(Received for publication, May 4, 1995; and in revised form, July 11, 1995)
From the
The extracellular matrix protein BM-40 (osteonectin, SPARC) has
recently been shown to be a major target for transglutaminase-catalyzed
cross-linking in differentiating cartilage. In the present study we
demonstrate that recombinant human BM-40 can be modified with
[H]putrescine in a 1:1 molar ratio by
transglutaminase
(tissue transglutaminase). Residues
Gln
and Gln
were identified as major amine
acceptor sites. This was confirmed with several mutant proteins,
including deletions in the N-terminal domain I of BM-40, site-directed
mutagenesis of the reactive glutamines, and fusion of the seven-amino
acid-long N-terminal sequence (APQQEAL) to an unrelated protein. The
results showed that the N-terminal target site is sufficient for
modification by transglutaminase but at a low level. For high
efficiency amine incorporation an intact domain I is required. The
conservation of at least one of the transglutaminase target glutamines
in the known vertebrate BM-40 sequences and their absence in an
invertebrate homologue point to an important, but yet unknown, role of
this modification in vertebrates.
Transglutaminases (EC 2.3.2.13) form a large protein family for
which at least six different gene products have been found in higher
vertebrates(1) . They catalyze a Ca-dependent
transfer reaction between the
-carboxamide group of a
peptide-bound glutamine residue and various primary
amines(2, 3, 4) . Most commonly,
-glutamyl-
-lysine cross-links are formed in or between
proteins by reaction with the
-amino group of lysine residues. The
function of transglutaminase
(tissue transglutaminase,
transglutaminase type II), which is of widespread occurrence in
vertebrates(1, 4, 5, 6) , is not
well understood. Its expression often correlates with cellular
differentiation(1, 4, 7) , and the enzyme may
have functions as different as GTP binding in receptor
signaling(8) , intracellular cross-linking in programmed cell
death(9) , and extracellular cross-linking in the assembly of
matrices(1) . It is also involved in wound healing and tissue
repair(10) . In general the function of transglutaminase
could be to stabilize protein molecules and protein complexes,
thus contributing to the stability of tissues. In skeletal tissues,
synthesis of transglutaminase
is strictly regulated and
correlates with chondrocyte differentiation and cartilage
mineralization(7, 11) . The enzyme is released by
hypertrophic chondrocytes (7) and activated by the elevated
Ca
concentration in the extracellular space (11) .
BM-40 (osteonectin, SPARC), which is co-expressed
with transglutaminase in maturing cartilage(7) , is
a major substrate protein for transglutaminase-catalyzed cross-linking
in this tissue(11) . BM-40 is widespread in extracellular
matrices(12, 13, 14) . It was initially
identified as a major component of bone(15, 16) , but
is also found in basement membranes (17) and is expressed in
platelets (18) and endothelial cells(19) . BM-40 has
been shown to bind collagen IV in basement membranes (20) and
thrombospondin when released from platelets(21) . The protein
has also been implicated in the mineralization of cartilage and bone
because of its affinity for hydroxyapatite (22) due to several
calcium-binding sites(23, 24) . This 35-kDa
extracellular matrix glycoprotein has been predicted to consist of four
domains, starting with the acidic N-terminal domain I, followed by a
cysteine-rich domain II, an
-helical domain III, and the
C-terminal EF hand domain IV ((23) ; see Fig. 3B). Several deletion and point mutants have
recently been used to localize the collagen IV and high affinity
calcium binding sites of BM-40 to domains III and IV(25) .
Tissue-specific post-translational modifications, such as glycosylation (26) or transglutaminase cross-linking(11) , may also
serve to regulate some of the diverse biological functions proposed for
BM-40(13, 14) .
Figure 3:
Design of mutant proteins and location of
transglutaminase amine acceptor sites. A, scheme representing
the mutations described in the text. Gray boxes indicate
domain I of BM-40 with the limits of the proposed -helical regions
(see B) marked by arrowheads. Numbers indicate sequence positions according to (30) . In mutant
QQ3,4-LA glutamines Gln
and Gln
were replaced
by Leu and Ala, respectively. Mutant
I has a precise deletion of
domain I (residues 1-52) but contains the additional N-terminal
residues APLA due to the ligation to the signal peptide of human BM-40
using an NheI restriction site(29) . Mutant
N
lacks residues 8-67 and contains an exchange of Asp
for Glu.
1III3-5 contains the N-terminal seven amino
acids of BM-40 fused to a domain of the
1 laminin chain containing
three EGF-like repeats. B, model of BM-40 according to (23) . Cylinders represent predicted
-helical domains, black dots indicate cysteines, and numbers indicate
sequence positions(30) . The amine acceptor sites of native
recombinant BM-40, Gln
and Gln
, and the
alternative site in mutant QQ3,4-LA, Gln
, are marked with
an asterisk.
