(Received for publication, May 8, 1995)
From the
Tissue and plasma transglutaminases cross-link human plasminogen
into high molecular weight complexes (Bendixen, E., Borth, W., and
Harpel, P. C.(1993) J. Biol. Chem. 268, 21962-21967). A
major cross-linking site in plasminogen involved in the tissue
transglutaminase-mediated polymerization process has been identified.
The Plasminogen is the circulating 90-kDa precursor of the serine
protease plasmin. Although best known as the primary fibrinolytic
enzyme in plasma, plasmin also degrades a broad spectrum of other
plasma proteins as well as proteins in the extracellular matrix of
vascular tissues (for a review, see Mayer(1990)). Plasmin also
activates procollagenases (Reich et al., 1988) and converts
latent transforming growth factor- Plasminogen consists of an NH We have recently found that plasminogen is a
substrate for transglutaminases from plasma as well as tissues. These
enzymes cross-link plasminogen into high molecular weight homopolymers
and also cross-link plasminogen to fibronectin, a major constituent of
the extracellular matrix of vascular tissues (Bendixen et al.,
1993). Transglutaminases are Ca We report here the identification of the major
cross-linking site in plasminogen involved in its
transglutaminase-catalyzed polymerization. The site has been identified
by comparison of HPLC
Separations of tryptic peptides derived from either cross-linked or
monomeric plasminogen were performed by reverse-phase HPLC on a
Hewlett-Packard 1084 B instrument. Columns (4 Edman degradation of selected peptides was performed
using an Applied Biosystems 477A sequencer with an on-line 120A HPLC
apparatus. The Normal-1 reaction and conversion cycles were used, and
initial yields of PTH-derivatives were 100-1000 pmol.
Plasminogen incubated as
described above was freed from reductant and treated with 0.5
mM 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole to
introduce a fluorescent label on the free sulfhydryl groups
(Toyo'oka and Imai, 1984). The remaining disulfide bridges were
then reduced with 10 mM DTT and alkylated with iodoacetamide
in 6 M guanidine HCl, 0.1 M Tris-HCl at pH 8.5. After tryptic digestion, the peptides were separated by
reverse-phase HPLC as described above. The column effluents were
monitored by recording the absorbance at 220 nm and by recording the
fluorescence (excitation at 374 nm and emission at 500 nm
(Toyo'oka and Imai, 1984)) using a Hitachi F-1000 fluorescence
spectrophotometer equipped with a 12-µl flow cell. Major
fluorescent pools identified from the fluorescence profile were
subjected to sequence analysis. The fluorescent Cys residues were
identified by HPLC as described by Chin and Wold(1993).
From previous studies, we know that plasminogen is readily
cross-linked into high molecular weight complexes by several
transglutaminases (Bendixen et al., 1993). The
When sequencing a
peptide containing an To minimize the differences
in the peptide profiles of cross-linked and native plasminogens, a
batch of 100 mg of plasminogen was divided in two equal portions. One
batch was incubated with guinea pig liver transglutaminase in the
presence of Ca
Figure 1:
Transglutaminase-catalyzed
cross-linking of plasminogen. Plasminogen was incubated with or without
guinea pig liver tissue transglutaminase as described under
``Materials and Methods.'' The reaction was stopped after 1 h
by the addition of EDTA, and aliquots were removed, mixed with reducing
SDS-PAGE sample buffer, and analyzed on a 10-20% gradient gel.
The equivalent of 2 µg of plasminogen was loaded in each lane. The
starting material is seen in lane1. Lane2 shows plasminogen incubated with only
Ca
DTT was used in the cross-linking
reaction to preserve the activity of guinea pig liver transglutaminase.
Plasminogen incubated with 0.5 mM DTT as described above but
in the absence of transglutaminase was found to contain 4.9 mol of S-carboxymethylcysteine/mol of protein ( To investigate whether this level of S-carboxymethylcysteine represented nearly full reduction of a
limited set of disulfide bridges or whether a large number of bridges
had randomly been reduced to a low extent, a sample of plasminogen was
incubated with 0.5 mM DTT. Free sulfhydryl groups were tagged
with a fluorescent label by reaction with
4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (Toyo'oka and
Imai, 1984), and after complete reduction and carboxamidomethylation,
tryptic peptides were separated by the procedures used for the main
digests (see below). It was found that the extent of reduction
described above resulted from a low level of reduction of randomly
distributed disulfide bridges in plasminogen (data not shown). To
estimate the amount of cross-links, aliquots of both cross-linked and
monomeric plasminogens were exhaustively digested with a mixture of
endo- and exopeptidases (Griffin et al., 1982). It was found
by amino acid analysis that 0.6-0.8 mol of
Both monomeric and
cross-linked plasminogens were digested with trypsin. To ensure
complete digestion, the two batches were first reduced with DTT and
alkylated with iodoacetic acid in the presence of guanidinium chloride.
