(Received for publication, March 23, 1995; and in revised form, June 1, 1995)
From the
Cathepsin C has been purified from human kidney by a modified
procedure. Human cathepsin C was isolated as pure protein with a pI
close to 6.0. The enzyme was shown to have a molecular mass of 200 kDa
and to consist of four identical subunits, each composed of three
different polypeptide chains, two of them disulfidebound. Their
NH-terminal amino acid sequences were determined. Two
chains showed pronounced similarity with the heavy and light chains of
other papain-like cysteine proteinases, whereas the third one
corresponded to the prosequence of the enzyme, thus showing that a
substantial part of the proregion remains bound in the mature enzyme.
The kinetics of substrate hydrolysis deviated substantially from
standard Michaelis-Menten kinetics, demonstrating substrate inhibition
at higher substrate concentrations. These data are explained by a
sequential cooperative interaction model, where an enzyme molecule can
bind up to four substrate molecules but where only the binary
enzyme-substrate complex is catalytically active. Substrate inhibition
was observed over the whole range of pH activity. From the pH activity
profile it can be concluded that at least three ionizable groups with
pK
values 4.2, 6.8, and 7.7 are involved
in substrate hydrolysis. Human cathepsin C thus appears to differ
qualitatively from other cysteine proteinases of different origin.
Cathepsin C, known also as dipeptidyl aminopeptidase I (DPPI), cathepsin J, or dipeptidyl transferase (EC 3.4.14.1), is a lysosomal cysteine peptidase (1, 2, 3) belonging to the papain family(4) . It is present in a variety of tissues from rat and human sources(5) . A survey of human tissues showed that spleen and kidney are the richest sources of cathepsin C. In addition, serum levels were highest in hepatic diseases, followed by peripheral arterial disease, thromboembolism, myocardial infarction, diabetes mellitus, and prostatic hypertrophy. Like other lysosomal cysteine proteinases cathepsin C is involved in intracellular protein degradation(1) , and it has been reported that cathepsin C activity is present at higher levels in cytotoxic lymphocytes and myeloid cells, indicating more specific but as yet unknown roles in these immune effector cells(6) .
Cathepsin C is active
against different substrates in the pH range between 3.5 and
8.0(7) . It cleaves primarily peptide and protein substrates
having an unsubstituted amino
terminus(8, 9, 10, 11, 12, 13, 14, 15) ,
although it can also degrade substrates with a blocked amino
terminus(16) . Early studies showed that the enzyme requires
halide ions and sulfhydryl reagents to achieve maximal hydrolytic
activity(11, 17) , indicating that it is a cysteine
proteinase. It is specifically inactivated by Gly-Phe-diazomethyl
ketone, a typical thiol-blocking reagent, with a K value of about 10 nM(18) . Recently, it has
been shown to be inhibited by E-64, another typical thiol-blocking
reagent, and leupeptin, both at high concentrations(2) .
Cathepsin C is also inhibited by rat stefin A and chicken cystatin, two
protein inhibitors of cysteine peptidases from the cystatin superfamily (2, 19) .
There are differing reports concerning the molecular mass and subunit composition of cathepsin C. Thus bovine cathepsin C was found to be an oligomeric enzyme of about 200 kDa, composed of eight subunits in the form of two tetramers with two different types of subunits(14) . The rat liver enzyme was also estimated to have a molecular mass of 200 kDa (12) , but more recently 160 kDa, consisting of two different subunits (2) . The enzyme from porcine spleen was isolated as a mercurial derivative of 56 kDa, which formed after reducing an active dimer of 110 kDa(20) .
The cDNA sequence of rat cathepsin C (21) is highly homologous to those of cathepsins B, H, L, and S and papain(22, 23, 24, 25, 26) . However, the propeptide region of cathepsin C is substantially longer than those of other cysteine peptidases but without pronounced amino acid sequence similarity to any of them(21) . Recently, partial amino acid sequences of rat (2, 21) and human (27) cathepsin C have been reported. The biosynthesis and processing of cathepsin C were investigated by pulse-chase experiments in cultured rat macrophages(28) , showing that the enzyme is first synthesized as procathepsin C with a molecular mass of 55 kDa. Within 1 h procathepsin C is cleaved and modified into mature cathepsin C having two chains of 25 and 7.8 kDa. Cathepsin C then oligomerizes just before entering the lysosomes. This behavior is unique among papain-like cysteine peptidases.
