(Received for publication, September 25, 1996, and in revised form, December 23, 1996)
From the Midkine (MK), a retinoic acid-inducible
growth/differentiation factor, serves as a substrate for tissue
transglutaminase (Kojima, S., Muramatsu, H., Amanuma, H., and
Muramatsu, T. 1995. J. Biol. Chem. 270, 9590-9596).
Upon incubation with transglutaminase MK forms multimers through
cross-linkages. Here, we report the following results. 1) Heparin
potentiated the multimer formation by MK. 2) The N- and C-terminal half
domains each formed a dimer through the action of transglutaminase. 3)
Gln42 or Gln44 in the N-terminal half and
Gln95 in the C-terminal half served as amine acceptors in
the cross-linking reaction, as judged from the incorporation of
putrescine into whole MK or each half domain, and the competitive
inhibition of the cross-linking by MK-derived peptides containing Gln
residue(s). The strongest inhibition was obtained with
Ala41-Pro51. 4) This peptide abolished the
biological activity of MK to enhance the plasminogen activator activity
in bovine aortic endothelial cells. The inhibition was limited against
the MK monomer, and not seen against the MK dimer, separated by gel
filtration chromatography. These results suggest that dimer formation
through transglutaminase-mediated cross-linking is an important step as
to the biological activity of MK.
Tissue type II transglutaminase (R-glutaminylpeptide: amine
Midkine (MK)1 and pleiotrophin (PTN)
constitute a new family of heparin-binding growth/differentiation
factors (15, 16). MK has been found as a product of a retinoic
acid-responsive gene (17), and exerts a variety of biological
activities; it enhances neurite outgrowth and the survival of various
embryonic neuron types (18-21), and is mitogenic for certain
fibroblastic cell lines (18, 19). In addition, recently, we found that
MK enhances the plasminogen activator (PA) activity in bovine aortic
endothelial cells (BAECs; Ref. 22). PTN, also called heparin-binding
growth-associated molecule (HB-GAM; Refs. 23 and 24), was found as
another neurite-promoting factor (25). PTN has been shown to be
mitogenic for endothelial cells (26) and to enhance tube formation
in vitro (27). Expression of these factors is strictly
controlled during the processes of differentiation and development
(28-30). MK is highly expressed in many human cancers (31) and
specifically localized in senile plaques of Alzheimer's disease (32),
and the overexpression of PTN in NIH3T3 cells results in transformation
of the cells (33), suggesting the involvement of MK and PTN not only in
normal development, but also in the pathogeneses of diseases (15,
16). MK is a 13-kDa heparin-binding polypeptide rich in basic amino acids and cysteine (17, 34), and exhibits 46% sequence identity with
PTN (15). Both MK and PTN are largely composed of two domains, called
the N-half (N1/2; Lys1-Gly59 in human MK) and
the C-half (C1/2; Ala60-Asp121 in human MK),
each of which contains a couple of intra-disulfide linkages (35, 36).
Of these two domains C1/2 is responsible for heparin-binding,
neurite outgrowth-promoting, and PA-enhancing activities (37, 38).
During the course of studying the PA-enhancing properties of MK, we
discovered that MK serves as a good substrate for tissue transglutaminase (22). BAECs constitutively produce and secrete MK,
which forms a transglutaminase-mediated complex in cultures. Prior to
this discovery, a 29-kDa MK-related protein, which is now recognized as
a dimer of MK, had been detected in a variety of tissues, such as the
lymphonode, spleen, testis, small intestine, stomach, lung, kidney, and
liver (39). Furthermore, Haynes and colleagues (40, 41) reported
similar dimer formation by MK in the developing brain. These lines of
accumulating evidence suggest that the cross-linking of MK by tissue
transglutaminase may occur and play an important role in MK biology
in vivo.
In the current study, we have investigated the mechanism underlying the
transglutaminase-mediated cross-linking reaction and the relevance of
dimer formation to MK activity.
The procedure for chemical synthesis of human MK
and its fragments including N1/2 and C1/2 domains was described in a
previous paper (42). Antibodies against the whole MK molecule, N1/2
domain, and C1/2 domain were produced in rabbits by injecting each
antigen subcutaneously into animals after homogenization with Freund's complete adjuvant (Sigma). Immunization and bleeding
were conducted biweekly. The IgG fraction was isolated from the sera
using protein A-Sepharose (Pharmacia Biotech Inc.). The antibodies were
shown to be immunospecific to each antigen by Western blotting as
reported previously (37) or as shown in Fig. 1. The antibody against whole MK recognized the C-terminal tail
(Thr105-Asp121) and thus recognized C1/2, but
not N1/2 (37). The anti-N1/2 antibody did not cross-react with C1/2
(Fig. 1, panel A). The antibody to C1/2 recognized C-domain
(Cys62-Cys104), which forms the compact
structure maintained by two disulfide bonds in C1/2 (Fig.