To investigate whether the cross-links in transglutaminase-treated
plasminogen could be identified by comparison of the tryptic peptide
profiles, identical aliquots of the digests were separated on Nucleosil
C-18 and Vydac C-18 columns and on Mono S and polysulfoethyl-A columns
using a variety of conditions for gradient elution (data not shown).
With the exception of a few peaks, the elution profiles of the peptides
obtained from plasminogen and cross-linked plasminogen were similar.
Representative peptide profiles obtained on Nucleosil C-18 are shown in Fig. 2(A (plasminogen) and B (cross-linked
plasminogen)). The heights of peaks P-1, P-2, and P-3 in Fig. 2A were markedly reduced in the peptide profile
obtained from cross-linked plasminogen. In Fig. 2B, the
corresponding peaks are X-1, X-2, and X-3. Furthermore, a single peak
(X-33) seen in the profile obtained from cross-linked plasminogen had
no counterpart in the profile obtained from monomeric plasminogen.
Therefore, this peak was expected to represent two peptides
cross-linked by an
Figure 2:
Separation of tryptic peptides derived
from reduced and carboxylated plasminogen. The peptides resulting from
tryptic digests of plasminogen (A) or plasminogen cross-linked
by tissue transglutaminase (B) were separated on Nucleosil
C-18. The column was eluted with a linear gradient of 0.1%
trifluoroacetic acid (solvent A) and of 90% acetonitrile, 0.075%
trifluoroacetic acid (solvent B). The labeled peaks were selected for
sequencing. Peak X-33, containing two peptides cross-linked by an
Figure 3:
Purification of the material in peak X-33.
Peak X-33, harboring an
Sequence analysis of the material in peaks P-1,
P-2, and P-3 showed that each peak contained a main sequence,
summarized in Table 1. A background of several minor peptides
that could not be clearly identified was present in these peaks as
well. As determined from the initial yields of PTH-derivatives, the
levels of the minor peptides in peaks P-1, P-2, and P-3 were
As shown in Fig. 1, virtually
all plasminogen had been cross-linked because only trace amounts of
monomeric plasminogen were present. If peak X-33 represents the major
site of cross-linking, peaks X-1, X-2, and X-3 would be expected to
disappear nearly completely. This was evidently not the case (Fig. 2B), but upon sequence analysis of peaks X-1,
X-2, and X-3, a set of NH Apart from the
differences in the peptide profiles described above, it can be seen
that the heights of several peaks with identical retention times in the
separations shown in Fig. 2(A and B) are not
the same. This could indicate different yields of certain
noncross-linked peptides in the two digests or the presence of
additional cross-linked peptides. To investigate this, we analyzed the
peptide profiles of both monomeric and cross-linked plasminogens by
dividing the eluates into 13 pools, as indicated in Fig. 2(A and B). Each of these pools was
subjected to reverse-phase HPLC on a Vydac C-18 column using shallow
gradients of acetonitrile. Differences between the profiles obtained
from plasminogen and cross-linked plasminogen were observed only in the
material from pools 2, 3, and 5 involving peaks P-1, P-2, P-3, X-1,
X-2, X-3, and X-33 discussed above. Furthermore, sequence analysis of
the relevant pools from Fig. 2B did not result in the
appearance of bis-PTH- Transglutaminase-catalyzed reactions result in the formation
of isopeptide bonds between certain Gln residues (acyl donor sites) and
Lys residues (acyl acceptor sites) in proteins. Potential acyl donor
sites in proteins have generally been identified by
transglutaminase-catalyzed incorporation of radioactive or fluorescent
amines followed by characterization of the relevant peptide (see
McDonagh et al.(1981), Aeschliemann et al. (1992),
and Gorman and Folk(1984)). It has recently been possible to
characterize potential acyl acceptor sites by incorporating
specifically designed Gln-containing fluorescent peptides into proteins
(Lorand et al., 1991). However, the identification of
Plasminogen is a substrate for transglutaminases (Bendixen et
al., 1993), and incubation of native plasminogen with guinea pig
liver tissue transglutaminase results in the formation of a polymer of
high molecular weight (Fig. 1). The content of
We
investigated whether the cross-linking site(s) could be identified by
comparison of the peptide profiles of tryptic digests of cross-linked
and monomeric plasminogens. Trypsin appeared to degrade cross-linked
and monomeric plasminogens equally well, suggesting that cross-linked
plasminogen retains a rather open structure, and the majority of the
peaks of the two profiles were easily paired (Fig. 2, A and B). Peptides were selected that were unique for
