(Received for publication, May 5, 1995; and in revised form, July 11, 1995)
From the
Thirteen recombinant A-crystallin mutants were constructed
that differed in the type of amino acid residue directly preceding the
sole amine donor lysine for transglutaminases in this protein. The
capacity of these mutants to be cross-linked to amine acceptor
substrates by tissue transglutaminase and factor XIII was assessed. Two
different biotinylated glutamine-containing oligopeptides were used as
amine acceptor probes. It appears that the type of residue preceding
the amine donor lysine has a considerable influence on the substrate
potential of
A-crystallin for transglutaminases. This influence
shows qualitatively similar trends for tissue transglutaminase and
factor XIII and is irrespective of the amine acceptor probe. In
general, glycine or aspartic acid before the amine donor lysine has the
strongest adverse effects on substrate reactivity, and proline,
histidine, and tryptophan are less favorable. Valine, arginine, and
phenylalanine, and to a more variable or somewhat lesser extent also
serine, alanine, leucine, tyrosine, and asparagine, have an enhancing
effect. This pattern of preference is largely in agreement with that
observed for the limited number of characterized amine donor lysines in
protein substrates for transglutaminases. It can be concluded that
tissue transglutaminase and factor XIII have a rather broad yet clearly
differentiated tolerance with respect to the residue preceding the
amine donor lysine substrate in native proteins.
Transglutaminases (TGs) ()form a ubiquitous family of
structurally and functionally related enzymes (for recent overviews see (1, 2, 3, 4) ). In man and other
mammals, five enzymatically active TGs have been identified: the
catalytic a subunit of plasma factor XIII, keratinocyte TG
(TGK or TGase1), tissue TG (TGC or TGase2), epidermal TG (TGE or
TGase3), and prostate TG (TGP or TGase4). These enzymes catalyze the
formation of isopeptide cross-links between glutamine and lysine
residues in a variety of proteins and can attach polyamines to
peptide-bound glutamines. Their most obvious function is to stabilize
biological structures. There is increasing evidence, however, that
especially TGC is involved in many additional, more subtle biological
processes, like apoptotic cell death (5) and tumor
progression(6) . Intriguing recent examples of unexpected
functional roles are the involvement of TGs in activation of
phospholipase A
(7) , in the regeneration of optic
nerves(8) , and as a GTP-binding protein in
-adrenergic receptor signaling(9) .
The
transglutaminase cross-linking reaction involves a
Ca-dependent acyl transfer that follows a double
displacement mechanism(10, 11) . In the first step, a
glutamine-containing acyl donor substrate binds to the enzyme.
Nucleophilic attack of the active site thiol of the enzyme on the
-carboxamide group of a glutamine residue in the substrate protein
leads to formation of an acyl-enzyme intermediate, under release of
ammonia. The amine donor substrate then binds, and the acyl group is
transferred to the primary amine, resulting in release of the
-glutamyl amine product. Elucidation of the three-dimensional
structure of factor XIII identified a catalytic Cys-His-Asp triad and
suggests how the glutamine and lysine substrate proteins can approach
each other in the active site cavity(12) . However, direct
information about the substrate binding regions of the TGs is still
lacking.
The number of proteins acting as glutaminyl substrates for TGs is restricted. Both primary structure and conformation of a protein appear to determine whether a glutamine residue can be reactive(10, 13, 14) . A considerable number of reactive glutamine residues have been identified in various substrate proteins for TGs(14, 15) . Accessibility of these glutamines in solvent-exposed surface regions or flexible extensions of the protein is a logical common feature. There is no obvious consensus sequence around these substrate glutamines, although some regularities can be discerned.
A common notion is that TGs are much less selective toward amine donor lysine residues in proteins than they are to the glutamine substrates (1, 3) . It appears indeed, on basis of studies with various primary amines, that TGC and factor XIII have a broad tolerance to structural differences in amine donor substrates(10, 11) . However, the modes of interaction of TGs with small amines and with native protein substrates are likely to be different(10, 16) . The limited number of identified amine donor lysine residues in substrate proteins for TGs(17, 18) precludes as yet an assessment of the substrate requirements in native proteins.
