Erickson1 1 Department of Biochemistry and Biophysics and2Department of Pathology and Laboratory Medicine, The University of North Carolina, Chapel Hill, NC, USA Testican-1, a secreted p
Trang 1Human proteoglycan testican-1 inhibits the lysosomal cysteine
protease cathepsin L
Jeffrey P Bocock1, Cora-Jean S Edgell2, Henry S Marr2and Ann H Erickson1
1
Department of Biochemistry and Biophysics and2Department of Pathology and Laboratory Medicine, The University of North Carolina, Chapel Hill, NC, USA
Testican-1, a secreted proteoglycan enriched in brain, has a
single thyropin domain that is highly homologous to
domains previously shown to inhibit cysteine proteases We
demonstrate that purified recombinant human testican-1 is a
strong competitive inhibitor of the lysosomal cysteine
pro-tease, cathepsin L, with a Kiof 0.7nM, but it does not inhibit
the structurally related lysosomal cysteine protease
cathep-sin B Testican-1 inhibition of cathepcathep-sin L is independent of
its chondroitin sulfate chains and is effective at both pH 5.5
and 7.2 At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease
to remain active longer, although the rate of proteolysis is reduced These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu
Keywords: cathepsin L; proteoglycan; protease; testican; thyropin
Testican is a proteoglycan first identified in human seminal
plasma [1] The cDNA was subsequently cloned from the
human testis [2], hence the name testican, and from human
vascular endothelial cells [3,4] and mouse brain [5] In both
human and mouse, testican mRNA is prominent in brain
and absent in certain other tissues Two additional human
homologues have been identified, 2 [6] and
testican-3 [7] The amino acid sequences of human and mouse
testican-1 are 94% identical, which argues for a significant
function for this proteoglycan [5]
Testican is a multidomain protein (Fig 1), including
three domains that have homology to inhibitors of three
different classes of proteases An N-terminal region of
testican-1 has been shown to inhibit membrane-type 1
matrix metalloproteinase activation of matrix
metallopro-teinase-2 [7] Adjacent to this domain is a follistatin-like
domain that includes a six-cysteine pattern with similarity to
Kazal domains found in serine protease inhibitors such as
pancreatic secretory trypsin inhibitor [8,9] The next domain
has homology to EF-hands and has been shown to bind
calcium when expressed as an independent domain [10]
Finally, near the C-terminus is a 64-amino acid domain
highly homologous to protein sequences shown to inhibit
cysteine proteases Such protease inhibition domains have
collectively been called thyropins [11] due to their homology
with a domain repeated 11 times in thyroglobulin, a precursor of thyroid hormones [12]
The cysteine protease inhibitory function of thyropin domains was established when a fragment of the class II invariant chain, that is normally part of the major histocompatibility complex (MHC), was isolated from human kidney bound to cathepsin L [13] The class II invariant chain exists in two alternatively spliced forms, p31 and p41 The latter form has a region which shares significant homology with the thyropin domain of thyro-globulin This domain of the p41 invariant chain was shown
to inhibit cathepsin L and to stabilize the active protease at
a pH which would normally denature the enzyme [13] Crystallography of this p41 domain complexed with cath-epsin L revealed that the domain assumes a wedge-shape conformation comprised of three loops stabilized by three disulfide bonds and is lodged in the active site of cathepsin L [14] Saxiphilin, a bullfrog serum protein that binds a neurotoxin [15], and equistatin, from a sea anemone [16], also have one or more thyropin domains Like p41, these proteins inhibit cathepsin L proteolytic activity [15,16], but
a mammalian proteoglycan has not been demonstrated to serve this role
Cathepsin L is a ubiquitously expressed protease that is normally efficiently segregated into lysosomes, where low
pH allows for optimal activity [17] When expression levels are increased, however, either during specific developmental stages, by cell transformation, or by ectopic expression from
a transfected plasmid, the proenzyme is secreted in signifi-cant amounts [18,19] In response to signaling events, active enzyme can also be released [20,21] In addition to mediating housekeeping proteolysis in the lysosome, the protease participates in developmental processes and anti-gen processing [22–24] Many studies also implicate extra-cellular cathepsin L in tumor biology [23,25], where the major role ascribed to secreted lysosomal proteases is degradation of extracellular matrix [26–30]
Correspondence to A H Erickson, Department of Biochemistry and
Biophysics, CB 7260, Mary Ellen Jones Building, The University of
North Carolina, Chapel Hill, NC 27599–7260, USA.