cross-linked plasminogen and that appeared in reduced quantities in the
profile of cross-linked plasminogen. As such, we found only one tryptic
peptide that was unique for cross-linked plasminogen, namely peak X-33 (Fig. 2B). From sequence analysis of the pure
cross-linked peptide ( Fig. 3and Table 1), in which the
cross-link was identified as bis-PTH- It was found
earlier (Bendixen et al., 1993) that fluorescent substrates
reacting with donor and acceptor sites become incorporated into
plasminogen. The initial incorporation took place in monomeric
plasminogen, but upon prolonged incubation, the substrates were also
found in polymerized plasminogen. We believe that these substrates
react with the donor and acceptor sites identified here and may become
released from plasminogen in the polymerization process. However, there
might be other reactive sites because incubation with dansylcadaverine
did not block the polymerization process and the polymeric product did
contain some fluorescent label (Bendixen et al., 1993). The
conformation of Glu-plasminogen has been described as a compact
ellipsoid, coiled up in a spiral in which the NH No high
resolution structure is available for kringle 3, but to illustrate the
putative location of Lys-298 and Gln-322, Fig. 4shows a model
of the homologous kringle 4 of plasminogen (Mulichak et al.,
1991) in which Lys-298 and Gln-322 have been modeled. Clearly, both the
donor and acceptor positions are located at the surface of the kringle,
spaced apart by
Figure 4:
Model of kringle 4 of plasminogen. The
positions of the two transglutaminase substrate sites Lys-298 and
Gln-322 found in kringle 3 have been substituted for Tyr-359 and
Ser-424 in kringle 4 to visualize the substrate sites in the kringle 3
domain. The three kringle disulfide bridges are indicated (local
numbering of the 80-residue kringle
structure).
Kringles 1, 4, and 5 mediate
binding of human plasminogen to fibrin and cellular surfaces (Thorsen et al., 1981; Lukas et al., 1983; Matsuka et
al., 1990; Wu et al., 1990). The present finding that the
cross-linking of plasminogen involves residues in kringle 3 is the
first indication of a specific function for this domain. Lys-298 is
conserved in plasminogen from all species sequenced so far (human
(Sottrup-Jensen et al., 1978; Forsgren et al., 1987),
rhesus monkey (Tomlinson et al., 1989), mouse (Degen et
al., 1990), cattle (Schaller et al., 1985), and pig
(Schaller et al., 1987)). Gln-322 is conserved in human,
rhesus monkey, and mouse plasminogen, being replaced by a Glu residue
in plasminogen from cattle and pig. Therefore, plasminogen from rhesus
monkey and mouse is a potential substrate for transglutaminases, but if
plasminogen from cattle and pig is a substrate for transglutaminases,
the acyl donor residue must be different from that of human
plasminogen. Polymeric plasminogen cross-linked by transglutaminases
maintains the characteristic behavior of 90-kDa plasminogen in the
sense that it binds to endothelial cells and is readily activable by
physiological plasminogen activators, and when activated, it shows full
plasmin activity (Bendixen et al., 1993). We suggest that
cross-linked plasminogen is physiologically important on cell surfaces
and in the extracellular matrix. Besides being cross-linked to itself,
plasminogen might become cross-linked to cell-surface and extracellular
matrix proteins because the plasminogen polymers possess one free donor
and acceptor site. The basement membrane proteins degraded by
plasmin are also substrates for transglutaminases, and it is possible
that plasminogen is cross-linked to some of these substrate proteins
and thus is able to locally become activated when proteolysis of the
extracellular matrix is required. Transglutaminase-mediated
cross-linking of plasminogen may be a novel mechanism by which plasmin
can interact with its specific target sites. These could include
fibrin, fibrinogen, the extracellular matrix, cell-surface proteins, or
circulating plasma proteins.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-(
-glutamyl)lysyl bridges of the polymer are formed
between Lys-298 and Gln-322. Both the acyl donor Gln residue and the
acyl acceptor Lys residue are located in the kringle 3 domain of
plasminogen, i.e. cross-linking of plasminogen by tissue
transglutaminase involves neither the catalytic domain nor the
lysine-dependent binding sites of plasminogen. This study documents
that kringle 3 contains a novel functional site with the potential to
participate in transglutaminase-mediated cross-linking interactions
with plasma, cell-surface, and extracellular proteins.
to its active form (Kojima et al., 1991) and is thereby actively involved in controlling
cell migration and tissue remodeling (Ossowski and Reich, 1983).
