(Received for publication, August 29, 1994; and in revised form, December 19, 1994)
From the
The structural basis for the biological specificity of human
cystatin C has been investigated. Cystatin C and other inhibitors
belonging to family 2 of the cystatin superfamily interact reversibly
with target peptidases, seemingly by independent affinity contributions
from a wedge-shaped binding region built from two loop-forming
inhibitor segments and a binding region corresponding to the N-terminal
segment of the inhibitor. Human cystatin C variants with Gly
substitutions for residues Arg-8, Leu-9, and/or Val-10 of the
N-terminal binding region, and/or the evolutionarily conserved Trp-106
in the wedge-shaped binding region, were produced by site-directed
mutagenesis and Escherichia coli expression. A total of 10
variants were isolated, structurally verified, and compared to
wild-type cystatin C with respect to inhibition of the mammalian
cysteine peptidases, cathepsins B, H, L, and S. Varying contributions
from the N-terminal binding region and the wedge-shaped binding region
to cystatin C affinity for the four target peptidases were observed.
Interactions from the side chains of residues in the N-terminal binding
region and Trp-106 are jointly responsible for the major part of
cystatin C affinity for cathepsin L and are also of considerable
importance for cathepsin B and H affinity. In contrast, for cathepsin S
inhibition these interactions are of lesser significance, as reflected
by a K value of 10
M for the cystatin C variant devoid of Arg-8, Leu-9,
Val-10, and Trp-106 side chains. The side chain of Val-10 is
responsible for most of the affinity contribution from the N-terminal
binding region, for all four enzymes. The contribution of the Arg-8
side chain is minor, but significant for cystatin C interaction with
cathepsin B. The Leu-9 side chain confers selectivity to the inhibition
of the target peptidases; it contributes to cathepsin B and L affinity
by factors of 200 and 50, respectively, to cathepsin S binding by a
factor of 5 only, and results in a 10-fold decreased affinity between
cystatin C and cathepsin H.
Cystatin C is comprised of one nonglycosylated 120-residue polypeptide chain (Grubb and Löfberg, 1982). It is ubiquitous in human tissues and body fluids (Abrahamson et al., 1986, 1990) and efficiently inhibits endogenous cysteine peptidases such as cathepsins B, H, L, and S (Barrett et al., 1984; Brömme et al., 1991). Cystatin C therefore appears to have a general protective function, to prevent connective tissue from destruction by intracellular enzymes leaking from dying cells or being misrouted for secretion from malignant cells (reviewed by Keppler et al.(1994)). Together with the other family 2 cystatins, cystatin D, S, SN, and SA, which have a more restricted distribution in human body fluids (Isemura et al., 1991; Freije et al., 1993), it may also be involved in defense against microbial infections. This seems likely because several parasitic protozoa, including, for example, the dysentery-causing Entamoeba histolytica and the pathogen in Chagas' disease, Trypanosoma cruzi, synthesize cysteine peptidases with crucial functions in parasite-host interaction (Luaces and Barrett, 1988; North et al., 1990). In addition, an antiviral function of cystatins is suggested from experiments with polio, herpes simplex, and corona virus-infected cell cultures (Korant et al., 1985; Björck et al., 1990; Collins and Grubb, 1991).
Cystatin C forms reversible 1:1 complexes
with target enzymes in competition with their substrates (Abrahamson et al., 1987a). From functional studies of cystatin C with
truncated N-terminal segments, it is clear that an enzyme binding
region located near the N-terminal end of the inhibitor is of major
importance for its typical tight-binding properties (Abrahamson et
al., 1987a, 1991). This region contains an evolutionarily
conserved glycine residue (Gly-11) that confers flexibility to the
preceding N-terminal segment, which is a prerequisite for optimal
enzyme binding of the segment (Hall et al., 1993). The
N-terminal region appears to contribute to endopeptidase binding via
interactions between side chains of the residues preceding the
evolutionarily conserved Gly-11 residue, Val-10, Leu-9, and, to a
smaller extent, Arg-8, and target enzyme substrate-binding subpockets
S, S
and S
, respectively (Hall et al., 1992; Lindahl et al., 1994). A similar
important effect of the corresponding residues in the N-terminal
segment of the avian family 2 homologue to human cystatin C, chicken
cystatin, has been reported (Abrahamson et al., 1987a;
Machleidt et al., 1989; Lindahl et al., 1992b).