1, panel B). This antibody showed strong, weak, and no cross-reactivity against C1/2, whole MK, and N1/2, respectively. Guinea pig liver transglutaminase and heparin sodium salt
were purchased from Takara Biochemicals (Ohtsu, Japan) and Nacalai
Tesque Inc. (Kyoto, Japan), respectively. Purified plasma factor XIII
was a generous gift from Dr. Y. Saito (Tokyo Institute of Technology,
Yokohama, Japan).
Intact (whole) MK, N1/2, or
C1/2 was incubated with transglutaminase in Hepes-buffered saline (129 mM NaCl, 5 mM KCl, 0.3 mM
Na2HPO4, 1 mM NaHCO3, 5 mM glucose, and 25 mM Hepes, pH 7.4) containing
10 mM CaCl2, 8 mM dithiothreitol,
0.3% (v/v) glycerol, and 3 mM
[14C]putrescine (0.45 µCi, Amersham), in a final volume
of 300 µl. The reaction mixture was incubated for the indicated time
at 37 °C and then the reaction was stopped by the addition of 600 µl of 16.7% trichloroacetic acid with 100 µl of 2% bovine serum
albumin. The precipitate was collected on Whatman GF/C glass filters
and washed three times with 2 ml of 10% trichloroacetic acid, and then
the radioactivity was measured by liquid scintillation counting. The
amount of putrescine incorporated into each sample was expressed as
moles of putrescine incorporated/mol of each sample.
Western blotting was performed as
described previously (22) using antibodies to intact MK (final 5 µg/ml), N1/2 (final 100 µg/ml), or C1/2 (final 100 µg/ml), and
goat anti-rabbit IgG antibodies conjugated with peroxidase (Jackson
ImmunoResearch Laboratories, Ltd., West Grove, PA). The signals were
detected with an Amersham (Buckinghamshire, United Kingdom) ECL
system.
BAECs were isolated and
grown in Gel filtration
chromatography was performed using a Superdex 75 HR 10/30 column and a
FPLC System (Pharmacia). The column was equilibrated and eluted with
Hepes-buffered saline buffer containing 1 M NaCl at the
flow rate of 0.5 ml/min. The high salt was included to suppress the
nonspecific adherence of MK to the apparatus (43). The absorbance at
280 nm was monitored, and fractions of 0.5 ml each collected.
Recently,
we found that MK serves as a substrate for tissue transglutaminase
(22). Incubation of recombinant murine MK with purified tissue
transglutaminase readily yielded SDS- and
The formation of
multimers indicates that both murine and human MK contain both Gln and
Lys residues that participate in the transglutaminase-mediated bridge
formation. Both murine and human MK contain 5 Gln residues/molecule
(15, 17). To determine the number of acyl-donating Gln sites, a
putrescine incorporation assay was performed using synthetic human MK.
MK was incubated overnight with transglutaminase in the presence of
[14C]putrescine, and then the number of putrescine
molecules incorporated into one molecule of MK was calculated from the
radioactivity. The result is shown in Fig. 3
(column 1). About 2 mol of putrescine was incorporated/mol
of MK, suggesting the presence of at least two different Gln sites in a
molecule. The incubation time and the concentration of putrescine
employed gave the maximum amount of putrescine incorporated as a
function of both the putrescine concentration and the incubation time
(data not shown). MK is structurally composed of two domains, called
N1/2 (Lys1-Gly59) and C1/2
(Ala60-Asp121; Ref. 15). To determine which
domain contains the Gln sites, the incorporation of putrescine into
synthetic N1/2 or C1/2 was measured. As can be seen in Fig. 3
(columns 2 and 3), both domains share one Gln
site each. This also suggested that N1/2 and C1/2 alone each might be
cross-linked by transglutaminase, as both domains contain many
acyl-acceptable Lys sites (9 in N1/2 and 14 in C1/2). This was proved
by the results of Western analyses performed following the incubation
of either N1/2 or C1/2 with transglutaminase (Fig. 4).
Although multimers larger than the tetramer were not observed much, a
significant amount of the dimer was detected for both N1/2 and C1/2
(lanes 2 and 3). In contrast, comparable amounts
of the dimer and tetramer were detected on the cross-linking of whole
MK molecules (lane 1). These reactions did not occur in the
absence of Ca2+ (data not shown). N1/2 and C1/2 contain two
and three Gln residues, respectively (15). Therefore, it is suggested
that either Gln42 or Gln44 in N1/2 and one of
the three Gln residues (amino acids 82, 93, and 95) in C1/2 serve as
acyl-donating sites. Plasma-derived FXIIIa catalyzed the incorporation
of [14C]putrescine into MK, but did not catalyze the
cross-linking of MK molecules, as assessed by Western blotting (data
not shown).
