzyme to substrate ratio of 1:2000 (w/w). This digestion was terminated by injecting the sample directly onto a
ProRPCTM HR5/10 column (Pharmacia) from which a sequential
elution of the generated domains II and III was obtained by a 35-min
linear gradient from 0 to 40% (v/v) 2-propanol in 0.1% (v/v)
trifluoroacetic acid at a flow rate of 300 µl min
1
(Fig. 1).
Peptide Mapping of Domains II and III after Trypsin
Degradation--
Reversed-phase HPLC purified domain II and III
(approximately 20 nmol) were taken to dryness by vacuum evaporation,
redissolved in 150 µl of 6 M guanidinium HCl, 0.5 M Tris-HCl, pH 8.0, and 2 mM EDTA. After
flushing the samples with argon, reduction proceeded for 2 h at
50 °C in the presence of 20 mM dithiothreitol. Generated thiol groups were subsequently alkylated by incubation with 50 mM iodoacetamide in the dark for 30 min after which the
alkylated domains were purified by gel filtration on a Superdex
75 column using 0.1 M NH4HCO3 as
solvent.
These preparations were directly subjected to trypsin cleavage by
addition of porcine trypsin at an enzyme to substrate ratio of 1:25
(w/w) followed by incubation overnight at 25 °C. The resulting degradation patterns were visualized by reversed-phase HPLC
chromatography using a Brownlee Aquapore OD-300 C18 column with a
linear gradient (50 min) from 0 to 70% (v/v) acetonitrile in 0.1 and
0.085% (v/v) trifluoroacetic acid, respectively. The flow rate was 250 µl min
1 and detection was at 214 and 280 nm. Identity
of the eluted polypeptides was revealed by a combination of
matrix-assisted laser desorption ionization mass spectrometry
(MALDI-MS) and amino acid composition analyses.
Enzymatic Deglycosylation of Purified uPAR and Isolated
Domains/Glycopeptides Thereof--
For structural analyses of the
glycan moieties, sequential deglycosylation of reversed-phase purified
domains of uPAR or isolated tryptic glycopeptides thereof was performed
in 10 µl of 50 mM NH4HCO3.
Samples were initially incubated for 18 h with 5 milliunits of
neuraminidase, followed by the withdrawal of 1 µl for MALDI-MS analysis. After subsequent addition of 125 milliunits of
N-glycanase, incubation was continued for another 18 h
and 1 µl was withdrawn for a second MALDI-MS analysis.
For functional binding studies of uPAR with differently processed
glycan moieties, intact uPAR (approximately 1 nmol) was incubated for
20 h at 37 °C in 50 µl of 50 mM Bistris-HCl, 45 mM NaCl, 5 mM EDTA (pH 7.1) containing either
50 milliunits of neuraminidase or 50 milliunits of
N-glycanase. The neuraminidase stock solution was pretreated
with 1 mM phenylmethylsulfonyl fluoride to avoid
proteolytic cleavage of uPAR by trace protease contamination in the
enzyme preparation.
The following experiments were conducted to explore whether the
accessibility of the glycan moiety on Asn52 in uPAR domain
I toward enzymatic hydrolysis by N-glycanase was affected
when the receptor was engaged in the formation of a bimolecular complex
with various uPA derivatives. Receptor complexes were formed by
preincubating uPAR (1 µM) for 5 min at room temperature with buffer alone or a 4-fold molar excess of either bovine serum albumin (as control) or various receptor binding uPA derivatives (pro-uPA, ATF, or GFD). Synthetic peptides (AE78 and AE79) were, however, preincubated with uPAR at a 20-fold molar excess. These mixtures were treated with 20 milliunits of neuraminidase and 100 milliunits of N-glycanase for 20 min at 37 °C, whereupon
further deglycosylation was prevented by boiling for 5 min. The
untreated sample also received the above deglycosylation mixture and
was boiled immediately. Domain I was subsequently generated by
incubation with 2 nM chymotrypsin for 10 min at room
temperature and 1 µl of these mixtures were finally subjected to
MALDI-MS analysis. These experiments were performed in 50 mM Bistris-HCl, 100 mM NaCl, 5 mM
EDTA (pH 7.1), including 2 mM APMSF, the presence of which
allows the subsequent chymotrypsin cleavage.