Computer docking based on the three-dimensional structures of chicken
cystatin and papain, in addition, strongly suggests that two
loop-forming cystatin segments, one located in the central part and the
other close to the C-terminal end of the cystatin polypeptide chain,
together form a second, wedge-shaped enzyme binding region (Bode et
al., 1988). These segments also contain residues that have been
conserved during the evolution of family 2 cystatins and correspond to
the human cystatin C segments Gln-55-Gly-59 and Pro-105-Trp-106.
Although the mechanism of cystatin interaction has been studied in some detail, it is still obscure what determines the biological specificity of the individual cystatins. Compared to cystatin C, cystatin S has for example 10,000,000-fold lower affinity for papain (Isemura et al., 1986; Lindahl et al., 1992a), and, unlike other human family 2 cystatins, cystatin D does not inhibit cathepsin B at all (Balbín et al., 1994). In the present study, we have used a site-directed mutagenesis approach to evaluate the relative importance of the N-terminal binding region to that of the wedge-shaped binding region for cystatin inhibition of mammalian cysteine peptidases, as well as to pinpoint which residues contribute most to target enzyme affinity by interactions with their substrate-binding subpockets.
To set up a mutagenesis protocol allowing rapid generation of cystatin C variants with amino acid substitutions in the N-terminal segment, two unique recognition sites in pHD313, for the restriction endonucleases ClaI and NcoI, were used. The ClaI-NcoI fragment, which includes the coding sequence for the OmpA signal peptide as well as cystatin C residues 1-15 (Fig. 1), was excised from the plasmid. Corresponding fragments containing predetermined mutations were generated by the polymerase chain reaction (PCR) using primer pairs hybridizing to sequences upstream from the ClaI site and encompassing the NcoI site, respectively (Fig. 1). The target sequence for the downstream primer was chosen to also include the codons for cystatin C residues 8-10, thereby allowing the desired mutations to be introduced in the PCR fragment by choosing a primer with appropriate nucleotide substitutions introduced at synthesis. The sequences of the upstream primer (206) and the downstream mutagenesis primers (209-214) are given in Table 1. PCR amplifications were performed in a Perkin-Elmer Cetus DNA Thermal Cycler using DNA polymerase and other reagents from the AmpliTaq kit (Perkin-Elmer Cetus), primers at 0.6 µM concentration, and 0.6 ng of uncleaved pHD313 DNA as template in a 100-µl reaction. The PCR cycle (1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C) was repeated 30 times. PCR products were purified by phenol/chloroform extractions and ethanol precipitation, cleaved with ClaI/NcoI, and ligated into ClaI/NcoI cut and dephosphorylated pHD313. Thereby mutated plasmids (named after the corresponding mutagenesis primer, e.g. pCmut209 for the V10G-cystatin C expression vector constructed with the aid of mutagenesis primer 209) were introduced in E. coli MC1061 (Casadaban and Cohen, 1980) that had been made competent for transformation by treatment with calcium chloride (Sambrook et al., 1989). Subclones of bacteria containing the expression plasmids were selected on ampicillin culture plates and grown overnight in LB medium containing 100 µg/ml ampicillin. Plasmid DNA was isolated from the cultures by a standard alkaline lysis procedure (Sambrook et al., 1989).
Figure 1:
Scheme of the mutagenesis procedure.
Unique restriction sites for ClaI, NcoI, BglII, EcoRI, and MvaI in the insert of the
human cystatin C gene insert of the expression vector pHD 313 are
indicated. Numbers at the top denote amino acids,
with the native cystatin C encoding region starting at 1 and ending at
120. The amino acid residues subjected to substitutions are also
numbered. Horizontal arrows show locations and directions of
oligonucleotides used: 209-214, for site-directed mutagenesis to
generate amino acid substitutions in the N-terminal cystatin C region
(including the recognition site for NcoI); 219, for
mutagenesis to generate the Trp-106 Gly substitution in the
C-terminal part of cystatin C (including the BglII site); 206,
as PCR primer together with any of the mutagenic oligonucleotides for
the N-terminal substitutions; 220, as PCR primer together with 219 for
the Trp-106
Gly substitution. PCR products were digested with
the indicated enzymes and introduced in pHD313 where the corresponding
wild-type segments had been excised. Oligonucleotides 206, 220, 078,
and 011 were used for sequencing to verify the correct insert sequences
of the resulting expression plasmids. For oligonucleotide sequences,
see Table 1.