In parallel with putrescine incorporation assays, the effect of
Gln-containing peptides on the cross-linking of intact MK, N1/2, and
C1/2 was examined. In the process of chemical synthesis of human MK, we
synthesized 13 segments that could be coupled to form N1/2 and C1/2,
and finally intact MK (42). Of these 13 segments, segment V
(Ala41-Pro51) contains both Gln42
and Gln44, and segments IX
(Thr78-Gly83), X
(Thr84-Gln93), and XI
(Cys94-Pro103) contain Gln82,
Gln93, and Gln95, respectively. The
cross-linking reaction was performed in the presence of these peptides
at the concentration of a 100-fold excess over moles of intact MK,
N1/2, or C1/2, and the formation of each dimer was assessed by Western
blotting. The results are shown in Fig. 5. Among the
four peptides tested, Ala41-Pro51 (lane
2) and Cys94-Pro103 (lane 5)
blocked the cross-linking reactions, whereas neither Thr78-Gly83 (lane 3) nor
Thr84-Gln93 (lane 4) affected the
reactions. The Ala41-Pro51 peptide showed a
complete inhibition against intact MK (panel A), N1/2
(panel B), and C1/2 (panel C). The
Cys94-Pro103 peptide also showed a complete
inhibition toward the cross-linking of intact MK (panel A)
and N1/2 (panel B), and partial inhibition against the
cross-linking of C1/2 (panel C). The retardation and smear
formation of each monomer observed in lanes 2 and
5 suggested that these peptides were preferentially
cross-linked to molecules in question and, thereby, prevented
cross-linking between monomer molecules. In this aspect, the results
showed that Cys94-Pro103 was incorporated into
MK or its fragment molecules more slowly than
Ala41-Pro51. Next, to determine which Gln
residue in Ala41-Pro51, namely either
Gln42 or Gln44, participated in the
cross-linking reaction, three mutant peptides were synthesized (Fig.
6); specifically, Gln42 was substituted with
Ala, Gln44 was substituted with Ala, and both
Gln42 and Gln44 were substituted with Ala, and
then the competitive inhibition was tested. As shown in Fig.
7 (lane 2), the intact peptide was readily
cross-linked to MK in a time-dependent manner and inhibited the cross-linking completely, consistent with the result in Fig. 5
(lane 2). Peptides in which one of the two Gln residues was substituted with Ala showed significant, but slower inhibition (lanes 3 and 4), suggesting that both
Gln42 and Gln44 can serve as amine acceptor
sites. In contrast, the double substituted peptide showed almost no
inhibition (lane 5), suggesting that the inhibition by the
peptides observed in lanes 2-4 was not due to the sequence
structure other than Gln residues. Similar inhibition was observed with
these peptides on the cross-linking of N1/2 or C1/2 (data not shown).
In conclusion, Gln42 and Gln44 in the N1/2
domain as well as Gln95 in the C1/2 domain were suggested
to function as potential amine-accepting sites in the
transglutaminase-mediated cross-linking of MK.
Next, we examined whether or not the cross-linking of MK
participated in the MK action of enhancing the PA level, using a Gln-containing peptide, Ala41-Pro51, that
showed the strongest inhibition against the cross-linking reaction
(Figs. 5 and 7). BAECs were incubated with MK with increasing amounts
of Ala41-Pro51, and then the PA levels were
determined. Indeed, the peptide suppressed the enhancement by MK (Fig.
8, curve B), whereas a mutant peptide, with
double substitution of two Gln residues with Ala, did not (curve
A). This suggested that interference of the cross-linking by
Ala41-Pro51 abolished the activity of MK added
to the culture medium, namely that cross-linking of the MK monomer by
cell surface transglutaminase might be required for the MK activity. If
this speculation is correct and if the inhibition by the peptide only
occurs at the step of the cross-linking, the once cross-linked dimer or
tetramer should enhance the PA level without undergoing inhibition by
the peptide. This was examined by means of an experiment in which the
monomer, dimer, and other multimers were separated by gel filtration
chromatography, followed by assaying for PA-enhancing activity in the
absence and presence of the Ala41-Pro51
peptide (Fig. 9, panel A). The PA-enhancing
activity (closed circles) was detected in fractions
corresponding to the peak of absorbance at 280 nm (dotted
line). From a comparison with the molecular standards used for the
gel filtration chromatography and from the results of Western blotting
of each fraction (panel B), three major peaks were
determined as the monomer, the dimer, and a mixture of the tetramer and
hexamer, from lower molecular weight fractions. As depicted by
open circles, Ala41-Pro51
suppressed the PA-enhancing activity only in the monomer fraction, i.e. not in the dimer or tetramer/hexamer fraction. This
supports the hypothesis that transglutaminase-mediated cross-linking of the MK monomer is required for its ability to enhance the PA level in
BAECs.