Real Time Biomolecular Interaction Analysis (BIA)--
The
effects of various glycosidase treatments of uPAR on its ligand binding
properties were determined by surface plasmon resonance using a BIAcore
2000TM equipment (Pharmacia Biosensor, Uppsala, Sweden).
The carboxymethylated dextran matrix (CM5 sensor chip) was preactivated
with
N-hydroxysuccinimide/N-ethyl-N'-(3-(diethylamino)propyl)carbodiimide, according to the manufacturers recommendations. Coupling of the ligands
was achieved by subsequent injection of uPA (20 µg/ml) or lectin AAA
(50 µg/ml) in 10 mM sodium acetate (pH 5.0) at a flow
rate of 5 µl min
1 for 6 min.
Just prior to the kinetic analyses by real time BIA, the various uPAR
preparations from the deglycosylations experiments were re-purified by
gel filtration on a Superdex 75 HR 3.2/30 column (Pharmacia, Uppsala,
Sweden) using 50 mM Bistris-HCl, 45 mM NaCl, 5 mM EDTA (pH 7.1) as eluent to remove any aggregated or
degraded material formed during the enzymatic deglycosylation (less
than 10%). Sensorgrams (resonance units versus time) were
recorded by the BIAcore 2000TM at a flow rate of 10 µl
min
1 at 5 °C, using 6 different concentrations of
these uPAR preparations in the range of 10-100 nM in
running buffer (50 mM Bistris-HCl, 45 mM NaCl,
5 mM EDTA (pH 7.1) including 0.005% surfactant P-20). To
reassure that no protein aggregation occurred while recording the
sensorgrams, the individual samples were retrieved after analysis using
the recovery function of BIAcore 2000TM and subjected to
analytical rechromatography on Superdex 75. After enzymatic
deglycosylation samples were kept at 5 °C and analyzed within
24 h. The sensor chip was regenerated at the end of each run by
injection of 0.1 M acetic acid containing 0.5 M NaCl.
Specific detection of glycopeptides containing
(1-6)-linked fucose
was performed by carbohydrate-specific surface plasmon resonance
detection using immobilized lectin AAA (35). In such experiments
regeneration of the sensor chip was obtained by injection of 100 mM fucose.
Data obtained from parallel mock-coupled flow cells (non-protein)
served as blank sensorgrams for subtraction of bulk refractive index
background. The obtained sensorgrams were analyzed by nonlinear least
squares curve fitting using BIAevaluation 2.0 software (Pharmacia Biasensor, Uppsala, Sweden) assuming single-site association and dissociation models.
Electrospray Ionization Mass Spectrometry (ESI-MS)--
ESI-MS
was performed on a Sciex API III triple quadrupole instrument (Sciex,
Thornhill, Canada) using an articulated ion-spray probe. The sample (2 mg/ml) was mixed with 1 volume of 1% (v/v) acetic acid in methanol and
subjected to ESI-MS at a flow-rate of 3 µl/min. A sprayer voltage of
5,000 volts and an orifice potential of 80 volts were used.
MALDI-MS--
MALDI spectra were recorded on a linear
time-of-flight instrument (VoyagerTM, PerSeptive Biosystems, MA)
equipped with a 1.2-m flight tube and a 337-nm nitrogen laser. To
minimize prompt as well as metastable fragmentation of sialic acids
during sample desorption (36), in particularly when
-cyano-4-hydroxycinnamic acid was used as matrix, laser power levels
were kept as low as possible, typically 1% above the threshold value
for desorption of matrix ions. Although metastable fragmentation of
sialic acids undoubtly occurs to a significant extent during this
desorption protocol, this would not give rise to artifacts in mass
assignments, since the time-of-flight instrument was operated in the
linear mode only, where fragment and parent ions possess identical
initial velocities (36, 37). The MS probes were precoated with a
thin-layer of matrix by deposition of 1 µl of freshly prepared matrix
solution (20 mg/ml in 98% (w/w) acetone) followed by fast solvent
evaporation. Sandwich sample preparation (38) occurred by consecutive
deposition of 1 µl of the following solutions to the precoated
target: 2% (v/v) trifluoroacetic acid, sample to be analyzed and
finally 15 mg/ml matrix dissolved in 50% (v/v) acetonitrile. After
1-2 min of crystallization, excess solvent was removed by aspiration and the probe surface was gently rinsed using 0.1% (v/v)
trifluoroacetic acid. The majority of samples were analyzed using
-cyano-4-hydroxycinnamic acid as matrix, but sinapinic acid was used
for intact uPAR to favor the formation of molecular ions
(MH+) as well as reducing the MS induced fragmentation of
sialic acid. One glycopeptide (T21) could, however, only be desorbed
after deposition in 2,5-dihydroxybenzoic acid (2 µl of 1:1 mixture of sample and matrix solution (10 mg/ml) was deposited on a clean MS probe
surface).