The same principle as described above for the generation of cystatin C variants with substitutions in the N-terminal region was applied for mutagenesis to give an expression vector for W106G-cystatin C. Unique recognition sites for the restriction enzymes BglII and EcoRI were taken advantage of (Fig. 1). The upstream, mutagenesis primer (called 219, Table 1) was designed to include the BglII site and span over the codon for cystatin C residue 106, allowing direct oligonucleotide introduction of the appropriate nucleotide substitution. The downstream primer used (``220,'' Table 1) corresponds to a vector sequence downstream from the EcoRI site (Fig. 1). Conditions for PCR amplification of a 357-base pair segment with this primer pair were the same as described above. Purification, digestion, and ligation of the PCR product into BglII/EcoRI cut and dephosphorylated pHD313, subsequent transformation of E. coli with the resulting plasmid (pCmut219), and plasmid isolation from selected subclones were also performed as above.
Expression vectors for
cystatin C variants with both substitutions in the N-terminal region
and the C-terminal substitution Trp-106 Gly, were constructed by
ligating the NcoI-ClaI-cut PCR fragments used for
generation of the N-terminal variants into the expression vector for
W106G-cystatin C, pCmut219, that previously had been digested with NcoI/ClaI and dephosphorylated. The resulting
expression plasmids were named pCmut209/219, pCmut210/219, etc., and
were introduced into E. coli MC1061 as described above.
Isolation of the different cystatin C variants from periplasmic extracts was accomplished by a previously detailed two-step procedure, including ion exchange chromatography on Q-Sepharose (Pharmacia Biotech Inc.) in ethanolamine buffer, pH 9.5 or 9.0, and gel chromatography on a Pharmacia FPLC Superdex 75 column in ammonium bicarbonate buffer (Hall et al., 1993). The salt-free solutions of the isolated protein variants were concentrated by ultrafiltration (Centricon 3; Amicon Corp.) to approximately 0.2 mg/ml when necessary and were stored frozen at -20 °C until used.
The isolated recombinant cystatin C variants were characterized by charge-separating agarose gel electrophoresis at pH 8.6 (Jeppsson et al., 1979), SDS-polyacrylamide electrophoresis after reduction in 16.5% gels with the buffer system described by Schägger and von Jagow (1987), and by automated N-terminal sequencing (Olafsson et al., 1990) using equipment and detailed procedures described earlier (Hall et al., 1993).
To one of the dilution series, equal portions of the protein variant under study was added to give a final concentration of approximately 2 µM. Samples in the other series, without protein, served as blanks at fluorescence measurements. The solutions were incubated at room temperature for at least 20 h before measurements of their tryptophan emission spectra. A Perkin-Elmer LS-50 fluorimeter was used at excitation and emission wavelengths of 295 and 350 nm, respectively. The corresponding bandwidths were 3 and 10 nm, the path lengths of the cells were 1 cm, and the temperature in the cell was 26 °C during measurements. To evaluate the data, the height of the emission peak at 350 nm after subtraction of the corresponding blank was related to that of the protein sample with no guanidinium chloride present.
Temperature
stability measurements of recombinant wild-type cystatin C and the
variants Cmut214, Cmut219, and Cmut219/214 were performed essentially
as earlier described (Abrahamson and Grubb, 1994). Solutions of the
cystatin C variants at 0.2-0.3 mg/ml concentration in 0.05 M NHHCO
buffer, pH 8.0, were incubated for
30 min at temperatures ranging from 30 to 95 °C. The samples were
centrifuged for 5 min at 12,000
g, and the
supernatants were thereafter analyzed by agarose gel electrophoresis,
and their relative immunoreactivity was measured. The immunoreactivity
was measured by single radial immunodiffusion (Mancini et al.,
1965) in plates containing 0.4% of the IgG fraction from a rabbit
antiserum raised against human cystatin C (DakoPatts, Copenhagen,
Denmark). The degree of temperature-caused denaturation was expressed
as the ratio between the immunoprecipitate area for each sample related
to that formed by a co-analyzed sample of the variant under study that
had been kept at 4 °C during the heat incubation of other samples.