As can be seen in Fig. 9
(panel B), several bands were detected on Western blotting
of the peak fractions. For example, the hexamer, tetramer, and dimer
were seen in fraction 23, which was determined to be composed of the
hexamer and tetramer, and the dimer and monomer were detected in
fraction 26, which was determined to be the dimer. As a reason for
this, we speculated that MK associated non-covalently in addition to
the covalent cross-linking by transglutaminase. To determine if such a
non-covalent association really occurs, we performed the cross-linking
reaction in the presence or absence of free Ca2+, and then
compared the chromatograms obtained on FPLC (Fig. 10). In the presence of free Ca2+, namely when transglutaminase
functioned, almost the same amounts of the monomer, the dimer, and the
tetramer/hexamer mixture were detected in fractions 36, 26, and 23, respectively (curve B, a solid line). However,
even when transglutaminase was blocked by chelating Ca2+,
almost the same peak was detected for fraction 26, and about a half
amount of the peak was detected for fraction 23, in addition to a peak
of the monomer for fraction 36 (curve A, a dashed
line). Furthermore, the PA-enhancing activity was detected in
accordance with the peak of multimers in addition to monomer (Fig.
11). Western blots of these fractions only showed the
monomer (data not shown), indicating that the peaks detected for
fractions 26 and 23 represented the non-covalently associated MK dimer,
tetramer, or hexamer, which dissociated upon exposure to SDS.
Therefore, under the current experimental conditions, MK forms
multimers via two distinct mechanisms, transglutaminase-mediated
covalent cross-linking and non-covalent association. We next examined
the effect of Ala41-Pro51 on this non-covalent
association. The cross-linking reaction was performed in the presence
of an excess of the peptide, and multimer formation was assessed by gel
filtration chromatography. As depicted by curve C (a
double-dashed line) in Fig. 10, almost no peak was detected
for fraction 26, although two small peaks were detected for fractions
22 and 23, suggesting that not only the cross-linking, but also the
non-covalent association was suppressed by the
Ala41-Pro51 peptide.
Retinol (vitamin A) and its derivatives (retinoids) have profound
effects on the regulation of cell growth and differentiation (44).
Using BAEC cultures, we found that retinoids induce the production of
PA (45), transglutaminase (46), transforming growth factor- MK was proved to be an excellent substrate for transglutaminase in that
1 mol of MK incorporated as much as 2 mol of
[14C]putrescine. MK formed the dimer, tetramer, and
hexamer on incubation with transglutaminase. Among multimers dimer
seemed to be dominant. Whereas the results in Figs. 5 and 7 suggest
that Gln42 and Gln44 in N1/2 as well as
Gln95 in C1/2 may function as potential amine-accepting
sites, the results in Fig. 3 suggest the existence of one Gln site in
each of N1/2 and C1/2. The most likely explanation for this difference is that although both Gln42 and Gln44 are
competent enough to serve as amine-accepting sites, only one of them is
exposed to the surface of the MK molecule. The second likely
explanation is that Gln42 and Gln44 serve
heterogeneously as amine-accepting sites. Namely, when Gln42 is used to form an We have concluded that dimerization of MK by cell surface
transglutaminase potentiates MK activity, based upon the fact that the
Ala41-Pro51 peptide inhibits both the
cross-linking and PA-enhancing activity of the MK monomer, with a
double mutated peptide as an inactive control. We did not obtain
additional proof utilizing an anti-transglutaminase antibody, because
the antibody could inhibit the formation of TGF- The formation of the non-covalently associated dimer and other
multimers explains why the MK monomer has always been detected on
Western blotting, even on incubation with transglutaminase overnight.
The Ala41-Pro51 peptide inhibited both the
cross-linking and the non-covalent association through being
incorporated into the Lys sites of MK molecules by transglutaminase. It
appears that the peptide cannot dissociate the dimer once formed via
either covalent cross-linking or even non-covalent association, as the
peptide did not affect the PA-enhancing activity in the dimer fraction
(Fig. 9). Once non-covalent association occurs between MK molecules,
the cross-linking sites are supposed to be hidden, and thus the MK
molecules might no longer act as a substrate for transglutaminase.
Therefore, the non-covalently associated dimer co-existed with the
cross-linked dimer, and the peptide was not cross-linked to the
non-covalently associated dimer and, thus, did not affect its activity.