Miscellaneous Analyses--
Amino acid analysis was performed on
a Waters amino acid analyzer with post-column
o-phthaldialdehyde detection. Acid hydrolysis was achieved
by incubation in vacuo at 110 °C for 20 h in 6 M HCl containing 0.05% (w/v) of both phenol and
3,3'-dithiodipropionic acid (39).
 |
RESULTS |
Generation and Purification of the Individual Domains of
uPAR--
The NH2-terminal domain I of uPAR was purified
by gel filtration after mild chymotrypsin treatment of a recombinant
uPAR (residues 1-277) secreted by CHO cells, as reported previously
(18). The remaining domain II + III (residues 88-277) was subsequently
treated with pepsin and subjected to reversed-phase HPLC purification, which yielded two distinct components representing isolated domains II
and III (Fig. 1). Amino acid composition
analysis and mass spectrometry (ESI and MALDI) confirmed the purity and
identity of these preparations of the individual domains of uPAR (Table I and Figs.
2 and 4). The pepsin treatment caused the
liberation of domains II and III due to the specific cleavage in the
interdomain linker region of the Glu183-Leu184
peptide bond, in accordance with the major substrate specificity of
this enzyme (40).

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Fig. 1.
Reversed-phase HPLC purification of uPAR
domains II and III generated by pepsin treatment. Purified uPAR
domain II + III (residues 88-277) obtained from chymotrypsin treatment
of intact uPAR was dissolved in 0.2 M acetic acid and
treated with low concentrations of pepsin. The enzymatic fragmentation
was terminated by the reversed phase HPLC chromatography shown. The
inset shows a Coomassie-stained polyacrylamide gel (10%)
after an SDS-polyacrylamide gel electrophoresis analysis of the
collected fractions after reduction and alkylation.
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Table I
Amino acid composition analyses of purified domains and glycopeptides
of uPAR
Values in parentheses correspond to the theoretical number of residues
according to the cDNA derived sequence of human uPAR (15).
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Fig. 2.
Analysis of intact and
N-glycanase treated domain I of uPAR by ESI-MS.
Purified uPAR domain I was treated with N-glycanase before
reversed-phase HPLC purification. Both the intact and the
deglycosylated domain I were amenable to ESI-MS from which the
reconstructed spectra are shown. The majority of the heterogeneity
observed originates from a variable degree of sialylation (mass of
N-acetylneuraminic acid is 291.2 Da). The theoretical mass
of the disulfide bonded domain I devoid of carbohydrate and corrected
for the Asn to Asp conversion introduced by N-glycanase
(upper panel) as well as the mass of the derived glycan
structure (lower panel) are shown.
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Although the theoretical molecular mass of the generated polypeptide
chain corresponding to domain II (10,838.9 Da) is only slightly larger
than that of domain III (10,185.3 Da) its migration in
SDS-polyacrylamide gel electrophoresis is considerably slower and more
heterogenous than that of domain III (Fig. 1, inset). As
both domains have two consensus motifs for N-linked
carbohydrate (Asn-Xaa-Thr/Ser) this might indicate that only one site
is processed in domain III and/or that domain II in general carries
more bulky carbohydrate moieties. The amount of
N-acetylglucosamine determined by amino acid composition
analysis also implies a more extensive glycosylation on domain II
(Table I).
Glycosylation of the Individual Domains as Assessed by ESI and
MALDI-MS--
Purified uPAR domain I was amenable to analysis by
ESI-MS, the spectra demonstrating that N-linked
glycosylation of Asn52 is the only post-translational
modification detected (Fig. 2 and Table I). By far the majority of this
glycosylation is accounted for by a biantennary complex-type
oligosaccharide carrying a deoxyhexose (most likely fucose) attached to
the chitobiose moiety. The presence of an
(1-6)-linked fucose
moiety in this complex-type carbohydrate is verified by lectin-based
surface plasmon resonance detection (35) using immobilized A. aurantia lectin. As shown in Fig. 3
only the glycosylated form of uPAR domain I binds specifically to the
lectin and this interaction is completely inhibited by 0.5 mM fucose but not by 1 mM mannose, 1 mM galactose, or 1 mM N-acetylglucosamine.