Due to difficulties in purification and procurement of tissues, human tissues could not be utilized to isolate cathepsins H, L, and S. However, comparative data available for the mammalian cysteine peptidases have shown no major differences in catalytic specificity in species variants of cathepsins B, H, L, or S (Mason, 1986a, 1986b; Shi et al., 1992; Xin et al., 1992).
Papain was used for active site titrations of the different cystatin C variants, as has been described in detail elsewhere (Hall et al., 1993). The active enzyme was purified from a commercial papain preparation (Sigma, type III) by affinity chromatography, at 4 °C, using the peptide H-Gly-Gly-Tyr-Arg-OH coupled to CNBr-activated Sepharose 4B (Pharmacia), according to the protocol described by Blumberg et al.(1970). The purified enzyme was approximately 75% active as determined by E-64 titrations (Barrett et al., 1982) and was stored frozen at -20 °C, resulting in no more than a 5% reduction of activity even after storage for 6 months.
Figure 2: Agarose gel electrophoresis of cystatin C variants. Electrophoresis of 8-µl samples was carried out in 1% agarose gel at pH 8.6. The point of application and the anode are indicated by an arrow and a plus sign, respectively. Lanes a and l contain 8 µl of a 1 mg/ml solution of isolated wild-type recombinant cystatin C. Lane b is variant Cmut209, followed to the right by Cmut210, Cmut211, Cmut212, Cmut214, Cmut219, Cmut219/209, Cmut219/210, Cmut219/211, and, in lane k, Cmut219/214. Human blood plasma, as a reference, is shown in the flanking lane to the left.
The charge difference expected for the recombinant
R8G- and (R8G,L9G,V10G)-variants compared to the wild-type inhibitor,
due to loss of the Arg-8 side chain, could be verified by a more anodal
mobility in agarose gel electrophoresis (Fig. 2). SDS-PAGE under
reducing conditions demonstrated that all variants had the same
mobility as wild-type recombinant cystatin C. For all, the SDS-PAGE
estimated M was slightly higher than the M
calculated from their sequences
(13,017-13,343), but identical with that obtained by the same
SDS-PAGE system for the native protein isolated from human urine
(Abrahamson et al., 1988). All variants and wild-type
recombinant cystatin C eluted by gel chromatography on a calibrated
Superdex 75 column at a position corresponding to a M
of 12,400. The five recombinant cystatin C variants were
subjected to 15 steps of automated Edman degradation. The released
phenylthiohydantoins verified in every position the expected N-terminal
sequences (data not shown). The combined results of the physiochemical
characterization of the variants, along with DNA sequencing of the
coding regions of the expression vectors present in the same bacterial
cultures as were used for expression, thus gave strong support for the
variants produced being those intended.
Expression of the five variants containing the
Trp-106 Gly substitution was induced in bacterial subclones
containing the corresponding plasmids, as verified by DNA sequencing,
and periplasmic extracts were collected. The five isolated Trp-106
Gly-containing variants are shown in Fig. 2(lanes
g-k). Again, the expected mobility difference due to removal
of the Arg-8 side chain, in the (R8G,W106G)- and (R8G,L9G,
V10G,W106G)-variants, could be verified by agarose gel electrophoresis.
Also, the SDS-PAGE mobilities and the elution profiles from the
calibrated Superdex 75 column coincided with those for wild-type
recombinant cystatin C, demonstrating that no proteolytic degradation
had occurred during the isolation procedure.
Figure 3:
Heat stability curves for cystatin C
variants. Samples of wild-type recombinant cystatin C and the variants
Cmut214, Cmut219, and Cmut219/214 were incubated for 30 min at various
temperatures in pH 8.0 buffer at a 0.2-0.3 mg/ml concentration.
The immunoreactivity of each incubated sample was determined by single
radial immunodiffusion and compared to that of a corresponding sample
stored on ice. Data for the L68Q-cystatin C variant (Abrahamson and
Grubb, 1994) have been added for comparison. , wild-type;
, Cmut214;
, Cmut219;
, Cmut219/214;
,
L68Q.