We cannot explain why in the presence of
Ala41-Pro51, the peak of the mixture of the
tetramer and hexamer split into the hexamer and tetramer (Fig. 10); nor
can we explain why the peptide did not inhibit non-covalent formation
of the hexamer and tetramer completely, although the peptide inhibited
the formation of the dimer completely. The non-covalent association may
be apt to occur when the concentration of MK is unphysiologically high. At a physiological concentration, the chances of MK associating non-covalently will be low, whereas cross-linking may occur at sites
where transglutaminase is exposed. Figs. 9 and 11 suggest that
dimer/oligomer formation alone is sufficient to stimulate increases in
PA-enhancing activity and that binding of MK to cells via cell surface
transglutaminase is not necessary. However, since plasma Factor XIIIa
is not able to cross-link MK molecules and tissue transglutaminase is
not released from the cells (46), the surface reaction may be
physiologically most important. The susceptibility of MK to
transglutaminase suggests a mechanism whereby the interaction of MK
with surface receptors and other surface-oriented structures could be
enzymatically altered. Tissue transglutaminase is distributed mainly
intracellularly and partially on the cell surface (11, 46), suggesting
either that newly synthesized MK is cross-linked before secretion or
that secreted MK is cross-linked on the cell surface as discussed
above. The results in Fig. 2 suggest that the latter reaction may
proceed very efficiently with the aid of heparan sulfate present on the cell surface. Recently, it was reported that hetero- or
homodimerization of cytokine and hormone receptors is important for the
emission of signals inside cells (50, 51). Together with the finding that the cross-linking is important for PA-enhancing activity, we
imagine that the MK dimer, cross-linked on the cell surface, may
activate its unidentified receptors by making them form the dimer. An experiment addressing this hypothesis is in progress.
The current study provided additional evidence for the hypothesis
concerning the mechanism whereby transglutaminase plays a role in the
regulation of cellular physiology through the cross-linking of
cytokines such as TGF- We thank Dr. Y. Saito for supplying
the Factor XIII as well as for comments on this study.
Laboratory of Gene Technology and Safety,
Department of
Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
-glutamyltransferase, EC 2.3.2.13) is a member of the
transglutaminase family that catalyzes
Ca2+-dependent acyl transfer reactions between
-carboxamide groups of the Gln residues in peptides and either
primary amines or
-amino groups of the Lys residues in peptides,
resulting in the formation of new
-amides of glutamic acid or
-(
-glutamyl)lysine bonds and ammonia (1, 2). The molecular
structure of tissue transglutaminase has been reported (3-5). Although
tissue transglutaminase is widely distributed in the body (6), its
physiological function is not well established compared those of other
members of the transglutaminase family, e.g. the formation
of cross-linkages between fibrin molecules by plasma Factor XIIIa (1,
2), and the formation of cross-linked envelopes during epidermal cell differentiation by tissue type I transglutaminase (7, 8). Recently,
tissue type II transglutaminase was implicated in the association of
proteases and protease inhibitors with the cell surface (9, 10), in the
activation of several cytokines (11, 12), in signal transduction (13),
and in the process of apoptosis (14).
Materials
Fig. 1.
Specificity of antibodies against N1/2 and
C1/2 domains. Panel A, after synthetic human MK C1/2 domain
(lane 1), N1/2 domain (lane 2), and intact
molecule (lane 3; 300 ng each) were subjected onto SDS-PAGE
on a 14% resolving gel, proteins were electrically transferred to
Immobilon P (Millipore) polyvinylidene difluoride membrane and the
membrane was blotted with 100 µg/ml anti-N1/2 antibody. The
antibody-reacted protein bands were visualized by peroxidase-conjugated
second antibody and ECL reagents. Panel B, after synthetic
C1/2 (lane 1), N1/2 (lane 2), intact MK
(lane 3), C-domain (lane 4), and C-terminal tail
(lane 5; 300 ng each) were subjected onto SDS-PAGE on a 14%
resolving gel, Western blotting was performed as in panel A
using anti-C1/2 antibody.
[View Larger Version of this Image (24K GIF file)]
-minimal essential medium containing 10% calf serum. The
levels of cellular PA activity were measured using the chromogenic
substrate, S-2403, as described previously (22), and expressed as
urokinase units/mg of protein in each sample.