The above results apply mainly to cathepsin C of non-human origin, and there is little information concerning the properties and structure of human cathepsin C(27, 29) . It has been reported that human cathepsin C is a glycoprotein with a molecular mass of 200 kDa consisting of eight subunits of 24 kDa(27) , while preliminary studies in this laboratory have indicated the existence of four subunits of 50 kDa. While differences in substrate specificity among human, bovine and porcine cathepsin C (27) have been reported, the detailed kinetics have not so far been studied for human cathepsin C.
We present new more detailed studies on the properties of human cathepsin C, its amino acid sequence analysis, characterization of its oligomeric structure, and kinetics of its interaction with synthetic substrates.
Figure 1: SDS-PAGE of human cathepsin C. Electrophoresis was performed as described under ``Experimental Procedures.'' A, human cathepsin C (2 µg) after several freezings (lane 1); , standard proteins (Low Molecular Weight Calibration Kit, Pharmacia) (lane 2). B, standard proteins (Low Molecular Weight Calibration Kit, Pharmacia) (lane 1); human cathepsin C (4 µg) after incubation at 100 °C and reduction with 2-mercaptoethanol for 5 min (lane 2); human cathepsin C (4 µg) after incubation at 100 °C for 5 min (lane 3); , human cathepsin C (4 µg) (lane 4); standard proteins (Prestained SDS-PAGE standards, broad range; Bio-Rad) (lane 5).
Figure 2: Electrophoretic titration curve of human cathepsin C. 40 µg of the purified protein was applied on a gel. The enzyme has isoelectric point 6.0 and migrates as a single band. Electrophoresis was performed as described under ``Experimental Procedures.''
Figure 3:
Position of the NH-terminal
amino acid sequences of the human cathepsin C polypeptide chains in
comparison with the rat preprocathepsin C. A, rat
preprocathepsin C amino acid sequence deduced from cDNA(21) . B, NH
-terminal amino acid sequences of rat
cathepsin C(2) . C, NH
-terminal amino acid
sequences of human cathepsin C. Identical amino acid residues of human
and rat cathepsin C are boxed.
Figure SI: Scheme 1.
Figure 4:
Substrate inhibition of human cathepsin C
modelled by negative cooperativity at pH 6.0. Experimental conditions
are described under ``Experimental Procedures.'' ,
experimental points. The dotted line is the theoretical curve
where ES
is inactive, calculated using , where d = e = 1 and f = 0. The dashed line is the theoretical curve
where ES
and ES
are inactive,
calculated using , where d = 1 and e = f = 0. The solid line is
the theoretical curve, where only ES is active, calculated
using , where d = e = f = 0. Equations in each case are fitted by nonlinear
regression analysis.
where v is the velocity of product formation, V is maximal velocity, and [S] is
substrate concentration. The results support the assumption that only
the binary enzyme-substrate complex is fully active, whereas all higher
complexes are inactive or almost inactive. As can be seen in Table 1, the binding of the second molecule is only slightly less
favorable than the binding of the first substrate molecule, whereas a
drastic drop in affinity is observed for the binding of the third
substrate molecule (Fig. 5), indicating a large negative
cooperativity effect. The binding of the fourth substrate molecule is
highly favorable,although the value of c is somewhat uncertain
due to the large error in calculations of the last parameter.
Figure 5:
Population of enzyme-substrate complexes
as a function of substrate concentration. Calculations have been made
on the basis of the model in Fig. SI. , the distribution
of the productive ES complex.
, the distribution of
inactive ES
complex. Formation of ES
is drastically decreased due to negative
cooperativity on binding the second substrate molecule.
, the ES
complex. Positive cooperativity is induced by ES
, and this complex is instantly transformed to ES
.
, the sum of all forms of
complexes.
In a further attempt to check whether the ternary and higher enzyme-substrate complexes are active, the data were fitted by the following equation, modified from that of Wong-Endrenyi(37) , to take into account reduced activity of higher complexes.
The parameters d, e, and f represent
coefficients by which the activities of the complexes ES, ES
and ES
are reduced due to substrate inhibition. Fitting to
the experimental data, for the case where the ternary complex is active (i.e.d > 0, e = f = 0), did not reduce the
error. The best
fit was obtained using , with d very close to zero
(<0.02). This indicates that the ternary complex has low or zero
activity, as proposed in Fig. SI. Similar results were obtained
for the systems where higher complexes were assumed to be active,
supporting the above conclusion that only ES has significant
activity.
The effect of pH on the rate of substrate hydrolysis was
studied in the pH range from 3.0 to 8.0. Substrate inhibition was
observed at all pH values, and the experimental data at each pH value
could be best described using . As can be seen from Table 1, all of the kinetic constants are strongly pH-dependent.