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Fig. 3.
Lectin based detection of fucose in uPAR
domain I by surface plasmon resonance. Lectin AAA from A. aurantia which recognize carbohydrate with (1-6)-linked fucose
was covalently immobilized onto the sensor chip used for surface
plasmon resonance studies. Subsequent interaction with the lectin was
measured in real time for 5 µM uPAR domain I (curve
1), 5 µM uPAR domain I treated with
N-glycanase, i.e. containing deglycosylated
domain I and the liberated glycan (curve 2), 5 µM uPAR domain I in the presence of 0.5 mM
fucose (curve 3), 1 mM mannose (curve
4), 1 mM galactose (curve 5), or 1 mM N-acetylglucosamine (curve 6), and
finally a buffer control (curve 7). Association of ligands
occurred for 350 s by injection of the relevant analyte after
250 s of buffer flow. Dissociation was initiated after 600 s.
Analyses were performed at 5 °C with a buffer flow of 10 µl/min.
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The ESI-MS spectrum also reveals an apparent microheterogeneity in the
sialylation pattern of domain I (Fig. 2, lower panel). Some
of this microheterogeneity may, however, be caused by ESI-MS induced
fragmentation, since increasing the orifice voltage of the MS probe
leads to a further fragmentation of the carbohydrate moiety.2 Analysis of uPAR
domain I by MALDI-MS after deposition in either
-cyano-4-hydroxycinnamic acid or sinapinic acid revealed a similar microheterogeneity in the sialylation pattern of uPAR domain I (data
not shown). Although laser-induced prompt fragmentation may in
principal generate some of this microheterogeneity, the predominant
fragmentation mechanism by metastable ion formation may not, since the
fragment and parent ions are not resolved by linear time-of-flight mass
spectrometry (36, 37).
As the analysis of isolated domains II and III by ESI-MS proved
unsuccessful, these domains were analyzed by MALDI-MS. The spectra
obtained (Fig. 4) reveal an average
carbohydrate load of 4900 Da on domain II compared with only 2900 Da on
domain III, thus further substantiating the suspected presence of an
unoccupied glycosylation site in domain III. Both domains contain
sialylated carbohydrates as demonstrated by the notable mass
differences recorded by MALDI-MS upon neuraminidase treatments (Fig.
4).

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Fig. 4.
MALDI-MS analyses of isolated domains II and
III of uPAR after various glycosidase treatments. Reversed-phase
purified domain II and domain III were treated with either
neuraminidase (middle panel) or N-glycanase
(upper panel). Due to a greater resistance of the
carbohydrate on domain II to endoglycosidases this domain was treated
with a mixture of N-glycanase, endoglycosidase F1, and
endoglycosidase F2 thus generating a mixture of completely
deglycosylated domain II (10,840.9 Da) and partial deglycosylated
domain II still carrying a proximal fucosylated
N-acetylglucosamine (11,189.2 Da). These preparations were
subsequently analyzed by MALDI-MS the spectra of which are shown along
with the theoretical mass of the disulfide bonded domains devoid of
carbohydrate and corrected for the Asn to Asp conversion introduced by
N-glycanase.
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Characterization of Glycopeptides Derived from Domains II and
III--
To obtain a detailed structural analysis of the
N-linked carbohydrates of uPAR domains II and III, including
the positive identification of the putative unoccupied glycosylation
site in domain III, the purified domains were reduced and
S-carboxamidomethylated before trypsin cleavage and
subsequent purification by reversed-phase HPLC (Fig.
5). The overall sequence coverage was
greater than 95% of uPAR1-277, when the data on the
identity of these HPLC purified peptides (Fig. 5, A and
B) were combined by those obtained for domain I (Fig. 2).
Only a single dipeptide (T13) and a single tripeptide (T18) thus
escaped identification by MALDI-MS or amino acid composition analysis.
Tryptic peptides encompassing both consensus motifs for
N-linked glycosylation present in uPAR domain II
(Asn162-Asp-Thr and Asn172-Thr-Thr) were indeed
recovered as the corresponding glycopeptides T20 and T21 (Fig.