Enhancement of the Cross-linking of MK by Heparin
-mercaptoethanol-resistant multimers, as detected by Western blotting (22). A similar result was
obtained with synthetic human MK (Fig. 2, panel
A). In BAECs, tissue transglutaminase functions in part on the
cell surface (11), suggesting that cross-linking of MK might also occur
in the cell surface milieu. On the other hand, MK is known to have strong heparin-binding activity (15). Therefore, we examined if the
heparin-binding activity of MK affected its cross-linking by tissue
transglutaminase. As shown in Fig. 2 (panel B), the inclusion of heparin in the reaction mixture significantly enhanced both the rate and the final degree of the formation of MK multimers by
transglutaminase.
Fig. 2.
Enhancement of transglutaminase-mediated
cross-linking of MK by heparin. Synthetic human MK (final: 3 µM) was incubated with 1.25 µM tissue
transglutaminase for the indicated time in the absence (panel
A) or presence (panel B) of 30 µg/ml heparin, and
then the formation of MK multimers was detected by Western blotting
after SDS-PAGE on 14% resolving gels. Each similar experiment was
repeated three times, and representative results are shown.
[View Larger Version of this Image (51K GIF file)]
Fig. 3.
Incorporation of
[14C]putrescine into intact MK, N1/2, and C1/2 by tissue
transglutaminase. Synthetic human MK, N1/2, or C1/2 (final: 3 µM each) was incubated at 37 °C for 16 h with 3 mM [14C]putrescine in the presence of 1.25 µM tissue transglutaminase in Hepes buffer containing 10 mM Ca2+. After the incubation, proteins in the
solution were precipitated by adding 10% trichloroacetic acid and
spotted onto filter paper discs, which were repeatedly washed.
Radioactivity precipitated on the discs was counted with a
scintillation counter and expressed as moles of putrescine
incorporated/mol of intact MK, N1/2, or C1/2. Column 1,
intact MK; column 2, N1/2; column 3, C1/2. Data represent averages ± S.D. (n = 3).
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
Cross-linking of intact MK, N1/2, and C1/2 by
transglutaminase. Synthetic human MK (final: 3 µM),
N1/2 (final: 6 µM), or C1/2 (final: 6 µM)
was incubated at 37 °C for 1 h with 1.25 µM
tissue transglutaminase, in a total volume of 24 µl, in Hepes buffer
containing 10 mM Ca2+. The reaction was
terminated by adding 50 mM EDTA, samples were separated by
SDS-PAGE on 14% resolving gels, and the formation of multimers was
assessed by Western blotting with an anti-intact MK antibody, anti-N1/2
antibody, or anti-C1/2 antibody. The exposure time was greatly
lengthened, compared with in Fig. 2. The 75-kDa band in lane
3 is a nonspecific band due to the first antibody. The
numbers at the right of each band are the numbers
of molecules cross-linked. Lane 1, intact MK; lane
2, N1/2; lane 3, C1/2. Each similar experiment was
repeated three times, and representative results are shown.
[View Larger Version of this Image (52K GIF file)]
Fig. 5.
Effects of Gln-containing peptides on the
cross-linking of intact MK, N1/2, and C1/2 by transglutaminase.
Synthetic human MK, N1/2, or C1/2 (final: 3 µM each) was
incubated for 30 min with 1.25 µM tissue transglutaminase
and 30 µg/ml heparin in the absence or presence of 300 µM Gln-containing peptides, and then the formation of
each dimer was detected by Western blotting after SDS-PAGE on 14%
resolving gels. Panel A, intact MK; panel B,
N1/2; panel C, C1/2. Lane 1, no competitor;
lane 2, Ala41-Pro51; lane
3, Thr78-Gly83; lane 4,
Thr84-Gln93; lane 5,
Cys94-Pro103. Each similar experiment was
repeated three times, and representative results are shown.
[View Larger Version of this Image (36K GIF file)]
Fig. 6.
Sequences of three mutant peptides. The
sequences of three mutant peptides derived from
Ala41-Pro51 are shown. Lane 1,
intact Ala41-Pro51, abbreviated as
QQ; lane 2, Gln42 Ala,
abbreviated as AQ; lane 3, Gln44
Ala, abbreviated as QA; lane 4, Gln42
Ala and Gln44
Ala, abbreviated as
AA.
[View Larger Version of this Image (23K GIF file)]
Fig. 7.
Effects of mutant peptides on the
cross-linking of MK by tissue transglutaminase. Synthetic human MK
(final: 3 µM) was incubated with 1.25 µM
tissue transglutaminase and 30 µg/ml heparin in the absence or
presence of 300 µM intact
Ala41-Pro51 peptide or mutant peptides, whose
sequences are presented in Fig. 6. After incubation for the indicated
time, the formation of MK dimer was detected by Western blotting.
Lane 1, no competitor; lane 2, intact
Ala41-Pro51, abbreviated as QQ;
lane 3, Gln42 Ala, abbreviated as
AQ; lane 4, Gln44
Ala,
abbreviated as QA; lane 5, Gln42
Ala and Gln44
Ala, abbreviated as AA. Each
similar experiment was repeated three times, and representative results
are shown.