Fax: + 1 919 966 2852, Tel.: +1 919 966 4694,
E-mail: ann_erickson@med.unc.edu
Abbreviations: BCIP, 5-bromo-4-chloro-3-indolylphosphate; MHC,
major histocompatibility complex; HEK 293, human embryonic
kidney (cells).
(Received 18 June 2003, revised 31 July 2003,
accepted 12 August 2003)
Trang 2Little is known about the function of testicans We have
determined that testican-1, which includes a thyropin
domain, is a competitive inhibitor of cathepsin L but not
of the related cysteine protease cathepsin B Inhibition is
independent of the chondroitinase ABC-sensitive
glycos-aminoglycan chains associated with this proteoglycan This
establishes a new role for testican-1 and provides the first
evidence that the protein backbone of a proteoglycan can
regulate lysosomal protease activity, thus expanding our
understanding of the role proteoglycans play in modulating
extracellular events
Experimental procedures
Materials
Human embryonic kidney 293 (HEK 293) cells were
obtained from ATCC (Manassas, VA, USA) Alkaline
phosphatase-conjugated goat antibodies to mouse
immu-noglobulins were purchased from Jackson
Immuno-Research, and mouse monoclonal antibodies specific for
the Myc epitope tag, Lipofectamine and Geneticin were
obtained from Invitrogen Centriprep concentrators were
from Millipore and Ni-nitrilotriacetic acid agarose was from
Qiagen Rainbow molecular mass markers were from
Amersham and Gelcode Blue Staining Reagent was
purchased from Pierce (Rockford, IL, USA)
5-Bromo-4-chloro-3-indolylphosphate (BCIP)/nitro blue tetrazolium
Color Development Substrate was obtained from Promega
Z-Phe-Arg-4-methyl-7-coumarin (Z-Phe-Arg-NHMec), E64
and Chondroitinase ABC were from Sigma-Aldrich
Fluo-trac 96-well microtiter plates were from Greiner Bio-One
(Longwood, FL, USA) Human cathepsin L, purified from
liver, was obtained from Athens Research, (Athens, GA,
USA) and human cathepsin B, purified from liver, was
from Calbiochem
Recombinant testican-1
A complete open reading frame cDNA for human
testican-1 less its last amino acid was assembled from several cDNA
clones and inserted between EcoRV and XhoI sites in the
Invitrogen expression plasmid, pcDNA3.1/MycHis, keep-ing the Myc epitope tag and the His6encoding DNA from the vector in frame at the 3¢-end of the testican-1 open reading frame The plasmid construct was cloned in Escherichia coliDH5a, and the intended cDNA insert was verified by sequencing This plasmid was transfected into HEK 293 cells using Lipofectamine according to the manufacturer’s recommendations Cells that had incorpor-ated plasmid DNA were selected in the presence of Geneticin at 250 lgÆmL)1 Expression of the recombinant gene was indicated by detecting the Myc epitope in culture fluid from the Geneticin-resistant cells by ELISA
Chondroitinase ABC treatment and purification
of recombinant testican-1 Conditioned Opti-MEM culture fluid was collected after
24 h from 810 cm2of confluent HEK 293 cells expressing recombinant testican-1 After pelleting cellular debris, the conditioned culture fluid was concentrated to 1 mL using a Centriprep concentrator designed to retain molecules larger than 10 kDa Half of the concentrated culture fluid was adjusted to basic pH by addition of pH 8 Tris/HCl to
40 mM and sodium acetate to 40 mM and treated with Chondroitinase ABC at 2 UÆmL)1 for 40 min at 37C Recombinant testican-1 was then purified by His6binding and elution from Ni-nitrilotriacetic acid agarose, as recom-mended by the manufacturer For molecular mass analyses, samples were reduced and denatured in the presence of
1 mMdithiothreitol and 2% SDS at 100C for 5 min and then resolved by standard PAGE using 12% acrylamide with 0.1% SDS Most of the full-length recombinant testican-1 expressed by HEK 293 cells possessed significant amounts of chondroitin sulfate that prevented the majority
of the protein from entering a 12% polyacrylamide gel Treatment with chondroitinase ABC reduced the effective mass, enabling the use of polyacrylamide gels stained with Gelcode Blue Staining Reagent to assess the purity of the recombinant protein isolated by Ni-nitrilotriacetic acid-affinity chromatography The size of the testican-Myc-His6 product was determined by probing gel blots with monoclonal antibodies specific for the recombinant, using