In the present study we have
identified Gln and Gln
within domain I as the
amine acceptor sites in BM-40 for
transglutaminase
-catalyzed cross-linking with the primary
amine putrescine. The interaction of transglutaminase with BM-40 was
analyzed by introducing mutations into domain I. The results indicated
that the N-terminal sequence APQQEAL alone is sufficient for
modification by transglutaminase but that the complete domain I of the
protein is required for efficient modification.
The transfection of human embryonic kidney cell clones 293 and the selection of stable cell clones by puromycin followed previous protocols(28) . The recombinant proteins were purified from serum-free culture medium on DEAE-cellulose and Superose 12 (HR 16/50, Pharmacia LKB Biotechnology Inc., Uppsala, Sweden) columns(25) . The purity of the proteins was analyzed by SDS-polyacrylamide gel electrophoresis and by Edman degradation which verified the novel N-terminal sequence APLAEA for mutant QQ3,4-LA.
The recombinant mutant proteins 1III3-5 and BM-40
QQ3,4-LA (150 µg; 8.3 and 5.0 nmol, respectively) were labeled with
[2,3-
H]putrescine in the
transglutaminase-catalyzed reaction at an enzyme/substrate molar ratio
of 1:10 and the labeled monomeric proteins purified by molecular sieve
chromatography on a Superose 6 column as described above. The labeled
proteins were reduced and carboxymethylated under denaturing conditions
and cleaved with endoproteinase Glu-C and trypsin, respectively, in 0.2 M ammonium carbonate at 23 °C and an enzyme/substrate
ratio of 1:100 for 16 h. The resulting peptides were separated by
reversed phase HPLC and the radioactivity was determined as described
above.
Radioactive peptides were sequenced using the Applied Biosystems Sequencer model 470A according to the manufacturer's instructions. The small volume of phenylthiohydantion derivative samples left over after on-line injection was collected in the instrument's fraction collector and used for radioactivity determination as described.
Figure 1:
Saturation of the
amine incorporation site(s) in BM-40. Increasing amounts of unlabeled
putrescine were added to the reaction mixture of recombinant BM-40 and
transglutaminase, whereas the concentration of
[
H]putrescine was left constant. The amount of
protein-bound radioactivity was measured as described(6) . The circles represent the mean of four determinations with bars indicating the deviation from the mean. Semilogarithmic
plots of the data (not shown) demonstrated that saturation was
reached.
For identification of the target
glutamine(s), the labeled monomeric protein was separated from
oligomers and the enzyme by gel filtration under denaturing conditions,
reduced and alkylated, and digested with trypsin. Separation of the
tryptic peptides by reversed phase HPLC and determination of
[H]putrescine content in the fractions showed two
peaks containing together about 90% of the applied label (Fig. 2A). Sequencing of these fractions showed that
they contained a mixture of peptides with the N-terminal sequence of
the protein as the predominant component in both pools. Pool 1 was
further cleaved with endoproteinase Asp-N, and separation of the
cleavage products by reversed phase HPLC revealed only one radioactive
peak containing 84% of the applied label (Fig. 2B).
This peak contained a long peptide starting with the N-terminal
sequence APQQE and a short peptide comprising positions 212-221
(IFPVHWQFGQ). Determination of radioactivity in the residual volume of
injection during sequencing showed
H label in cycles 3 and
4, but not in cycles 7 and 10. This demonstrated that only Gln
and Gln
are substrate sites for
transglutaminase
. However, as the N-terminal peptide was
too long to be sequenced entirely, the second radioactive pool of
tryptic peptides, which apparently contained a similar labeled
fragment, was further cleaved with endoproteinase Glu-C. The
chromatogram of the reversed phase HPLC separations of these peptides
showed one radioactive peak with 75% of the applied radioactivity (Fig. 2C). This peak contained only the peptide
APQQEALPDETEVVEE, and label was again found only in cycles 3 and 4. The
relative recoveries of radioactivity in cycle 3 and 4, corresponding to
Gln
and Gln
, varied in the different sequencer
runs. Although with the long peptide of pool 1 slightly more
radioactivity was found associated with Gln
than
Gln
, the reverse was true for the short peptide derived
from cleavage of pool 2. The radioactivity released during sequencing
in cycle 4, on the other hand, was too high to be explained by
carryover of the phenylthiohydantoin-derivative from cycle 3.
Therefore, [
H]putrescine was attached to either
Gln
or Gln
of human BM-40 probably to a similar
extent. However, because reversed phase HPLC could apparently not
separate peptides labeled at either Gln
or Gln
,
we were always sequencing mixtures of both.
Figure 2:
Radioactivity profiles of reversed phase
separations of peptides derived from cleavage of
[H]putrescine-labeled human recombinant BM-40.
[
H]Putrescine was incorporated into native
recombinant BM-40 by transglutaminase
. The labeled protein
was purified by molecular sieve chromatography, reduced and alkylated,
and digested with trypsin. The separation of the tryptic peptides,
resulting in two pools (1 and 2) of labeled
fragments, is shown in A. The separation of products of
endoproteinase Asp-N cleavage of pool 1 and endoproteinase Glu-C
cleavage of pool 2 are shown in B and C,
respectively.