[View Larger Version of this Image (38K GIF file)]
Fig. 8.
Suppression of MK activity by the
A41-P51 peptide. Confluent BAEC cultures
were incubated for 16 h in serum-free -minimal essential medium
containing 0.1% bovine serum albumin with 100 ng/ml (7.7 nM) synthetic human MK in the presence of increasing amounts of either the intact Ala41-Pro51 peptide
or a mutant peptide, in which both the Gln42 and
Gln44 residues were substituted with Ala. Cellular lysates
were prepared in 0.5% Triton X-100 in 0.1 M Tris-HCl, pH
8.1, and the PA level of each cell lysate was measured using a
chromogenic substrate and expressed as urokinase (UK)
units/mg of protein in the sample. Curves A and
B, MK-treated cells; curves C and D,
unstimulated cells. Curves A and D, mutant
peptide; curves B and C,
Ala41-Pro51 peptide. Each value represents the
average ± S.D. (n = 3). Each similar experiment
was repeated twice, and representative results are shown.
[View Larger Version of this Image (24K GIF file)]
Fig. 9.
PA-enhancing activity of the MK dimer.
Synthetic human MK (final: 3 µM) was incubated at
37 °C for 1 h with 1.25 µM tissue
transglutaminase (in a total volume of 1.25 ml), in Hepes buffer
containing 10 mM Ca2+ and 30 µg/ml heparin,
and the reaction was terminated by the addition of 50 mM
EDTA. Samples were concentrated 12.5-fold using an Ultrafree C3LGC
micro-concentrator (Millipore; molecular weight cut-off, 10,000) and
then applied to a Superdex 75 HR 10/30 gel filtration column. The
column was eluted with Hepes buffer containing 1 M NaCl,
using a FPLC system, at the flow rate of 0.5 ml/min. The absorbance at
280 nm was monitored and shown as a dotted line (panel A).
0.5-ml fractions were collected, concentrated to 24 µl, and then
either examined for PA-enhancing activity in the absence (closed
circles) or presence (open circles) of 24 µg/ml (15.2 µM) Ala41-Pro51 peptide, as
described in Fig. 8 (panel A), or assessed the molecular species of MK by Western blotting (panel B). The PA activity
levels plotted are averages (n = 3). Each similar
experiment was repeated twice, representative results being
shown.
[View Larger Version of this Image (30K GIF file)]
Fig. 10.
Effect of the
Ala41-Pro51 peptide on the non-covalent
association of MK. Synthetic human MK was incubated with tissue
transglutaminase as described in Fig. 9 in the absence (curves
A and B) or presence (curve C) of 475 µg/ml (300 µM) Ala41-Pro51
peptide. Ca2+ was chelated by the addition of EDTA either
before (curve A) or after (curves B and
C) the incubation. Samples were concentrated and then
subjected to the gel filtration chromatography, using a FPLC system.
The absorbance at 280 nm was monitored and expressed. Each similar
experiment was repeated twice, representative results being
shown.
[View Larger Version of this Image (23K GIF file)]
Fig. 11.
PA-enhancing activity of the non-covalently
associated MK dimer. Synthetic human MK was incubated with tissue
transglutaminase as described in Fig. 10 after the addition of EDTA.
Samples were concentrated, fractionated through a Superdex 75 HR 10/30
gel filtration column, and eluted as described in Fig. 9. The
absorbance at 280 nm was monitored and shown as a dashed
line. 0.5-ml fractions were collected, concentrated to 24 µl,
and then examined for PA-enhancing activity. The PA activity levels
plotted are averages (n = 3). Each similar experiment
was repeated twice, representative results being shown.
[View Larger Version of this Image (25K GIF file)]
(TGF-
; Ref. 47), and MK (22), and that these retinoic acid-inducible
factors interact with each other. In retinoic acid-treated BAECs, PA
and transglutaminase are required to promote the activation of latent
TGF-
(11, 47), whereas MK and TGF-
regulate PA activity,
respectively, in the opposite way (11, 22). The present paper describes
an additional relationship between transglutaminase and MK.