-terminal 77-residue
domain, five internally homologous kringle domains, and a
COOH-terminally located serine protease domain (Sottrup-Jensen et
al., 1978). Some of the kringle domains are able to bind
COOH-terminal Lys residues found in several plasma and cell-surface
proteins (Harpel et al., 1985). This is likely to be the basis
for the specific interaction of plasminogen with fibrin (Thorsen et
al., 1981; Suenson et al., 1984; Harpel et al.,
1985), fibronectin (Salonen et al., 1985), thrombospondin
(Silverstein et al., 1984), and the extracellular matrix of
vascular tissues (Knudsen et al., 1986) as well as a number of
vascular cell surfaces (Miles et al., 1988; Hajjar et
al., 1986). It has become a basic concept that the interaction of
plasminogen with protein matrices and cell surfaces plays a key role in
the regulation of plasminogen activation, so plasmin activity is
restricted to sites where its proteolytic activity is required
(Nachman, 1992).
-dependent enzymes
that catalyze the cross-linking of certain Gln and Lys side chains in
proteins, resulting in the formation of
-(
-glutamyl)lysyl
bridges. Transglutaminases are ubiquitously found in tissues and body
fluids (for a review, see Lorand and Conrad (1984)). The best
characterized transglutaminase is plasma transglutaminase Factor XIIIa,
formed from its precursor, Factor XIII, by activation with thrombin. It
cross-links polymerized fibrin into a tight matrix during the final
steps of blood coagulation (Lorand et al., 1980) and also
cross-links
-antiplasmin to the fibrin clot (Sakata
and Aoki, 1980). The physiological roles of tissue transglutaminases
are not understood. However, since several of their substrates are
constituents of the extracellular matrix and basement membrane of
vascular tissues, transglutaminases are believed to play a major role
in the formation and stabilization of these matrices (Martinez et
al., 1989; Kinsella et al., 1990; Sane et al.,
1991; Greenberg et al., 1991; Aeschliemann et al.,
1992). It has previously been suggested that plasminogen cross-linked
into high molecular weight homopolymers may also be cross-linked to the
extracellular matrix as well and that this cross-linking of plasminogen
may represent a novel mechanism by which plasmin activity is targeted
to the extracellular matrix of vascular tissues (Bendixen et
al., 1993).
(
)profiles of tryptic
peptides of monomeric and cross-linked plasminogens, and from sequence
analysis, it is found to be constituted by Lys-298 and Gln-322 located
in the kringle 3 domain.
Proteins and Reagents
Human plasminogen (90 kDa)
was purified as described earlier (Sottrup-Jensen et al.,
1978). Guinea pig liver transglutaminase (75 kDa) was from Sigma.
1-Chloro-3-(4-tosylamido)-4-phenyl-2-butanone-treated trypsin was from
Worthington. Standard chemicals were analytical grade and were obtained
from Merck, Fluka, and Sigma. -(
-Glutamyl)lysine was from
Sigma. Packing materials for reverse-phase and ion-exchange HPLC were
from Machery Nagel, Phase Separations, and PolyLC, Inc. A Mono S HR 5/5
column was from Pharmacia Biotech Inc. Reagents and solvents for
sequence analysis were from Applied Biosystems, Inc.
Transglutaminase-catalyzed Cross-linking of
Plasminogen
550 nmol (50 mg) of human plasminogen was incubated
with 2.75 nmol (0.208 mg) of guinea pig liver tissue transglutaminase
in the presence of 10 mM Ca, 0.5 mM
DTT, and 50 mM Tris-buffered saline at pH 7.5. The total
volume was 50 ml. The reaction was started by the addition of
transglutaminase, proceeded for 1 h at 20 °C with end-over-end
mixing, and was stopped by the addition of 20 mM EDTA.