5A and Tables I and II), as
predicted from the previous analyses of the purified receptor domain II
(Fig. 4). Inspection of the MALDI-MS spectra recorded after
neuraminidase or N-glycanase treatments of glycopeptide T20
(Asn162) demonstrated that this glycan moiety is dominated
by sialylated tri- and tetraantennary complex-type carbohydrate (Fig.
6). Similar experiments revealed that the
second glycosylation site in uPAR domain II located at
Asn172 (T21) primarily carried the smaller sialylated
biantennary complex-type carbohydrate including an additional
deoxyhexose most likely as a core fucosylation (Fig.
7). Carbohydrate-specific surface plasmon resonance analysis, similar to that conducted for domain I in Fig. 3,
confirmed the presence of fucose in purified intact domain II (data not
shown).

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Fig. 5.
Reversed-phase HPLC peptide mapping of
domains II and III after trypsin treatment. The elution profiles
of tryptic fragments derived from S-carboxyamidomethylated
domains II (upper panel) and III (lower panel)
are shown as visualized by absorbance at 214 and 280 nm. The elution
position of the uncleaved domains are indicated by arrows.
The identity of the eluted peptides were revealed by a combination of
MALDI-MS and amino acid composition analysis and is represented in the
figures by the following number code. Theoretical trypsin cleavage of
the human uPAR cDNA derived sequence yields 28 fragments, numbered
T1 to T28; if a peak contains more than one peptide it will be assigned
by two numbers, i.e. T1 + T2; if a peptide on the other hand
arises as a consequence of incomplete cleavage as an example between T1
and T2 it will be denoted T(1 + 2).
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Table II
Characterization of tryptic glycopeptides by mass spectrometry
Masses were determined by ESI-MS (domain I) or MALDI-MS (glycopeptides)
and are shown along with the theoretical masses in parentheses (when
cysteine is present in the glycopeptides the mass of
S-carboxamidomethyl cysteine is used). One atomic mass unit
has been subtracted from the masses recorded of each molecular ion
(MH+) by MALDI-MS due to the association of one proton during
ionization. The relative abundance of the various glycoforms were
estimated by comparison of peak intensities (peak heights) obtained
within a single MALDI-MS spectrum for each glycopeptid after
neuraminidase treatment.
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Fig. 6.
MALDI-MS spectra of the tryptic glycopeptide
T20 from domain II. MALDI-MS spectra were recorded for HPLC
purified T20 (residues 146-169) before and after treatment with
neuraminidase and N-glycanase. The average molecular mass of
the deglycosylated T20 is 2697.96 Da (corrected for
S-carboxyamidomethylation of cysteine and the Asn to Asp
conversion by N-glycanase). An interpretation of the masses
obtained for the neuraminidase-treated glycopeptide is shown in the
middle spectrum. Peaks of low intensity with masses
corresponding to an identical carbohydrate moiety carrying an
additional core fucosylation (mass increase of 146.1 Da) have been
highlighted. , N-acetylhexosamine; , hexose (most
likely mannose); , hexose (most likely galactose).
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Fig. 7.
MALDI-MS spectra of the tryptic glycopeptide
T21 from domain II. MALDI-MS spectra were recorded for HPLC
purified T21 (residues 170-175) before and after treatment with
neuraminidase and N-glycanase. The average molecular mass of
the deglycosylated T21 is 783.86 Da (corrected for
S-carboxyamidomethylation of cysteine and the Asn to Asp
conversion by N-glycanase). Since the
NH2-terminal S-carboxyamidomethyl cysteine
undergoes an intramolecular cyclization generating the corresponding
thiazine derivative, the actual average molecular mass is 766.83 Da
(compare with upper panel). An interpretation of the masses
obtained for the neuraminidase-treated glycopeptide is shown in the
middle spectrum. : N-acetylhexoseamine; ,
hexose (most likely mannose); , hexose (most likely galactose); ,
deoxyhexose (most likely fucose).