To fill this gap we apply
site-directed mutagenesis to study the primary structure requirements
around amine donor lysines in proteins, using A-crystallin as a
model. This demonstrated already that TGC accepts a variety of residues
flanking the substrate lysine in
A-crystallin, albeit to different
extents(17) . The present work focuses on a more detailed
analysis of the influence of the amino acid side chain present directly
NH
-terminal of the substrate lysine. On the basis of
studies with synthetic peptides it has been proposed that this side
chain might be a major determinant in acyl enzyme-lysine substrate
interaction(10, 19, 20) . We wanted,
moreover, to compare the reactivity of TGC and factor XIII with the
various mutant substrate proteins, because these enzymes have been
reported to have markedly different specificities(10) . Because
first substrates may predicate amine specificity(10) , we also
assessed the influence of using different acyl donor substrates.
Figure 1:
Degenerated linkers used to construct
the mutant A-crystallins. The wild-type (wt) HhaI-BamHI fragment from clone pET8c
A
encodes the three carboxyl-terminal amino acid residues of bovine
A-crystallin (positions 171-173) and contains the
translation termination codon. Substituting this wild-type HhaI-BamHI fragment with the degenerated HhaI-BamHI linkers 1-4 generated
expression clones for the mutant
A-crystallins, introducing an
amine donor lysine at position 173 and a variable residue in front of
it.
To quantitate the reactivity of the proteins toward the
amine acceptor probes, two samples (10 µl) of each incubation were
analyzed in parallel by SDS-polyacrylamide gel electrophoresis. One gel
was used for staining with Coomassie Brilliant Blue (CBB) and the other
for streptavidin blot analysis(22) . These pairs of gels were
prepared in triplicate. All CBB-stained gels and streptavidin blots
were scanned twice with an LKB-Bromma laser densitometer. Measurements
were in the linear range of densities. For each sample the average and
standard deviation for the CBB staining
and the streptavidin reaction were calculated. The streptavidin values
for each mutant were normalized using the corresponding CBB densities
to correct for gel loading variations and differences in protein
concentrations between the samples.
The aim of the present work was to study the influence on TG
reactivity of the type of amino acid side chain present directly
NH-terminal of the substrate lysine in a native protein. To
that end we constructed 13 mutant clones of bovine
A-crystallin,
encoding for proteins having the COOH-terminal sequence
Ala-Leu-Xaa-Lys-Gly-OH in stead of the wild-type sequence
Ala-Pro-Ser-Ser-OH. The introduction of a COOH-terminal or penultimate
lysine residue in
A-crystallin makes this protein a good substrate
for TGC(17) . The eight COOH-terminal residues indeed form a
flexible extension from the surface of this homomultimeric
protein(23) . Residue Xaa at position 172 in the mutants was
occupied by different residues, as presented in Fig. 1, covering
the various types of amino acid side chains. The glycine residue behind
the introduced amine donor lysine was added because this has been shown
to enhance the substrate capacity of the mutant proteins and thus would
facilitate analysis(17) . The presence of leucine rather than
the wild-type proline before the variable residue Xaa has little
influence on the TG reactivity (17) and was chosen because this
enabled us to include four mutant clones from our previous series in
the present study.
In our previous work we only assessed the
reactivity of the mutants toward TGC and used a single biotinylated
peptide as the amine acceptor probe. Considering that TGC and factor
XIII react to different extents with a particular substrate
protein(13, 24) , we now wanted to compare the
reactivity of the mutant proteins with both TGC and factor XIII.
Binding of the amine acceptor substrate induces conformational changes
in the enzyme, creating a suitable binding place for the amine donor
protein(10) . It thus can be envisaged that different types of
amine acceptor substrates may modulate the binding of the mutated amine
donor substrates to different extents. We therefore used two different
amine acceptor probes: a biotinylated hexapeptide TVQQEL, as used in
our previous work(22) , and a biotinylated heptapeptide
PGGQQIV, patterned on the amine acceptor sequence in
fibronectin(25) . Finally, to improve the quantitative
assessment of the TG reactions, we now used purified mutant
A-crystallins rather than crude water-soluble E. coli extracts as in our previous study(17) .