Fig 1 Alignment of the cathepsin-inhibitory domain of mouse p41 invariant chain with homologous domains of mouse and human testican-1 Identical residues are shown on a shaded background The location of the thyropin domain within testican-1 is illustrated relative to the other known domains of testican-1 (not drawn to scale) Residues 1–21 comprise the signal peptide [45] The following domain (residues 25–84) is unique to the three testicans This region of testican-1 is responsible for the inhibition of a membrane-type metalloproteinase [7] Residues 86–183 have similarity
to follistatin domains [55], with a six cysteine Kazal-like sequence Residues 197–312 comprise an extracellular calcium-binding (EC) module [10] Thyropin domain homology occurs between residues 310 and 379 [11], comprised of exons 9 and 10 Following the thyropin domain is a region enriched for acidic residues Twelve of the 13 amino acids within five amino acids of the C-terminus are negatively charged The serines at 383 and
388 in this domain may have chondroitin or heparan sulfate attached [1], which is designated here as GAG for glycosaminoglycans.
Trang 3alkaline phosphatase-conjugated goat antibodies to mouse
immunoglobulins as the secondary antibody, and localizing
the bound alkaline phosphatase activity as a blue
precipi-tate using BCIP/nitro blue tetrazolium Color Development
Substrate as recommended by the manufacturer The
protein concentrations were determined using Bio-Rad
Protein Assay reagent 500–006 in a microtiter plate assay
using bovine serum albumin for the standard curve
Cathepsin L active site titration
Cathepsin L was diluted in buffer consisting of 340 mM
sodium acetate pH 5.5 and 1 mMEDTA, and incubated on
ice for 5 min with 5 mM dithiothreitol to activate the
enzyme [15] The concentration of active cathepsin L in
the preparation used for these studies was determined by
titration with increasing amounts of the stoichiometric
inhibitor, E64, at a constant Z-Phe-Arg-NHMec substrate
concentration of 6 lM [31] Liberated fluorophore was
detected by excitation at 355 nm and emission at 460 nm
using a fluorescence microplate reader andFLUOSTAR2000
analysis software from BMG Labtechnologies (Durham,
NC, USA)
Testican-1 inhibition of cathepsin L
Cathepsin L was preactivated in the same buffer utilized for
active site titration, as described above Active cathepsin L
(0.2 nM) and varying concentrations (423 pM)100 nM) of
recombinant testican-1 were incubated at room temperature
for 20 min to allow for complex formation The
tempera-ture was reduced to 0C to synchronize the reactions and
substrate was added to 6 lM Reaction mixtures were then
incubated at 30C for 10 min The substrate conversion
was monitored as described above The effect of testican-1
on cathepsin B was similarly assayed at a final enzyme
concentration of 2 nM in a reaction buffer consisting of
50 mM sodium acetate, pH 5.0, 100 mM NaCl, 1 mM
EDTA, 5 mMdithiothreitol, and 6 lMZ-Phe-Arg-NHMec
as substrate [15]
Determination of inhibition constant
Two approaches were used to determine inhibition
con-stants In the first approach, the enzyme and inhibitor were
preincubated in the reaction buffer to allow complex
formation, as above Nonlinear regression analysis of
testican-1 titration data obtained on assay of residual
enzyme activity was used to determine the inhibition
constant Kidue to the tight binding of the protease by the
inhibitor and the possibility of modification of the inhibitor
by the enzyme [32] These data were fitted to the theoretical
equation for competitive inhibition:
0ÞþðI0ÞþKif½ðE0ÞþðI0ÞþKi24ðE0ÞðI0Þg1=2
2ðE0Þ where a is the experimentally determined residual
enzyme activity in the presence of inhibitor, E0 is the
initial concentration of enzyme, and I0 is the initial
concentration of inhibitor [33] For these studies,
chondroitinase ABC-treated testican-1 was utilized
because the preparation purity could be assayed readily
by gel electrophoresis
To compare the ability of testican-1 to inhibit cathepsin L
at pH 5.5 and 7.2, an alternative method for determination