Fluorescence emission spectra in the presence of varying concentrations (0-6 M) of guanidinium chloride were analyzed for wild-type cystatin C and the variant with most extensive N-terminal substitution, (R8G,L9G,V10G)-cystatin C. For every guanidinium chloride concentration analyzed, the spectra for wild-type cystatin C and the variant were highly similar (data not shown). The calculated denaturation curves revealed an unfolding event between 3 and 4 M guanidinium chloride for both, but due to a large solvent effect resulting in a more than 4-fold tryptophan fluorescence increase at 350 nm for samples equilibrated in 6 M guanidinium chloride compared to those analyzed with no guanidinium chloride present, the transition midpoints for the denaturation process could just be approximated. The estimated midpoints were in 3.75 M and 3.25 M guanidinium chloride for wild-type and (R8G,L9G,V10G)-cystatin C, respectively.
Taken together, the stability tests indicated that neither extensive substitutions in the N-terminal segment nor removal of the Trp-106 side chain significantly affect the overall structure of cystatin C.
1. The N-terminal enzyme binding region of cystatin C is
generally important for inhibition, but its contribution to target
enzyme affinity varies for the different peptidases. It is essential
for cathepsin B inhibition (removal of Arg-8, Leu-9, and Val-10 side
chains results in a >4000-fold affinity decrease, and the resulting K value is >1 µM), also of
considerable importance for cathepsin L inhibition (corresponds to 4
orders of magnitude in affinity contribution), but of relatively low
importance for cathepsin H and S inhibition (affinity contribution
approximately 2 orders of magnitude for both). Cystatin C lacking the
N-terminal binding region retains a considerable affinity for
cathepsins H, L, and S (K
10
-10
M).
2. The evolutionarily conserved Trp-106 residue is generally
important for the enzyme affinity of cystatin C. Its affinity
contribution to cystatin C inhibition corresponds to 3 orders of
magnitude or more for cathepsins B, H, and L, but it contributes less
to the interaction with cathepsin S (affinity contribution
approximately 2 orders of magnitude). The relative contribution of the
Trp-106 side chain is 1 order of magnitude lower than that of the
entire N-terminal binding region in the cystatin C inhibition of
cathepsin L, 1 order of magnitude higher for cathepsin H, and equally
important as that of the N-terminal binding region for the inhibition
of cathepsin S and probably also cathepsin B (K values >1 µM regardless of side chain removal for
Trp-106 or for all residues in the N-terminal binding region).
3.
For the enzyme affinity conferred by the wedge-shaped binding region,
the Trp-106 residue contributes differently in interactions with the
four enzymes studied. In the cystatin C inhibition of cathepsin B, the
Trp-106 side chain is responsible for a substantial fraction of the
affinity contribution by this region, but for the inhibition of
cathepsin S it is of less importance. Consequently, the remainder of
the wedge-shaped binding region including the evolutionarily conserved
loop-forming segment Gln-55-Gly-59 is of considerable importance for
the cystatin C inhibition of cathepsin S (K = 9 nM for (R8G,L9G,V10G,W106G)-cystatin C
inhibition of cathepsin S, compared to >1 µM for all
other enzyme interactions), but its direct contribution to the
inhibitor's affinity for cathepsin B seems minor.
Thus, the structural basis for the differences in cystatin C affinity for the four target peptidases is complex, with varying binding contributions from the N-terminal binding region and the wedge-shaped binding region for the investigated peptidases, and also with different parts of the wedge-shaped binding region contributing to varying extents for the different enzymes. The observed variation probably reflects decisive general differences in the three-dimensional structures of the active site clefts of the enzymes (Björk et al., 1994) and indicates that the general orientation of cystatin C at enzyme interaction differs with the enzyme.
Concerning individual residues in the N-terminal binding region, the main conclusions from the inhibition results for the cystatin C variants with single substitutions in the N-terminal binding region (Table 2) are as follows.
The functional results for the
cystatin C variants with double or triple substitutions in the
N-terminal binding region support the data obtained for the variants
with single substitutions and, hence, demonstrate that the side chains
of the Arg-8, Leu-9, and Val-10 residues contribute to target enzyme
affinity in a simple additive fashion. For the cathepsin S interaction,
for example, the Val-10 Gly, Leu-9
Gly, and Arg-8
Gly substitutions in cystatin C results in affinity decreases of
factors 30, 5, and 1.3, respectively, while the triple substitution
results in a 150-fold affinity decrease, approximately equal to the
product of the factors for the three variants with single
substitutions.
