Báo cáo khoa học: Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator potx

Tetranectin binds hepatocyte growth factor and tissue-type
plasminogen activator
Uffe B. Westergaard, Mikkel H. Andersen, Christian W. Heegaard, Sergey N. Fedosov
and Torben E. Petersen
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark
In the search for new ligands for the plasminogen kringle 4
binding-protein tetranectin, it has been found by ligand blot
analysis and ELISA that tetranectin specifically bound to the
plasminogen-like hepatocyte growth factor and tissue-type
plasminogen activator. The dissociation constants of these
complexes were found to be within the same order of mag-
nitude as the one for the plasminogen-tetranectin complex.
The study also revealed that tetranectin did not interact
with the kindred proteins: macrophage-stimulating protein,
urokinase-type plasminogen activator and prothrombin. In
order to examine the function of tetranectin, a kinetic
analysis of the tPA-catalysed plasminogen activation was
performed. The kinetic parameters of the tetranectin-
stimulated enhancement of tPA were comparable to fibri-
nogen fragments, which are so far the best inducer of
tPA-catalysed plasminogen activation. The enhanced
activation was suggested to be caused by tetranectin’s ability
to bind and accumulate tPA in an active conformation.
Keywords: tetranectin; plasminogen; hepatocyte growth
factor; tissue-type plasminogen activator.
Tetranectin (TN) is a homotrimeric C-type lectin [1]. It was
originally purified due to its specific affinity towards the
kringle-4 domain of plasminogen (Plg) [2]. Each of the three
20 kDa-monomers consists of an N-terminal heparin-bind-
ing domain, an a-helical domain involved in the trimeriza-
tion through a triple coiled coil structure, and a C-terminal

carbohydrate recognition domain responsible for the bind-
ing to Plg [3,4]. Interaction between the carbohydrate
recognition domain and Plg is both lysine and calcium
sensitive, each of which practically abolished the binding
between TN and Plg [3].
TN was originally isolated from plasma [2] but it shows a
wide tissue distribution. Predominantly, TN was found in
the secretory cells of endocrine tissue like pituitary, thyroid,
parathyroid glands, and the liver, pancreas, and adrenal
medulla [5]. A distinct accumulation of TN was observed in
the surrounding extracellular matrix of various carcinomas
[6–8] where it colocalized with Plg [9]. Whether the TN
originated from plasma or it was produced by the surround-
ing tissue is still unknown. However, it has been established
that the TN concentration in plasma decreased parallel with
the growth of the tumour and this characteristic is considered
to be an indication of poor survival prognosis [10].
Although the biological function of TN is still uncertain,
there has been some speculation. One suggestion is that TN
forms a link between the extracellular matrix and Plg by
linking Plg to sulphated polysaccharides, thus enabling a
local tissue remodelling. Therefore, TN may be involved in
events leading to the proteolysis of matrix proteins, as
activated Plg is believed to play a key role in the degradation
of the extracellular matrix. Another possibility is that TN
stimulates mineralization during osteogenesis [11] and may
participate in myogenesis during embryonic development as
well as muscle regeneration [12]. Thus, the TN-employing
mechanisms are likely to be generally applicable to tissue
remodelling and not just a characteristic feature of tumour

invasion.
At the present stage TN has been reported to bind
apolipoprotein(a) [13], fibrin [14], Plg, and some sulphated
polysaccharides [15]. Here evidence is presented of two novel
TN-binding proteins: hepatocyte growth factor (HGF) and
tissue-type plasminogen activator (tPA), whereas three
related proteins: macrophage-stimulating protein (MSP),
urokinase-type plasminogen activator (uPA), and pro-
thrombin, appeared to be incapable of TN-binding.
Experimental procedures
Expression and purification of recombinant tetranectin
Using the previously cloned TN cDNA [16] from a murine
lung cDNA library as template in a polymerase chain
reaction the coding sequence of murine TN was amplified
andinsertedintopPICZa-vector (Invitrogen, Netherlands)
containing a signal peptide, a myc-epitope for tracing, and
six histidine residues for purification at the N-terminal end
of the expressed protein. Additionally, the sequence of a
coagulation factor X
a
cleaving site (Ile-Glu-Gly-ArgflGly)
was inserted at the 5¢-end of the TN-sequence to facilitate
Correspondence to T. E. Petersen, Protein Chemistry Laboratory,
Department of Molecular and Structural Biology, University of
Aarhus, Gustav Wieds Vej10 C, DK-8000 Aarhus C, Denmark.
Fax: + 45 86 13 65 97, Tel.: + 45 89 42 50 94,
E-mail:
Abbreviations: TN, tetranectin; Plg, plasminogen; HGF, hepatocyte
growth factor; tPA, tissue-type plasminogen activator; uPA,
urokinase-type plasminogen activator; MSP, macrophage-