Figure 4:
Transglutaminase-catalyzed
incorporation of [
H]putrescine into BM-40 and the
different mutant proteins. The incorporation of
[
H]putrescine into human recombinant BM-40 and
the mutants QQ3,4-LA,
I,
N, and
1III3-5 by
transglutaminase
was carried out at 37 °C for 45 min in
the presence of 5 mM Ca
as described
previously(6) . The results are shown as a mean of 6-18
independent measurements, and the bars indicate the standard
deviation from the mean.
In order to determine
the modified glutamines in these mutant proteins we isolated and
digested the [H]putrescine-labeled monomeric
proteins. Mutant
1III3-5 yielded several radioactive peaks
after cleavage with trypsin and separation of the peptides by reversed
phase HPLC. All of these peaks contained peptides of various length
starting at the N-terminal sequence APQQ with a total yield of
55%
of the radioactivity applied to the column. Radioactivity was released
only in sequencing cycles 3 and 4, indicating that
[
H]putrescine was cross-linked to the same
glutamine residues as in genuine BM-40. The
[
H]putrescine-labeled mutant QQ3,4-LA was cleaved
with endoproteinase Glu-C. When the resulting HPLC peaks were screened
for radioactivity, only a single peak was found to be radioactive with
a yield of about 40%. This peak included only one Gln-containing
peptide, VSVGANPVQVE (residues 24-34), in addition to smaller
amounts of the peptides RDEDNNLLTE, and DNNLLTE. Only cycle 9 released
radioactivity, indicating that Gln
was the residue labeled
with [
H]putrescine.
Human recombinant BM-40 was used to determine the amine
incorporation site for transglutaminase-catalyzed
modification. Proteolytic digestion of
[
H]putrescine-labeled BM-40 yielded labeled
peptides of variable length that were derived from the N terminus and
had the label attached exclusively to Gln
and
Gln
. The transglutaminase did not show a clear preference
for either of these glutamine residues but modified only one of them in
each BM-40 molecule as indicated by the 1:1 ratio of putrescine
incorporation under saturating conditions. Two adjacent glutamine
residues were identified as amine acceptor sites in several other
proteins, including fibronectin (34) and fibrinogen
-chain (35) using factor XIIIa, plasminogen activator
inhibitor-2(36) , and
A3-crystallin (37) using
the guinea pig liver enzyme (transglutaminase
), and
involucrin (38) using the keratinocyte enzyme
(transglutaminase
). Transglutaminases often show a
preference for one of the two residues, although exclusive modification
of one of the adjacent residues has not been observed with proteins
(discussed in (33) ). However, in a study using short peptides
patterned on the N-terminal sequence of fibronectin, EAQQIV, only the
first Q has been shown to be an amine acceptor in the factor XIIIa or
transglutaminase
-catalyzed modification with
monodansylcadaverine(39) . Both, Gln
and Gln
are conserved in human, mouse, bovine, and frog BM-40, whereas in
rat and chicken only one of the two Gln residues is retained (Table 1). The BM-40 homologue from the nematode Caenorhabditis elegans lacks these glutamine residues as well
as showing no significant overall sequence similarity in the N-terminal
domain I(40) . The conservation in higher vertebrates of the
unique N terminus of BM-40, which contains the transglutaminase
cross-linking site and binding sites for
Ca
(24) , suggests a specific function of this
domain in these species. This could be related to the need for
cross-linking of BM-40 in calcifying cartilaginous and osseous tissues,
which are absent in almost all invertebrates.
When the N-terminal
sequence of BM-40 was grafted onto an entirely different protein, the
EGF-like repeats 3-5 of domain III of laminin 1-chain, this
sequence was still modified by transglutaminase
at the same
glutamine sites as genuine BM-40, but the amount of label incorporated
was distinctly reduced and the substrate specificity factor (k
/K
(app)) was only half of
that with recombinant BM-40 as a substrate. A drastic reduction in
putrescine incorporation was also found with mutant
N, in which
most of domain I of BM-40 was missing and the transglutaminase target
site was directly connected to domain II. This shows that an N-terminal
sequence of BM-40, consisting of seven amino acids, is sufficient to be
recognized and modified. However, because the level of modification is
much lower than with intact BM-40, the remainder of domain I also seems
to play an important role in the interaction between transglutaminase
and BM-40. Possibly other amino acids in this domain also contribute to
enzyme recognition. The results with mutant QQ3,4-LA indicated that
binding of the enzyme to domain I of BM-40 can give rise to the
modification of another accessible Gln (Gln
) when the
major target residues are lacking. However, the level of modification
was significantly lower than for intact BM-40, indicating that the
proper distance between the transglutaminase binding site and the
target residue is crucial for efficient modification or that the proper
conformation of the target sequence depends on the rest of domain I. No
sequence similarity is apparent between the N-terminal target sequence
and the immediate surroundings of Gln
, the substitute
target in mutant QQ3,4-LA. However, the comparison of known
transglutaminase target site sequences (33) does not reveal a
clear sequence motif which may serve as a signal sequence for
modification.