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Only a single glycopeptide (T24) was detected in the tryptic peptide
map of uPAR domain III (Fig. 5B and Table I) and accordingly its sequence encompass one of the two glycosylation motifs present in
this domain: Asn200-Ser-Thr. Subsequent glycoprofiling by
MALDI-MS demonstrated that the glycan attached to Asn200
primarily consisted of sialylated tri- and tetraantennary complex-type carbohydrate, approximately one-third of which contain an additional deoxyhexose presumably representing a traditional core fucosylation (Fig. 8). Accordingly, isolated domain
III bound immobilized lectin AAA from A. aurantia as
detected by surface plasmon resonance (data not shown). The average
molecular mass of this glycan (3,075 Da) is comparable to the mass
difference (2,875 Da) observed upon N-glycanase treatment of
intact uPAR domain III (Fig. 4). The second cognate glycosylation motif
present in uPAR domain III (Asn233-Gln-Ser) must therefore
be unoccupied and the corresponding tryptic peptide (T26) was
accordingly recovered quantitatively without carbohydrate as assessed
by both amino acid composition analysis (Table I) and MALDI-MS (Table
II).

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Fig. 8.
MALDI-MS spectra of the tryptic glycopeptide
T24 from domain III. MALDI-MS spectra were recorded for HPLC
purified T24 (residues 199-216) before and after treatment with
neuraminidase and N-glycanase. The average molecular mass of
the deglycosylated T21 is 2071.18 Da (corrected for
S-carboxyamidomethylation of cysteine and the Asn to Asp
conversion by N-glycanase). As indicated in Fig.
5B, this fraction also contains the incomplete tryptic
peptide T(23 + 24), the corrected molecular mass of which is 2898.14 Da. The NH2-terminal glutamine of T(23 + 24) is partly
converted to pyroglutamic acid (2881.11 Da). The characteristic double
peaks (±17 Da) corresponding to the incomplete tryptic peptide T(23 + 24) are indicated in the individual spectra by an asterisk
(*). An interpretation of the masses obtained for the
neuraminidase-treated glycopeptide T24 is shown in the middle
spectrum. , N-acetylhexoseamine; , hexose (most
likely mannose); : hexose (most likely galactose); , deoxyhexose
(most likely fucose).
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In conclusion, site-specific microheterogeneity was evident from the
analyses of all isolated tryptic glycopeptides. The glycan moieties on
Asn162 and Asn200 were dominated by sialylated
tri- and tetraantennary complex-type, whereas the glycan moieties on
Asn52 and Asn172 primarily consisted of the
smaller sialylated biantennary complex-type carbohydrate. A fifth
potential glycosylation site located at Asn233 was not
processed at all.
Functional Impact of uPAR Glycosylation on Its Ligand Binding
Affinity--
As noted previously (18) the only carbohydrate
accessible to cleavage by N-glycanase on native intact uPAR
is that on domain I (Asn52). To test whether this property
is retained within bimolecular receptor-ligand complexes, we subjected
preformed complexes between uPAR and pro-uPA, ATF, GFD, or a 17-mer
synthetic peptide antagonist AE78 (AEPMPHSLNFSQYLWYT) to a
combined enzymatic deglycosylation using neuraminidase and
N-glycanase. When analyzed by MALDI-MS it was evident that
the accessibility of the proximal N-acetylglucosamine linkage to Asn52 to enzymatic hydrolysis is severely
hampered by the presence of all ligands tested (Fig.
9 and Table
III). In contrast, the terminal sialic
acids on this carbohydrate retained their sensitivity to neuraminidase
(data not shown).

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Fig. 9.
Ligand induced protection on enzymatic
deglycosylation of uPAR domain I as revealed by MALDI-MS spectra.
Intact uPAR was preincubated with the ligands indicated before
deglycosylation was attempted by incubation with a mixture of
neuraminidase and N-glycanase. Domain I (residues 1-87) was
finally released by a brief chymotrypsin treatment before the entire
mixture was subjected to MALDI-MS. * indicates deglycosylated uPAR
domain I, whereas ** represents the various glycoforms of uPAR domain
I. The relative peak intensities corresponding to the glycosylated
domain I (**) differ from that recorded previously by ESI-MS (Fig. 2),
presumably because the activity of neuraminidase is not immediately
and/or completely destroyed by the subsequent boiling. AE78
is a peptide antagonist of the uPA-uPAR interaction (AEPMPHSLNFSQYLWYT)
and AE79 is an inactive scrambled version thereof
(AEWSNLMQPYYPSTHFL). BSA, bovine serum albumin.