stimulating protein.
(Received 26 July 2002, revised 4 February 2003,
accepted 28 February 2003)
Eur. J. Biochem. 270, 1850–1854 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03549.x
cleavage of the N-terminal service peptides (myc,His
6
)after
protein purification. The construct was transfected into the
protease-deficient SMD1168 strain of Pichia pastoris as
described by the manufacturer.
Positive clones were initially grown in shake flasks on
glycerol to gain a high cell density. Afterwards, the carbon
source was changed to 0.5% methanol, which induced
expression of TN. The fermentation was carried out as
outlined by the manufacturer. The cell free culture medium
was saturated with ammonium sulphate to 95% and
centrifuged. The precipitate was dissolved in 50 m
M
Na
2
HPO
4
pH 7.4, 0.14 m
M
NaCl and dialyzed overnight
at 4 °C to decrease the salt concentration. From preliminary
experiments, the sample was expected to contain a mixture
of two TN-forms: myc-His
6
-TN and TN without the service

peptides (the latter one to be prevailing). Therefore, the myc-
His
6
-TN form was separated from the other by adsorption
on a Ni-matrix, and TN was purified as described below.
The dialyzed sample was mixed with Ni-NTA-Sepharose,
loaded into a column, and washed with 50 m
M
Na
2
HPO
4
pH 7.4, 0.5
M
NaCl. The run through and the wash
fractions were collected and dialyzed overnight at 4 °Cto
remove salt. The obtained preparations were adsorbed on
an S-Sepharose column and washed until a stable baseline
was reached. TN was eluted with 50 m
M
Na
2
HPO
4
pH 7.4,
0.5
M
NaCl and concentrated by ultrafiltration.
The identity of the purified protein was confirmed by
N-terminal sequencing and showed the sequence

GESPTPKAKK…, which compared to the native sequence
(ESPTPKAKK…) only including an additional N-terminal
glycine. The concentration of TN stocks was determined by
aminoacidanalysis.
Ligand blot analysis using
125
I-labelled tetranectin
Three micrograms of bovine Plg, 4 lg human tPA, 2 lg
human uPA, 2 lg human HGF (294-HGN, R&D Systems
Europe,UK),3lg bovine prothrombin, and 2 lg human
MSP (352-MS, R&D Systems) were dissolved in the
Laemmli-buffer containing 2% SDS and subjected to
SDS/PAGE. Subsequent electroblotting was carried out
on Immobilon-P transfer membranes (Millipore, Bedford,
MA, USA). The membrane was blocked with 5% BSA,
0.05% Tween 20 and 1 m
M
EDTA in 50 m
M
Na
2
HPO
4
pH 7.4 for 2 h. The blot was then incubated overnight
at 4 °Cwith
125
I-labelled TN corresponding to
100 000 cpmÆmL
)1
. TN was labelled with

125
I according
to the chloramine T method. After washing 3 · 15 min
with 0.05% Tween 20 and 1 m
M
EDTA in 50 m
M
Na
2
HPO
4
pH 7.4 the blot was dried and visualized by
autoradiography.
Concentration-dependent binding assays
ELISA-trays (96-well) were coated with 100 lL2lgÆmL
)1
bovine Plg, human tPA, human uPA, bovine prothrombin,
or monoclonal anti-human HGF (MAB694, R&D Sys-
tems)at4 °C overnight. The wells were then blocked for 1 h
at 37 °Cwith200lL 0.5% gelatine and 1 m
M
EDTA
dissolved in 50 m
M
Na
2
HPO
4
pH 7.4, 0.14 m
M

NaCl. The
wells were washed briefly three times with 200 lLof
50 m
M
Na
2
HPO
4
pH 7.4, 0.14 m
M
NaCl, 0.05% Tween 20,
1m
M
EDTA buffer between every incubation. There was
an additional step in the case of HGF when the antihuman
HGF-containing wells were incubated with 100 lLof
1 lgÆmL
)1
human HGF for 2 h at 37 °C. All incubations
were performed in blocking buffers. The compounds
immobilized in the wells were then exposed to TN, which
concentration varied from 0 to 50 lgÆmL
)1
in 100 lL. The
incubation continued for 2 h at 37 °C. After washing, the
wells were treated with 100 lL custom-made (DAKO,
Roskilde, DK), specificity-checked rabbit anti-murine TN
serum (1 : 1000) for 1 h at 37 °C. Then 100 lLof
peroxidase-conjugated porcine anti-rabbit IgG (1 : 2000)
was added and incubated for one hour at 37 °C. Finally, the