SDS-PAGE Analysis
For SDS-PAGE, the Tris/glycine
system of Laemmli(1970) was used with 0.5 80
100-mm
10-20% gradient gels.
Determination of
-(
-Glutamyl)lysine
200-µg aliquots of
cross-linked plasminogen or untreated plasminogen were digested with a
mixture of endo- and exopeptidases using the procedure of Griffin et al.(1982). Because
-(
-glutamyl)lysine elutes
close to methionine in the amino acid analysis system used
(Sottrup-Jensen, 1993), samples were oxidized with performic acid prior
to analysis. The concentrations of the plasminogen solutions were
determined by amino acid analysis after hydrolysis with 6 M HCl for 18 h at 110 °C.
Protein Digestion, Peptide Separation, and Sequence
Analysis
Plasminogen cross-linked as described above or native
plasminogen was reduced by adding 5 mM DTT in the presence of
6 M guanidinium chloride at pH 9.0. Reduction proceeded at 20
°C for 30 min, where after all free sulfhydryl groups were blocked
by reaction with 20 mM iodoacetic acid for 30 min at pH 8.0.
The reduced and alkylated samples were desalted by repeated dialysis
against 50 mM NHHCO
, and digestion of
plasminogen (37 °C, 12 h) was performed by adding trypsin (1:100,
w/w) in 50 mM NH
HCO
(total volume of
150 ml). The peptide material was freeze-dried, redissolved in 25 ml of
0.1% trifluoroacetic acid, and stored as aliquots at -20 °C.
250 mm) were
packed with 5-µm Nucleosil C-18 or 10-µm Vydac C-18 material
and eluted with gradients of acetonitrile in 0.1% trifluoroacetic acid
at 50 °C at a flow rate of 1 ml/min. The elution profiles were
monitored at 220 nm. For cation-exchange chromatography, an instrument
assembled from Pharmacia Biotech HPLC modules was used. Separations
were performed on a 4
120-mm polysulfoethyl-A column and on a
Mono S column using a gradient of NaCl in 25% (v/v) acetonitrile, 5
mM phosphoric acid (Alpert, 1988; Crimmins et al.,
1989). Peptides were collected manually and dried in a Speed-Vac
centrifuge.
Characterization of the Status of the Disulfide Bridges
in Plasminogen Exposed to DTT
A sample of plasminogen incubated
without transglutaminase under the conditions used for cross-linking
was desalted on Sephadex G-25 and immediately treated with 20 mM iodoacetamide at pH 8.0 for 1 h to block free sulfhydryl groups.
The content of S-carboxymethylcysteine was determined after
acid hydrolysis (Sottrup-Jensen, 1993).Visualization of Three-dimensional Structures of
Proteins
RasMol version 2.4 (Roger Sayle, Glaxo Research and
Development) was run under Microsoft Windows and used to display the
three-dimensional structure of human kringle 4 (Mulichak et
al., 1991). The program was obtained by anonymous FTP
(ftp.dcs.ed.ac.uk), and the coordinate file was from Brookhaven Protein
Data Bank.
-(
-glutamyl)lysyl cross-links are stable in the currently
used methods for reduction, digestion, and peptide separation.
Therefore, to locate the sites of cross-linking, tryptic HPLC profiles
of plasminogen cross-linked with guinea pig liver tissue
transglutaminase were compared with those of untreated plasminogen. The
novel peptides found in the digest of cross-linked plasminogen must
contain the sites of cross-linking. Consequently, the yields of
peptides that harbor the reactive Gln and Lys residues must be reduced
when compared with those of untreated plasminogen.
-(
-glutamyl)lysyl bridge, two
NH
-terminal sequences of equimolar ratio are expected.
Because the isopeptide bond is not cleaved during Edman degradation,
the cross-linked pair of residues is released as
bis-PTH-
-(
-glutamyl)lysine, which is easily distinguished
from the normal PTH-derivatives in the standard HPLC separation
(Sottrup-Jensen et al., 1990).
, while the other was incubated with
Ca
only. The molar ratio of transglutaminase to
plasminogen was 1:200, so peptides of transglutaminase origin would not
be expected to interfere with the interpretation of the peptide
profiles of tryptic plasminogen digests. Fig. 1shows a reducing
SDS-PAGE analysis of plasminogen incubated with or without
transglutaminase. Plasminogen incubated with transglutaminase and
Ca
was essentially completely cross-linked after 1 h
of incubation and was retained at the top of the stacking gel (lane3), while untreated plasminogen was still in the 90-kDa
form (lane2), identical to the starting material (lane1).