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Table III
Ligand protection towards enzymatic deglycosylation of Asn52 on
intact uPAR
Purified uPAR was incubated with 20 milliunits of neuraminidase and 100 milliunits of N-glycanase for 20 min, whereupon domain I was
liberated by limited chymotrypsin cleavage and analyzed subsequently by
MALDI-MS in the mixture. A relatively short incubation time was chosen
to minimize dissociation of preformed complexes. This condition yields
a 75% deglycosylation efficiency of uPAR domain I in the absence of
added ligands, which defines the maximal (100%) deglycosylation
efficiency achievable under these experimental conditions,
cf. the equation presented in Footnote b. Representative
MALDI-MS spectra for some of these experiments are shown in Fig. 9.
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Defined preparations of intact uPAR containing truncated carbohydrate
moieties were prepared by treatment with neuraminidase and
N-glycanase under nondenaturing conditions. The manipulated proteins were purified by gel filtration and the extent of
deglycosylation assessed by MALDI-MS (Table
IV). The binding kinetics between immobilized uPA and these uPAR preparations were measured directly by
real time BIA using surface plasmon resonance (Table IV). The association and dissociation rate constants measured for the
interaction between uPA and uPAR were comparable to those determined
previously (31, 34, 42) and correspond to a dissociation constant of approximately 0.6 nM. Enzymatic removal of the sialic acids
on uPAR by neuraminidase treatment did not change the binding kinetics significantly, whereas the specific removal of the entire carbohydrate moiety on Asn52 in uPAR domain I by
N-glycanase treatment led to an approximately 5-fold
increase in the dissociation constant (Kd of 3.2 nM versus 0.6 nM). This increase was
caused by a combined effect on the association rate (a 3-fold decrease)
and the dissociation rate (a 2-fold increase).
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Table IV
Kinetic and affinity constants for the interaction between immobilized
uPA and intact uPAR subjected to various glycosidase treatments
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DISCUSSION |
Several reports have proposed a possible functional role of the
carbohydrate moieties of uPAR on its ligand binding properties (26, 28,
29). In the present study we have therefore examined the glycosylation
pattern of a human soluble uPAR expressed in CHO cells. This
recombinant protein escapes attachment to the plasma membrane due to a
COOH-terminal truncation (residues 278-313), which eliminates the
signal sequence responsible for the normal glycolipid anchoring of the
receptor (16). A similar soluble uPAR protein (presumably representing
residues 1-283) is also secreted in vivo from peripheral
blood cells affected by the disease paroxysmal nocturnal hemoglobinuria
(43). The uPA binding properties of the soluble uPA receptor expressed
in CHO cells are not impaired by the absence of the glycolipid moiety
(44). In the present report we demonstrate that although the cDNA
derived sequence of uPAR contains 5 consensus motifs for
N-linked glycosylation (15), only 4 of these are actually
utilized in the soluble uPAR secreted by CHO cells. The glycosylation
profile determined for this recombinant uPAR is summarized in Fig.
10. The presence of O-linked
carbohydrate in uPAR is precluded by comparison of masses derived from
the cDNA sequence and those determined for the individual uPAR
domains after treatment with N-glycanase (Figs. 2 and 4). In
addition, no galactosamine was detected by amino acid composition analysis of intact uPAR after acid hydrolysis (16, 22). The degree of
microheterogeneity observed at the individual N-linked glycosylation sites in uPAR is comparable to that observed for other
proteins expressed by CHO cells including tissue plasminogen activator
(9, 45) and plasminogen (8), the most striking difference being the
absence or very low abundance of high-mannose and hybrid-type
oligosaccharides in the present uPAR preparation.

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Fig. 10.
Deduced glycosylation profile of recombinant
human uPAR expressed in CHO cells. The glycosylation profile as
determined by mass spectrometry for the 5 potential carbohydrate
attachment sites on human uPAR is shown for the desialylated
recombinant protein (residues 1-277) expressed in CHO cells (see Table
II). , N-acetylhexoseamine; , hexose (most likely
mannose); , hexose (most likely galactose); and , deoxyhexose
(most likely fucose).