TN-positive samples were visualized by a coloured reaction
with o-phenylenediamine. The reaction was stopped with
100 lL2
M
H
2
SO
4
, and the absorbance at 490 nm gave a
relative content of bound TN.
Plasminogen activation assay
The ability of TN to enhance the plasminogen activation
potential of tPA was investigated in a coupled reaction
assay by measuring hydrolysis of the plasmin substrate S-
2251 (H-D-Val-Leu-Lys-p-nitroaniline). The proteolytic
cleavage of the substrate resulting in release of the yellow
p-nitroaniline was followed spectroscopically at 405 nm.
The plasminogen activation assay was carried out in
100 lL0.1
M
Tris pH 7.4, 0.02% Tween 80, 5 l
M
TN,
and 0.5 m
M
S-2251 at varying concentrations of bovine
plasminogen (0.1–2.0 l
M
). The reaction was initiated by
addition of 10 n

M
human tPA, and the appearing
plasmin activity was followed for 3 min at 37 °C. Two
independent experiments were made for each Plg con-
centration.
The activation of Plg and the subsequent hydrolysis of the
plasmin substrate can be described in the simplest case by
the following scheme:
tPA þ Plg !
K
1
tPA-Plg À!
k
1
tPA þ Pln ð1Þ
Pln þ S !
K
2
Pln-S À!
k
2
Pln þ P ð2Þ
where Pln represents plasmin, S and P represent the plasmin
substrate S-2251 and the yellowish product P, respectively,
K
1
and K
2
are the Michaelis constants (K
m

)ofthe
corresponding enzymes, k
1
and k
2
are their catalytic
constants (k
cat
). The process was carried out at
[tPA] << [Plg] and [Plg] % constant during the time
of the reaction. The amidolytic activity of tPA
(tPA + S fi tPA + P) can be ignored because of its
low efficiency when compared with Pln.
The collected data (t,p) was transformed and analyzed
according to a previously published model [17] that gives a
linear dependence of y on t
2
y ¼ y
0
þ v
a
t
2
where y ¼ 2
K
2
k
2
ln S
0

e
p=K
2
=ðS
0
À p Þ
ÀÁ
and y
0
is the error in
determination of the zero point (y
0
% 0). The transforma-
tion of P to y was carried out with the known values of
K
2
¼ 250 l
M
and k
2
¼ 1000 min
)1
[18] and the substrate
concentration from this experiment s
0
¼ 500 l
M
. The slope
Ó FEBS 2003 Tetranectin binds HGF and tPA (Eur. J. Biochem. 270) 1851
v

a
is equal to the velocity at a given [Plg]. A number of
([Plg],v
a
) pairs were obtained and fitted to the equation
v
a
¼ðk
1
½tPA
0
½PlgÞ=ðK
1
þ½PlgÞ which enabled calcula-
tion of the kinetic parameters K
m
and k
cat
of tPA towards
Plg, see [17] for details.
Results
Purification of the recombinant tetranectin
The recombinant TN was expressed as a mixture of two
forms (myc-His
6
-TN and TN) due to occasional N-terminal
cleavage of the protein at the X
a
-site in the yeast. The form
without service peptides (TN) prevailed and it was therefore

separated from myc-His
6
-TN by absorption of the latter on
Ni-NTA-sepharose. The run through fraction was subjected
to S-Sepharose, which enabled TN purification. Two intense
bands each with a relative molecular mass of approximately
20 kDa were visualized on SDS/PAGE (Fig. 1). These
bands corresponded to TN on Western blot (data not
shown). Amino acid sequencing of the N-terminus revealed
that the myc-epitope and the histidine-tag were cleaved off
at the coagulation factor X
a
-site upon secretion leaving the
recombinant TN of the desired length. An additional
cleavage occurred after Lys10 as well. From N-terminal
sequence analysis it was estimated that the purified protein
contained a 1 : 4 mixture of full-length and N-terminally
cleaved TN.
Ligand blot analysis
In an attempt to identify new compounds capable of TN-
binding, several candidates were subjected to ligand blot
analysis. The proteins were chosen in the light of their
contents of kringle domains. Plg with five kringle domains
served as a positive control for the binding of TN. The
plasminogen-like growth factors HGF and MSP are very
similar to Plg in their domain structure and each of them
contains four kringle domains. Prothrombin has two kringle
domains and the structures of plasminogen activators tPA
and uPA include two and one kringles, respectively.
The ligand blot analysis (Fig. 1) revealed binding of TN