In the present investigation, we have tried to elucidate the
structural basis for the biological specificity of cystatin C by
functional studies of inhibitor variants with Gly substitutions for
residues Arg-8, Leu-9, Val-10, and/or Trp-106. The Arg-Leu-Val residues
were investigated because they, by several lines of evidence, seem to
be responsible for the important contribution to target peptidase
affinity that is conferred by an N-terminal binding region of family 2
cystatins like human cystatin C and chicken cystatin (Abrahamson et
al., 1987, 1991; Bode et al., 1988, 1990; Machleidt et al., 1989; Hall et al., 1992; Lindahl et
al., 1992b). They are also good candidates for residues conferring
specificity to the inhibitory reaction, since their side chains
interact with the nonprimed substrate-binding subpockets of several
cysteine peptidases (Hall et al., 1992; Lindahl et
al., 1994) and, hence, should have the capacity to reflect an
enzyme's specificity for substrates. Studies of low molecular
weight substrates and peptidyl inhibitors have indicated that the
residues offering greatest discrimination between the four cysteine
peptidases studied in the present work are located in P,
P
, and P
(Mason and Wilcox, 1993). At target
enzyme interaction, cystatin C should have the evolutionarily conserved
Gly-11 residue in the P
position (Bode et al.,
1988, 1990) and thus lacks the major determinant for discrimination
between cathepsins B, L, and S. For substrate interactions, Val is
accepted in P
by all four enzymes, as is Leu in
P
. Thus, the N-terminal segment of wild-type cystatin C
with its Val-10 and Leu-9 residues could be regarded as a valuable
sequence conferring general cysteine peptidase inhibitory properties to
the protein, provided that it interacts with all target enzymes.
Indeed, our present results (Table 2) demonstrate that the
N-terminal segment can interact with all four mammalian cysteine
peptidases. The Trp-106
Gly substitution was included in the
present investigation primarily to enable studies of the effects of
substitutions in the N-terminal segment of cystatin C for its
interactions with cathepsins L and S, because the affinities of
wild-type cystatin C for these enzymes are too high to allow reliable
measurement of complex dissociation. It seemed likely that removal of
the Trp-106 side chain would lower the overall enzyme affinity of
cystatin C as this residue is directly involved in cystatin binding to
several plant cysteine peptidases (Lindahl et al., 1988,
1992a), but without affecting interactions from the seemingly
independent N-terminal binding region of the inhibitor (Hall et
al., 1993). Our present data (Table 2) demonstrate that this
approach was fruitful. The strategy to completely remove the side
chains of the residues studied by Gly substitutions seemed plausible
since the N-terminal segment of human cystatin C, as well as that of
chicken cystatin, has no ordered structure according to NMR studies
(Dieckmann et al., 1993). (
)Furthermore, the Trp
residue is located in a hairpin loop and has a side chain that is
solvent-exposed according to x-ray crystallography and NMR (Bode et
al., 1988; Dieckmann et al., 1993),
meaning
that increased polypeptide chain flexibility caused by total removal of
these side chains by Gly substitutions would be unlikely to affect the
overall structure of the inhibitor. Our protein stability studies of
the generated cystatin C variants indicate that the latter assumption
is valid; no gross stability differences could be seen by comparison
with wild-type cystatin C.