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The latter observation has a bearing on the recently reported
interaction between a recombinant uPAR and the cation independent mannose 6-phosphate receptor, since this complex formation was assumed
to occur through a specific interaction with the carbohydrate moieties
of uPAR (46). The recombinant uPAR analyzed in the present study also
binds a purified mannose 6-phosphate receptor in ligand-blotting
experiments via a mechanism that exhibited complete inhibition by
preincubation with 1 mM free mannose 6-phosphate (data not
shown). It is therefore most likely that the observed interaction with
the mannose 6-phosphate receptor is governed by a very low abundance
glycosylation variant in the present uPAR preparation carrying
phosphorylated high-mannose or hybrid-type oligosaccharides. The
relative abundance of such immature carbohydrates may, however, vary
among uPAR preparations produced in different laboratories, since
carbohydrate processing of recombinant glycoproteins is sensitive to
several environmental conditions during large scale production,
including the accumulation of ammonium ions (47).
Since the NH2-terminal domain I of uPAR (residues 1-87)
plays a predominant role for uPA binding (17, 21, 31, 33, 44), we were
particularly interested in testing a possible functional impact of the
glycosylation at Asn52 on ligand binding. As a rule,
carbohydrates can be considered as bulky hydrophilic structures that
may shield a significant portion of the solvent accessible molecular
surface area from participation in protein-protein interactions. In
this context it is therefore notable that the sole N-linked
glycosylation site of uPAR domain I predominantly carries the smaller
biantennary oligosaccharides (Fig. 10) and is located in close
proximity of the ligand-binding site for uPA. The immediate vicinity of
the N-linked oligosaccharide attached to Asn52
and the ligand-binding site for uPA was demonstrated by the reduced accessibility of this particular glycan to enzymatic hydrolysis by
N-glycanase under nondenaturing conditions, when the
receptor was engaged in ligand binding to pro-uPA, ATF, or GFD (Table
III and Fig. 9). Even the small 17-mer peptide antagonist (AE78) was capable of enforcing a similar constrain on the specific
deglycosylation of Asn52 upon interaction with its cognate
ligand-binding site on uPAR (Table III and Fig. 9). Consistent with
this topological relationship is the recent finding by site-specific
photoaffinity labeling, that replacement of the single phenylalanine in
AE78 with either p-benzoylphenylalanine or
4'-(trifluoromethyl-diazirinyl)-phenylalanine led to a photoprobe that
inserted specifically into uPAR domain I upon UV-light exposure (31).
The primary target residue for this photoaffinity labeling of uPAR has
very recently been identified as Arg53 in domain
I.3 Being juxtaposed to the
glycosylation of Asn52 this interaction site provides a
reasonable molecular basis for the observed protection against
deglycosylation of domain I in preformed uPAR·AE78 complexes. In
addition, Tyr57 has also been shown to participate directly
in the formation of the receptor-ligand binding interface as
demonstrated by protein-protein footprinting analyses (33).
A moderate negative modulation of the binding kinetics of the uPA-uPAR
interaction was observed after enzymatic removal of the biantennary
oligosaccharides attached to Asn52 (Table IV). Although the
molecular mechanism responsible for this relationship remains to be
elucidated, these studies suggest, that an altered processing of the
carbohydrate moiety attached to Asn52 of uPAR domain I may
in theory influence the binding kinetics of the uPA-uPAR interaction.
Consistent with this speculation is the finding that prevention of the
glycosylation of domain I by site-directed mutagenesis
(Asn52
Gln) in a glycolipid-anchored human uPAR
expressed in murine LB6 cells led to a 4-5-fold increase in the
apparent Kd for the cellular binding of uPA,
compared with cells transfected with a wild type uPAR (29).
Differentiation of monocytes and monocyte-like cell lines is
accompanied by a decrease in their affinity toward uPA as well as an
increase in the molecular size heterogeneity of their surface-exposed
uPAR molecules (26, 28, 48). A shift in the glycan processing
particularly at Asn52 to the more bulky tri- and
tetraantennary oligosaccharides could thus play a role for the in
vivo modulation of the cellular binding of uPA observed upon
monocyte differentiation. In future studies it should therefore be
interesting to explore the binding kinetics between uPA and various
recombinant uPAR molecules secreted by different expression systems,
including baculovirus, Picia pastoris, Saccharomyces
cerevisiae, and Aspergillus niger to test whether a
correlation exists between the binding kinetics and the size of the
glycan moiety attached to Asn52.
We are indebted to Dr. Arne Jensen (Dept.
Protein Chemistry, University of Copenhagen, Denmark) for amino acid
analyses and are grateful to Dr. Vincent Ellis (Thrombosis Research
Institute, London, UK) for helpful discussions. The technical
assistance from Helle Hymøller Hald and John Post is also greatly
acknowledged.