The pH dependence of the second order rate constant for substrate
hydrolysis, k/K
, exhibits a
bell-shaped profile (Fig. 6), indicating that at least two
ionizable groups of cathepsin C are involved in the interaction. In a
detailed analysis, the reaction rate was measured every 0.2 pH unit as
described under ``Experimental Procedures.'' The pH activity
data profile obtained in this way was best fitted by the equation
corresponding to a model with three different dissociable groups of the
enzyme involved in substrate hydrolysis(40) .
Figure 6:
pH dependence of k/K
for hydrolysis
of Gly-Phe-4M
NA by human cathepsin C. The solid line represents the best fit of to experimental data for
every 0.2 pH unit, determined at low substrate concentration. The
equation gives pK
values 4.2, 6.8, and
7.7.
where (k/K
)
represents the limiting value of k
/K
. In Fig. 6the best fit, corresponding to the pK
values 4.2 ± 0.05, 6.8 ± 0.1, and 7.7 ± 0.1
is shown.
A number of isolation procedures have been described for the purification of cathepsin C from different mammalian tissues(12, 17, 20) , but only two used human tissues as a starting material(27, 29) . We isolated cathepsin C from human kidney, known to be a rich source of lysosomal cysteine peptidases. In our purification scheme the successive affinity chromatography steps used by McGuire and co-workers (27) are replaced by cation exchange and covalent chromatographies, which resulted in a higher yield of pure enzyme as shown by sequence homogeneity and electrophoresis (Fig. 2).
Several attempts
have been made to determine the oligomeric structure of cathepsin C,
with results differing in the number and size of the enzyme subunits.
When dissociation of bovine cathepsin C was studied in the presence of
guanidinium chloride and urea(14) , an intermediate with a
molecular mass of 53 kDa was observed, which could be further
dissociated into smaller subunits with molecular mass of 24.5 kDa,
suggesting that cathepsin C consists of eight subunits. Furthermore,
two different NH-terminal amino acids were found,
suggesting that two different types of subunits are present in the
enzyme. Similar conclusions were reported for the human spleen enzyme (27) and for the rat enzyme(28) , since these
researchers found that the enzyme is a 200-kDa protein composed of
24-kDa subunits, as determined by SDS-PAGE. In addition, another band
was detected on SDS-PAGE, corresponding to the light chain of the
enzyme(28) . The molecular mass of 200 kDa, as we determined
for the native cathepsin C by gel filtration, is in good agreement with
these results, as well as the molecular mass of 23 kDa and <10 kDa
of the subunits, determined by SDS-PAGE under nonreducing conditions (Fig. 1B). However, three different components with
molecular masses 23, 16, and <10 kDa were obtained by SDS-PAGE under
reducing conditions, indicating that at least two components are
connected with disulfide bonds. These results are in agreement with
those reported for rat liver cathepsin C(2) . They reported a
molecular weight of 160 kDa with two different kinds of subunits,
present in a 1:1 molar ratio. One of the subunits was found to be a
glycoprotein, which could under denaturing conditions dissociate into
two components with molecular masses of 19-24 kDa and 6 kDa. The
NH
-terminal sequence of the larger component was found to
be very similar to those of rat cathepsins B, H, and L, whereas that of
the smaller component exhibited considerable similarity to those of the
light chains of these cathepsins. The other subunit was also found to
be glycosylated and exhibited a molecular mass of 17 kDa on SDS-PAGE,
but its NH
-terminal amino acid sequence did not show any
similarity to the partial sequences of other cathepsins. Moreover, the
alignment of the NH
-terminal sequences of rat and human
cathepsin C with that of rat procathepsin C (Fig. 3) revealed
that the sequences are highly similar. Furthermore, in the mature
enzyme, the 16- or 17-kDa fragment(2) , which corresponded to
the proregion of the enzyme (Fig. 3), was present, indicating
that a substantial part of the proregion still remains bound in the
mature enzyme. This is further supported by the finding that monomeric
procathepsin C (55 kDa) is only slightly larger than the monomeric form
of the mature enzyme (Fig. 1A) or of the intermediate
of 53 kDa(14) , which presumably corresponded to the monomeric
form. Although this finding is unusual for lysosomal cysteine
proteinases, where only cathepsin H is a slight
exception(41, 42) , it is well known that large parts
of proregions can remain bound to the active form of an enzyme.
Examples are the various serine proteinases from the blood coagulation
cascade(43, 44) . From the results presented here we
conclude that cathepsin C is an oligomeric enzyme, consisting of four
identical subunits, each composed of three different polypeptide
chains.