to Plg, tPA, and HGF, whereas uPA, prothrombin, and
MSP showed no binding properties towards TN. Distinct
bands of Plg and HGF were visible after a short time of
exposure. The tPA band was somewhat weaker, but it
became clearer after longer exposure.
Concentration-dependent binding assays
To validate the results from the ligand blot analysis the
interactions were tested by ELISA. The solid-phase binding
assays revealed a concentration-dependent binding of TN to
Plg, HGF, and tPA. As expected, no specific binding was
observed for uPA and prothrombin (Fig. 2). MSP was not
included in this assay. Table 1 presents the summarized data
from the ELISA, where the specific affinities are compared
to unspecific binding, and the measured dissociation
constants K
d
are listed for Plg, HGF, and tPA.
Fig. 1. SDS/PAGE/Ligand blot analysis. The samples were reduced
prior to electrophoresis. Left: SDS/PAGE indicates two bands of TN
as a result of N-terminal cleavage. Right: Lanes 1–6 were loaded with
plasminogen, tPA, uPA, HGF, prothrombin, and MSP, respectively.
The blot shows TN-binding to plasminogen and HGF. However,
longer exposure revealed binding to tPA as well.
Fig. 2. Concentration-dependent binding of ligands to TN. The curves
correspond to plasminogen (s), tPA (n), HGF (e), uPA (h), and
prothrombin (,). Each point is the mean of quadruplicate determi-
nations. In the case of uPA and prothrombin, no K
d
could be deter-
mined.

Table 1. Summary of TN affinity assays. The column ÔBound TNÕ
corresponds to the amount of TN detected in the wells coated with a
potential ligand; Unspecific binding corresponds to the amount of TN
detected in the uncoated wells. The percentages are standardized
according to plasminogen (100%). Calculation of the K
d
is based on
Michaelis–Menten kinetics. Dissociation constants for the binding of
TN to plasminogen and tPA are based on three independent experi-
ments of quadruplicate measurements. For the TN-HGF binding, the
K
d
is based on one experiment of quadruplicate measurements. In the
case of uPA and prothrombin, one and two independent experiments
of quadruplicate measurements were performed, respectively.
Bound
TN (%)
Unspecific
binding (%) K
d
(l
M
)
Plg 100.0 ± 9.5 6.2 ± 3.2 0.33 ± 0.05
HGF 84.3 ± 8.7 11.8 ± 0.5 0.49
tPA 97.2 ± 14.5 8.7 ± 4.7 0.28 ± 0.09
uPA 14.2 ± 0.4 9.0 ± 1.0 –
Prothrombin 17.4 ± 5.8 8.1 ± 1.1 –
1852 U. B. Westergaard et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Two different experiments complemented and supported

each other, thus confirming the affinities of the novel
TN-binding proteins.
Plasminogen activation assay
It was found in the preliminary experiments that the
amidolytic activities of TN-Pln and TN-tPA complexes
towards the substrate S-2251 were equal to those of Pln and
tPA and that no difference was found in the uPA-mediated
activation of Plg with or without the inclusion of TN. This
validates the direct application of Eqn (1) for calculation
of v
a
at different Plg-concentrations. The final data are
presented in Fig. 3 as a plot of the reaction velocity v
a
vs. the
concentration of Plg. The analysis reveals the kinetic
parameters of the activation of Plg by tPA in the presence
of TN: K
m
¼ 0.28 l
M
and k
cat
¼ 10 min
)1
. In the control
experiment the k
cat
could not be determined. However,
when approximating k

cat
to 10 min
)1
, K
m
can be calculated
to 2.5 l
M
.
Discussion
The results presented here show evidence for TN-binding to
HGF and tPA by two independent methods, thereby
adding two kringle-containing proteins to the list of
TN-ligands. The affinity of the recombinant TN to Plg
(comparable to the one of the natural TN) demonstrates
that the recombinant protein has been correctly folded in
P. pastoris and hence is relevant for the affinity assays. Lack
of interaction between TN and uPA indicates that TN does
not have a general affinity towards kringle domains.
Moreover, TN does not bind MSP, a growth factor
structurally similar to HGF.
The calculated K
d
for Plg’s binding to TN
(0.33 ± 0.05 l
M
) is consistent with the previously pub-
lished dissociation constants of 0.5 l
M
and 0.2 l