The 13 mutant
A-crystallins and the wild-type protein were constructed,
expressed, and isolated as described under ``Materials and
Methods.'' The nature of the mutations was ascertained by
sequencing of the mutant clones. As shown in Fig. 2D,
the wild-type and mutant
A-crystallins were effectively purified
by the employed procedure. Samples of the purified
A-crystallins
were reacted with TGC and the hexapeptide probe (Fig. 2A), TGC and the heptapeptide probe (Fig. 2B), and factor XIII and the hexapeptide probe (Fig. 2C). Inspection of the resulting streptavidin
blots and comparison with the CBB-stained pattern in Fig. 2D immediately reveal conspicuous differences in the reactivity of
the various mutants. A quantitative representation of these
reactivities was obtained by densitometric screening of the blots (Fig. 3). The obtained values are normalized by relating them to
the densities of the corresponding CBB-stained bands, thus correcting
for differences in protein concentrations and loading variations. All
measurements were performed in duplicate on three independently
analyzed samples.
Figure 2:
Variation in lysine substrate capacity of
mutant A-crystallins for TGC and factor XIII. Streptavidin blots
are shown after reaction of the wild-type (wt) and mutant
proteins with TGC and biotinylated hexapeptide (A), TGC and
biotinylated heptapeptide (B), and factor XIII and
biotinylated hexapeptide (C). D gives a corresponding
Coomassie Brilliant Blue-stained pattern. The different residues
preceding the amine donor lysine in the mutants are indicated by the
one-letter notation above and below the lanes. Lane x contains a mutant
A-crystallin
that is identical to mutant G, apart from the presence of an additional
serine at the COOH terminus (Leu-Gly-Lys-Gly-Ser-OH). This mutant was
obtained by screening clones obtained with the degenerated linker 3
from (17) .
Figure 3:
Comparison of the TG-mediated
incorporation of amine acceptor probes in mutant A-crystallins. A, incorporation of biotinylated hexapeptide by TGC (cf.Fig. 2A). B, incorporation of
biotinylated heptapeptide by TGC (cf.Fig. 2B). C, incorporation of
biotinylated hexapeptide by factor XIII (cf.Fig. 2C). The bars indicate the relation
between streptavidin staining and the amount of mutant protein at an
arbitrary scale as determined by densitometric scanning. For further
details, see ``Materials and
Methods.''
Fig. 2and Fig. 3reveal some features that are quite consistent, independent of the probe and type of TG used. The presence of glycine before the amine donor lysine is unfavorable for the cross-linking of mutant and probe, although less so for factor XIII. The aliphatic side chains of alanine, valine, and leucine seem to be favorable, but reactivity does not increase proportionally to the length of the side chain. The TGs seem to be reasonably tolerant to the structure-breaking effect of proline. The size and positive charge of arginine have a positive effect in all instances, but histidine is of intermediate efficacy. The bulky indole side chain of tryptophan is less favorable than the phenyl ring of phenylalanine. The addition of the hydroxyl group in tyrosine slightly decreases the substrate activity. Serine generally makes the mutants good substrates, but compared with alanine the presence of the hydroxyl group variably influences the substrate capacity. Finally, the negative charge of aspartic acid, as compared with the carboxamide group of asparagine, clearly has an adverse effect.
Taken together, it thus appears that both TGC and factor XIII have a broad tolerance toward various types of side chains preceding the amine donor lysine. Apart from glycine and the negative charge of aspartic acid, a broad but variable acceptance is exhibited for aliphatic, aromatic, polar, and positively charged side chains of various sizes. Although differences in reactivity for TGC are measured when using the hexapeptide or heptapeptide probes (Fig. 3, compare A and B), it appears that the overall pattern is remarkably similar. This allows the conclusion that in this system the first substrate does not greatly influence the amine specificity of the TG reaction. The same holds true for the comparison of TGC and factor XIII, although the negative influence of tryptophan is conspicuous for factor XIII (Fig. 3, A and C). However, more detailed kinetic measurements, which we could not perform with our assay, may still reveal more profound differences between the two TGs. Our findings are in agreement with earlier evidence that TGC and factor XIII have very similar amine binding modes(10, 19) . The observation that TGC has a broader substrate specificity than factor XIII (10) seems not simply attributable to different effects of the residue preceding the amine donor lysine.