The kinetics of substrate hydrolysis showed a significant
decrease in reaction rate at high substrate concentration, thus
deviating from the simple Michaelis-Menten kinetics. As further shown,
this can be explained in terms of substrate inhibition. A similar
observation was also made for beef spleen cathepsin C(45) ,
although Heinrich and Fruton used different substrates. This phenomenon
can be explained by cooperativity between the subunits upon substrate
binding. Kinetically, the experimental data could be best interpreted
by the model in which an enzyme molecule can bind up to four substrate
molecules and where only the binary enzyme-substrate complex is active (Fig. SI). This model for substrate inhibition is also
consistent with molecular weight and sequence data, such that each of
the four identical subunits has an active site similar to those of
other cysteine proteinases. Although the arrangement of subunits in the
cathepsin C molecule is unknown, the model suggests that all four
substrate binding sites have equal initial affinities for substrate. A
theoretical model, corresponding to the tetrahedral arrangement of
subunits (37) gave a slightly better result than the
corresponding models for the linear and square geometry of the
subunits. All attempts to fit other models, including the simple
Adair-Pauling (39) and Koshland (36) models, resulted
in substantially larger error and significant
deviations from the experimental data. As shown in Table 1,
binding of the first substrate molecule to any of the four binding
sites has little effect on the affinity for the second substrate
molecule. Increasing substrate concentration, however, enables an
enzyme molecule to bind additional substrate molecules. Binding of the
second substrate molecule seems not to be productive and, in addition,
it reduces the catalytic activity at the first site (Fig. 4, Table 1). In addition, binding of the second substrate molecule
drastically decreases the binding affinity for the third substrate
molecule, exhibiting a large negative cooperativity effect. In Fig. 5it is seen that, over the range of experimental substrate
concentrations, the enzyme exists predominantly as ES and ES
. Calculations for the population of
enzyme-substrate complexes have been made on the basis of the model in Fig. SI. In contrast, binding of the fourth substrate molecule
is highly favorable, indicating positive cooperativity (Table 1).
As a consequence, the population of ES
is
negligible. However, binding of these additional substrate molecules
has no effect on overall enzyme activity, since it is clear from Fig. 5that the inhibition of activity is largely, if not
entirely, due to formation of ES
.
McGuire etal.(27) observed normal Michaelis-Menten behavior for the hydrolysis of a similar substrate in a similar concentration range, which could be explained by the fact that they were using frozen enzyme, which was shown to irreversibly lose its quaternary structure (Fig. 1A).
The kinetics of
substrate hydrolysis, investigated between pH 3.0 and 8.0, showed
substrate inhibition over the whole pH range (Table 1),
indicating that this phenomenon is not an artifact but a general
feature of this enzyme. From a detailed investigation of the pH
activity profile three pK values of 4.2, 6.6, and
7.7 were determined (Fig. 6). The three-dimensional structure of
cathepsin C is not known, and therefore any assignment of these values
to particular groups of the enzyme would be speculative. Nevertheless,
cathepsin C exhibits considerable sequence homology with other
papain-like cysteine proteinases, including the conserved Cys-25 and
His-159 (papain numbering), which are essential for the catalytic
activity(46) . However, the pK
values for
the formation and decomposition of the thiolate-imidazolium ion pair,
which is the reactive component, are 3.4 and 8.3 in papain or 3.5 and
8.6 in cathepsin B(40, 47) . Therefore it is very
likely that the pK
values we observed belong to
neighboring charged groups, which are involved in the catalytic
activity of cathepsin C. As suggested by Brocklehurst(48) , use
of substrates in the extreme pH regions, where cysteine proteinases are
only slightly active, may well mask the ionization of the reactive ion
pair. Cathepsin C exhibits some additional similarities with lysosomal
cysteine proteinases. It has a pH optimum around 6 (Fig. 6, Table 1), typical for the lysosomal cysteine
proteinases(49, 50) , and is extremely unstable at
neutral pH (2) , a property characteristic of cathepsins B and
L(51, 52) . Substrate-induced inhibition was
demonstrated also at pH values where the enzyme is stable (Table 1), confirming that the inhibition is not associated with
protein instability.
In conclusion, cathepsin C, isolated from human kidneys, exhibits a number of structural similarities with other lysosomal papain-like cysteine proteinases. However, some important differences from other members of the papain family have been shown: (i) cathepsin C is an oligomeric enzyme, consisting of four identical subunits; (ii) each of these subunits is composed of three different polypeptide chains, two of them disulfide-bound; (iii) the kinetics of substrate hydrolysis deviate from Michaelis-Menten kinetics, reflecting both negative and positive cooperative effects of substrate binding between the subunits; (iv) substrate binding leads to inhibition of hydrolysis, observed over the whole pH range of enzyme activity.