M
[3,13]. The values of K
d
for HGF (0.49 l
M
)andtPA
(0.28 ± 0.09 l
M
) are within the same order of magnitude
pointing to the same type of binding for all three ligands. In
other words, the carbohydrate recognition domain is very
likely to be responsible for the binding of HGF and tPA,
although this needs further clarification. Differences in K
d
are insignificant and may be caused by the nature of the
primary and secondary antibodies used for detection.
It has long been known that TN stimulates the activating
cleavage of Plg by tPA, though, no details about the process
have been known [2]. The present study verifies the
activating ability of TN and shows that the presence of
TN increases the association between tPA and Plg by
10-fold. The values of K
m
and k
cat
calculated for the
TN–tPA complex were similar to the parameters for the
tPA activation of Plg in the presence of fibrinogen fragments
(K
m

¼ 0.1 l
M
and k
cat
¼ 25 min
)1
) [17]. This implies that
TN may act in the same way as fibrinogen fragments. The
experimental data were, however, not conclusive concerning
the exact mechanism behind the enhancement. Recently, we
have reported that bovine tPA exists in equilibrium between
two different conformations, where only a minor part of
tPA-molecules can bind and cleave Plg. Fibrinogen frag-
ments bind only the active form of tPA and thereby poises
the equilibrium towards accumulation of the active confor-
mation. The net result is an increased concentration of
active tPA capable of cleaving Plg [17]. An analogous
mechanism was suggested for human tPA, though, conver-
sion between inactive and active tPA occurred rapidly and
was difficult to detect by conventional measurements. It
seems possible that the TN-induced activation of tPA is
similar to the one of fibrinogen fragments. Another
explanation suggests that TN is able to bind the active
form of tPA and Plg simultaneously and by bringing the
two components together, it acts as a cofactor in the
activation of Plg to plasmin. The ability of TN to
accumulate by one or another mechanism the active tPA
in the extracellular matrix enables a higher control of the
local plasmin activity.
HGF is a mitogen for a variety of cells including epithelial

and endothelial cells, melanocytes, keratinocytes, and
hepatocytes. Additionally, the cytokine stimulates cell
motility and morphogenesis. HGF has also been shown to
take part in angiogenesis. HGF is a 90-kDa glycoprotein
composed of a 60-kDa a chain and a 30-kDa b chain
covalently linked by a disulphide bond. The a chain
contains an N-terminal heparin-binding hairpin-loop and
four kringle domains. The b chain is homologous to serine
proteases, but lacks proteolytic activity due to mutations in
the catalytic triad. HGF is secreted as a biologically inactive
single-chain form and extracellular processing is required to
obtain the active two-chain HGF. This is accomplished by a
specific cleavage at Arg494-Val495, similar to the Arg-Val
cleavage required for activation of Plg to plasmin [19].
There is evidence that single-chain HGF can be activated
along two or more pathways. In response to a tissue injury,
epithelial cells produce HGF activator, a 34-kDa serine
protease, that once it has been activated by thrombin, can
process single-chain HGF [20]. Another pathway involves
the plasminogen activators tPA and uPA. It has been shown
in in vitro bioassays that tPA and uPA can convert single-
chain HGF into biologically active HGF [21]. Taking the
activation of HGF by tPA together with the new findings
Fig. 3. tPA-catalysed plasminogen activation in the presence of TN. The
curve represents a plot of the reaction velocity v
a
vs. the concentration
of plasminogen fitted to a Michaelis equation. Data obtained in the
presence of TN (s) and control data without TN (d) are based on two
or more measurements. The kinetic analysis revealed that TN has a

resemblance to fibrinogen fragments in respect to the enhancement of
tPA-catalysed plasminogen activation.
Ó FEBS 2003 Tetranectin binds HGF and tPA (Eur. J. Biochem. 270) 1853
that TN can both bind and enhance tPA activity, it is
tempting to hypothesize whether TN is involved in the
regulation of HGF as well. Testing this hypothesis would
seem evident. However, several unsuccessful attempts were
made to express recombinant single-chain HGF as the
commercially available HGF is in an already activated form.
The colocalized expression of HGF and tPA in the murine
olfactory system tells in favour of this suggestion [22].
Acknowledgements
This work was supported by a grant from Novo Nordisk A/S with a
scholarship to UBW.
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