Our functional data for the 10 cystatin C
variants indicate general differences in the modes of interaction with
the four mammalian cysteine peptidases. Cathepsins L and S, for
example, are both very tightly bound by wild-type cystatin C with K values < 10
M (Barrett et al., 1984; Brömme et
al., 1991). In the cystatin C interaction with cathepsin L, the
N-terminal binding region and the Trp-106 residue of the wedge-shaped
binding region both contribute considerably, resulting in affinity
contributions corresponding to more than 3 orders of magnitude for each
and a poor inhibitory activity of (R8G, L9G,V10G,W106G)-cystatin C (K
1 µM). For the inhibition of
cathepsin S, both the N-terminal binding region and Trp-106 of cystatin
C are of lesser significance, and retained good inhibition by the
(R8G,L9G,V10G,W106G)-variant (K
9 nM)
shows that the remainder of the wedge-shaped binding region including
the conserved loop-forming segment Gln-55-Gly-59 is of considerable
importance. A relatively low importance of the loop-forming
Gln-55-Gly-59 segment of the wedge-shaped binding region for the
cystatin C interaction with cathepsin L can thus be inferred. This
conclusion is supported by results from a site-directed mutagenesis
study of residues in the corresponding first hairpin loop of the
wedge-shaped binding region of chicken cystatin, demonstrating that a
number of different substitutions in this region had almost no effect
on cathepsin L inhibition (Auerswald et al., 1992). However,
such substitutions significantly affected the chicken cystatin
inhibition of cathepsin B. Since the cathepsin B affinity for wild-type
cystatin C is at least 100-fold lower than those displayed by
cathepsins L and S, our results for this enzyme do not allow any
general conclusions beyond the statement that both the N-terminal
binding region and the Trp-106 residue are of crucial importance for
the cystatin C inhibition of cathepsin B. But it seems likely that the
mode of cystatin interaction with cathepsin B is different and probably
follows a two-step binding mechanism, because of the extra occluding
loop of the enzyme that covers the S`
substrate pocket and
which needs to be displaced in order to allow optimal inhibitor binding
(Musil et al., 1991; Björk et
al., 1994).
Our previous studies of a cystatin C variant devoid
of the 10 N-terminal residues ([des-1-10]cystatin C)
have demonstrated that the N-terminal binding region contributes
decisively to cathepsin B and L affinity (by 3 orders of magnitude) but
not to that for cathepsin H (6-fold affinity decrease at removal of the
N-terminal decapeptide, only) (Abrahamson et al., 1991). The
data from the Gly-substituted cystatin C variants in the present study
are fully consistent with these earlier results for cathepsins B and L,
demonstrating that conclusions concerning the importance of an
N-terminal binding region deduced from studies of N-terminally
truncated forms of different cystatins should be valid at least for
these two enzymes. For cathepsin H, however, the small functional
difference between [des-1-10]- and wild-type cystatin C
previously noted seems to underestimate the importance of the
N-terminal binding region in intact cystatin C for this peptidase. The
data of the present study rather indicate that the N-terminal binding
region of cystatin C contributes significantly to cathepsin H
inhibition (120-fold) and agrees well with recent data demonstrating
that cathepsin H has endopeptidase activity (Xin et al., 1992)
and therefore should have an extended substrate cleft with the capacity
to accommodate the N-terminal binding region of cystatins. Although
this interaction of the N-terminal region is of less importance for
inhibition of cathepsin H than for inhibition of cathepsins B and L, it
seems to be physiologically relevant since it improves inhibition from
a K of 10
to 10
M, i.e. to considerably below the cystatin C
concentration in all investigated human body fluids (Abrahamson et
al., 1986).
The purpose of the present study was to collect
structure-function data for cystatin C, with the long-term goal to use
protein engineering methodology for development of protein inhibitors
specific for physiologically relevant cysteine peptidases. Access to
specific cystatins should be of importance to allow studies of the
importance of individual cysteine peptidases in biological systems and
would have the advantage over synthetic low molecular inhibitors
designed for this purpose that they also could be used in transfection
experiments to establish overproducing cell lines or transgenic
animals. Specific cystatins will, in addition, most probably be devoid
of the general cytotoxic properties displayed by several low molecular
weight inhibitors. The present work has produced some practically
useful results for the cystatin engineering work. For example, the
N-terminal binding region should be the primary target for
modifications aiming at selectivity. Especially Leu-9 seems to be a
most promising target for such selectivity-determining substitutions. A
systematic exploration with guidance of data for enzyme specificity for
the P position in substrates to generate specific
inhibitors for cathepsin B, H, and L might be rewarding. In addition, a
recent study indicates that selectivity for cathepsin B inhibition can
be achieved by the P
substitution Val-10
Arg
(Lindahl et al., 1994). For specific cathepsin S inhibition,
the properties of (R8G,L9G, V10G,W106G)-cystatin C produced in the
present work demonstrate that this variant is more selective for
cathepsin S than any known wild-type cystatin and might be useful for
studies in complex biological systems.