The present series of mutants is meant to highlight the importance for TG reactivity of the residue directly preceding the substrate lysine in a protein. Although this residue may be the most influential(10, 17, 19) , other nearby residues further modulate the reactivity. As an indication to that end, we included another mutant in lane x in Fig. 2. This mutant only differs from mutant G (Leu-Gly-Lys-Gly-OH) in having an additional COOH-terminal serine (Leu-Gly-Lys-Gly-Ser-OH). It appears that this mutant consistently has an enhanced reactivity as compared with mutant G (Fig. 3). This is in agreement with the suggestion that the active site of TGs corresponds in size to at least 9-10 residues of the polypeptide in their substrates(10) .
How do our
observations compare with the information available about the influence
of the NH-terminally neighboring residue of amine donor
lysines in peptides and proteins? Extensive studies with synthetic
peptides have demonstrated that replacing a leucine for a glycine
residue in front of the amine donor lysine significantly enhanced the
substrate activity toward TGC and factor
XIII(10, 19) . Other hydrophobic amino acids in this
position might provide a similar effect. Analogs of Xaa-Lys sequences,
where Xaa is Ala, Phe, Leu, Val, or Trp, indeed all denoted high
specificity in the TG reaction(20) . This led to the idea that
the major force in acyl enzyme-lysine peptide interaction is provided
by the secondary hydrophobic binding through the side chain of the
residue adjacent to and on the amino side of the substrate
lysine(10, 19) . Our observations are in agreement
with a positive effect of various hydrophobic residues. However, they
also indicate that under our conditions, basic and uncharged polar side
chains in front of the amine donor lysine can be equally effective in
directing its proper orientation in the active site of TGs and hence in
determining its cross-linking capacity.
Only a very small number of
amine donor lysines have been characterized in substrate proteins for
TGs. Fourteen identified substrate lysines for
TGC(26, 27, 28, 29, 30, 31, 32) have
recently been tabulated(17) . Another lysine substrate for TGC
has since been identified in Alzheimer amyloid protein A4, located
in the sequence -SNKG-(33) . For factor XIII the
substrate lysine in the fibrin
chain has been characterized long
ago (-GAKQ-)(34) . Hohl et al.(35) were the first to isolate isodipeptides of an in
situ cross-linked protein, (i.e. loricrin from the
cornified cell envelope). This revealed the internal (-SVKY-)
and the COOH-terminal (-PSK) lysine residues as substrates for
the epidermal transglutaminases. Recently, Steinert and Marekov (18) identified another 12 cross-linked lysine residues from
the cornified envelope. These involved the sequences -YQKKQ- and
-QQKQ- in loricrin, -QQKQ- and -KQK in SPR1,
-KSK in SPR2, -QDKVKA- and -PVKG- in
prepro-elafin, and -GSKS-, -GTKS-, and -SSKQ- in
keratins 1, 2e, and 10, respectively. Among this total of 30
characterized amine donor residues, the residues directly preceding the
substrate lysines are Gln (6 times), Lys (5 times), Ser (5 times), Ala
(3 times), Thr (3 times), Val (3 times), Arg (2 times), Leu (1 time),
Asn (1 time), and Asp (1 time). This listing combines results from
various TGs and from a limited number of proteins but nevertheless
suggests a preference for uncharged and basic polar residues, as well
as for the smaller aliphatic ones. It is noteworthy that the residues
found in our studies to negatively influence TG reactivity, like Asp,
Gly, Pro, His, and Trp, indeed appear to be largely avoided. These data
thus are in accord with the preferences that we established for our
mutant proteins.
Most of the current insight in the basis for specificity of TG-catalyzed isodipeptide bond formation stems from the work of Folk and co-workers on small model substrates(10, 19, 20) . It has been emphasized (10) that little has been learned yet about the manner in which the TGs operate on macromolecular substrates in biological systems. Using site-directed mutagenesis, we now show that the residue preceding an accessible amine donor lysine in a native protein has considerable influence on TG cross-linking potential. This corroborates and extends the results of earlier work on synthetic peptides(19, 20) . Further studies are required to reveal the possible modulating effects of native protein conformation in comparison with small model substrates on substrate specificity and mechanism of action of TGs.