, while lane3 shows plasminogen
cross-linked by tissue transglutaminase. HMWplg.,
high molecular weight plasminogen.
10% of the
content of halfcystine).
-(
-glutamyl)lysine was present per mol of cross-linked
plasminogen, while untreated plasminogen, as expected, did not contain
the isopeptide. The estimation of the amount of cross-link in
plasminogen is not accurate because the small peak corresponding to
-(
-glutamyl)lysine is located in the valley between the huge
peaks corresponding to valine and isoleucine (Sottrup-Jensen, 1993),
but the data suggest that cross-linking results in the introduction of
only 1 mol of isopeptide bond/mol of plasminogen.
-(
-glutamyl)lysyl isopeptide bond. Peak
X-33 was further purified by cation-exchange chromatography (Fig. 3).
-(
-glutamyl)lysyl bond, was further purified by
cation-exchange chromatography (see Fig. 3).
-(
-glutamyl)lysyl bond, was separated
by cation-exchange chromatography. A polysulfoethyl-A column was eluted
with a linear gradient of 5 mM H
PO
,
25% (v/v) acetonitrile (solvent A) and of 5 mM H
PO
, 25% (v/v) acetonitrile, 1 M NaCl (solvent B) at a flow rate of 1
ml/min.
20%
of that of each major sequence. As shown in Table 1, peak X-33
was found to consist of the stretches Thr-291-Arg-306 and
Arg-312-Arg-324, cross-linked by an isopeptide bond involving
Lys-298 and Gln-322. As expected, upon sequence analysis, the yields of
the cross-linked mates were similar, and the cross-linking prevented
tryptic cleavage at Lys-298. In the peptide profile from plasminogen
peptides, residues 291-306 were recovered as the major peptides
in peaks P-2 and P-1, while residues 312-324 were recovered as
the major peptide in peak P-3.
termini similar to those
representing the minor components in peaks P-1, P-2, and P-3 were
revealed. In peaks X-1, X-2, and X-3, the major sequences of peaks P-1,
P-2, and P-3 were recognized, respectively, but their levels were only
20% of the average level of peptides present. Hence, we estimate
that the pair Lys-298 and Gln-322 has been utilized in the
cross-linking process to an extent of
95%.
-(
-glutamyl)lysine. Hence, the
cross-link between Lys-298 and Gln-322 identified above is likely to be
the only major cross-link in transglutaminase-polymerized plasminogen.
-(
-glutamyl)lysyl cross-links in proteins is not
straightforward, as documented by the variety of procedures employed
(Chen and Doolittle, 1970; Cottrell et al., 1979; Kimura and
Aoki, 1986; Purves et al., 1987; Pucci et al., 1988;
Sottrup-Jensen et al., 1990; Hohl et al., 1991).
-(
-glutamyl)lysine was estimated at 0.6-0.8 mol/mol of
plasminogen, which, in view of the near absence of monomeric
plasminogen after treatment with transglutaminase, is likely to reflect
the introduction of only 1 mol of cross-link/mol of plasminogen.
-(
-glutamyl)lysine, we
conclude that Lys-298 and Gln-322 are cross-linked by an
-(
-glutamyl)lysyl isopeptide bond. From the digest of
monomeric plasminogen, we have characterized the corresponding
noncross-linked peptides and found that these peptides are present in
the digest of cross-linked plasminogen in very low amounts. Because the
peptide separations have been performed under a variety of conditions
and because only one cross-linked peptide was identified in
cross-linked plasminogen, we conclude that Lys-298 and Gln-322, both
located in the kringle 3 domain, constitute the acyl acceptor and acyl
donor sites in the cross-linking process, respectively, and that these
residues have been utilized to an extent of
95%.
-terminal
portion is in close proximity to the serine protease portion (Banyai
and Patthy, 1984; Ponting et al., 1992; Mangel et
al., 1990). According to this model, the kringle 3 domain
protrudes from the compact plasminogen molecule. Our results
substantiate this proposal by showing that part of the surface of
kringle 3 is exposed for transglutaminase-mediated polymerization.
Furthermore, the lack of oligomers (Fig. 1) (Bendixen et
al., 1993) in the cross-linking of plasminogen points to highly
specific protein-protein interactions in the system.
13 Å.
We thank Lene Kristensen for excellent technical
assistance and Jesper Haaning for help with display of protein
structures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.