-(
-glutamyl) lysine bridge
between a Lys residue in another MK molecule, or putrescine in the
present experiment, neighboring Gln44 becomes inaccessible
for further bridge formation due to stereohindrance by the MK molecule
cross-linked to Gln42. Conversely, when Gln44
is cross-linked to another MK molecule, Gln42 becomes no
longer accessible. Stereohindrance will also happen when Factor XIIIa
is used as the enzyme. As Factor XIIIa is larger than tissue
transglutaminase (~300 kDa versus 80 kDa), the
cross-linking of MK molecules may not be performed by Factor XIIIa,
even though it can stimulate putrescine incorporation into MK
molecules. It is notable that these three acyl-donating Gln residues
are conserved among the MK/PTN family, whereas the other two Gln
residues are not (48). As can be seen in Fig. 4, the dimer and tetramer
of MK C1/2 migrated slower than those of N1/2. This might be due to
that C1/2 is much more highly charged being basic. The
three-dimensional structure of MK C1/2 recently clarified by NMR
spectroscopy2 is consistent with the
results of the present investigation. In C1/2, basic amino acids, which
are expected to form the heparin binding site (Arg81,
Lys86, and Lys87), are clustered on one side.
Site-directed mutagenesis of these amino acids resulted in decreased
heparin-binding and neurite-promoting activities.3 Gln95, which was
shown to be involved in the dimerization, is located on the opposite
side. This distinct localization of the heparin binding site and the
cross-linking site will permit the cross-linking between two MK
molecules after binding to heparin. Thus, heparin may potentiate the
cross-linking with transglutaminase by stabilizing the conformation of
dimer. In addition, heparin increases the amount of products, too, by
preventing the loss of MK molecules from the reaction mixture that
happens due to static adherence of MK molecules to the vessel wall
(43). Because of this reason and because we supposed the physiological
reaction on the cell surface as discussed below, we analyzed the dimer
formation in the presence of heparin. We are currently trying to
determine the amine-donating Lys sites in the MK molecule. It is also
of great importance to clarify the structural requirements for the substrate for transglutaminase, i.e. the role of flanking
sequences in the Ala41-Pro51 peptide, and to
compare it with the result obtained with fibronectin-derived sequence
(49).
, which counteracts
MK activity (22). Since the activation of latent TGF-
is required
for transglutaminase to localize latent TGF-
on the surface, the
inclusion of an anti-transglutaminase antibody in the culture medium
prevents the formation of active TGF-
(11). Hence, the peptide was a
strong tool to prove our hypothesis, although the specificity of the
inhibition by the peptide was critical to obtain a conclusion. As a
receptor interacting site(s) is located in C1/2 (37, 38), there is a
low possibility that the Ala41-Pro51 peptide
competes with the binding of MK to its receptor(s). Actually, the dimer
exerted the PA-enhancing activity in the presence of this peptide. As
Factor XIIIa functions only in putrescine incorporation, it is possible
that Factor XIIIa might be used to cross-link peptide to MK molecule,
serving as a control for this issue. It is true that when the
PA-enhancing effect of the MK monomer is blocked by
Ala41-Pro51, cross-linking of the MK monomer,
especially formation of the dimer, is suppressed completely. However,
it is possible that other transglutaminase-mediated cross-linking
reactions were affected by the Ala41-Pro51
peptide, and that this caused the inhibition of MK activity by the
peptide. In this context, we need to examine the specificity of the
inhibition by this peptide. Although the peptide inhibited the
cross-linking of intact MK, N1/2, and C1/2 by 80% at a concentration of as much as a 50-fold molar excess, a 200-fold molar excess of this
peptide caused only weak (less than 5%) inhibition of the
cross-linking of pro-SPAI (data not shown; Ref. 10). We are now
investigating whether the peptide affects the cross-linking of other
hitherto known substrates for transglutaminase. Nevertheless, we
believe that the present conclusion is correct, because in the
experiment in Fig. 9, whether an inhibitory effect of the peptide was
observed or not depended only upon whether activity was derived from
the monomer or dimer, and all other conditions were the same.
Therefore, no matter what other cross-linking this peptide may
interfere with, or if MK is cross-linked to other proteins such as
matrix components and if the peptide blocks such cross-linking, we
think that the results in Fig. 9 strongly suggest the relevance of the
formation of the MK dimer.
(11), interleukin 2 (12) and, now, MK.
*
This work was supported in part by a grant for the
"Biodesign Research Program" from RIKEN, by Grants-in-aid 08780689 (to S. K.) and 08457035 (to T. M.) from the Japanese Ministry of
Education, Science and Culture, and by grants from the Ito Foundation
(to S. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom all correspondence should be addressed. Tel.:
81-298-36-3522; Fax: 81-298-36-9050; E-mail:
kojima{at}rtc.riken.go.jp.
1
The abbreviations used are: MK, midkine; PTN,
pleiotrophin; PA, plasminogen activator; BAECs, bovine aortic
endothelial cells; N1/2, N-terminal half; C1/2, C-terminal half;
TGF-, transforming growth factor-
.
2
F. Inagaki, personal communication.
3
N. Asai, unpublished result.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.