of inhibition constants was necessary to avoid cathepsin L inactivation that would occur during a preincubation at neutral pH The reactions were initiated by addition of cathepsin L to 0.2 nM into buffer containing a final concentration of 5 mM dithiothreitol, varying concentra-tions of Z-Phe-Arg-NHMec, and varying concentraconcentra-tions of testican-1 To make it possible to detect any change in affinity should the enzyme be allosteric, we chose to emphasize substrate concentrations below the Km [34] The pH 7.2 buffer was 50 mMsodium phosphate pH 7.2,
100 mM NaCl and 1 mM EDTA [15] Reactions were monitored fluorometrically every 20 s for up to 20 min A lag phase up to 100 s was observed to be required for the enzyme to react completely with dithiothreitol and the reaction mixture to warm to assay temperature The subsequent linear region of each curve was utilized to create the Lineweaver–Burk plots Ki was determined as the x-intercept of a plot of the slopes of these lines vs inhibitor concentration [34]
Results
Testican-1 purity Recombinant testican-1 purified by His6affinity chromato-graphy from the conditioned culture fluid of transfected HEK 293 cells before and after treatment with chondroi-tinase ABC was resolved by SDS/PAGE and visualized by Coomassie staining and immunoblotting (Fig 2 insert) The most abundant proteins in the conditioned culture fluid (lane 1) were absent after Ni-nitrilotriacetic acid affinity chromatography (lane 2) Testican-1 purified after chond-roitinase treatment migrated with a relative molecular mass
of 50–60 kDa and was shown to contain the Myc epitope by the Western blot The mass is consistent with that expected for the recombinant polypeptide less its signal sequence (51 kDa), plus varying amounts of O-linked oligosaccharide that has been reported to be attached in the calcium binding domain [10] Testican-1 purified before chond-roitinase ABC treatment had the same protein profile but was less intense (data not shown) Treatment with chond-roitinase increased the amount of protein entering the gel by 2.6-fold, indicating that at least 60% of the testican had chondroitin sulfate chains removed by the chondroitinase treatment
Cathepsin L is inhibited by testican-1
To determine whether purified testican-1 could inhibit cathepsin L proteolytic activity, the enzyme was preincu-bated with various concentrations of recombinant testican-1
to allow complex formation prior to assay for cleavage of a synthetic peptide substrate Greater than 50% of cathep-sin L activity was lost at an inhibitor to enzyme ratio of
2 : 1, while nearly 80% was lost at a 10 : 1 ratio (Fig 2) This dramatic decrease in enzyme activity at low concentra-tions of inhibitor indicates that inhibitor binding is tight
To determine whether the inhibition of cathepsin L was
Trang 4affected by chondroitin sulfate chains on testican-1,
cath-epsin L activity was also assayed in the presence of
testican-1 that had not been treated with chondroitinase ABC prior
to purification There was no change in the efficiency of
cathepsin L inhibition (Fig 2), indicating that chondroitin
sulfate associated with testican-1 does not mediate or
prevent the inhibition of cathepsin L
The inhibition constant, Ki, at pH 5.5 was determined to
be 0.7nM using nonlinear regression analysis of enzyme
activity remaining after cathepsin L had been preincubated
with testican-1 to allow enzyme-inhibitor complexes to
form The data fit the theoretical equation for competitive
inhibition [33] with an R2value of greater than 0.9 The Km
at pH 5.5 was calculated to be 8.5 lM, which is consistent
with the reported value of 7 lM [31] for this substrate,
although others have reported a lower Km[35]
Testican-1 does not inhibit cathepsin B
Certain thyropin domain-containing proteins have been
found to inhibit the endopeptidase activity of cysteine
proteases other than cathepsin L [15,16] Therefore, to
determine whether testican-1 could inhibit cathepsin B, the
enzyme was assayed at 2 nMin the presence of up to 200 nM
testican-1 The mean residual activity for cathepsin B was
91.8 ± 8.9%, n¼ 34 Thus, no significant inhibition of
cathepsin B by testican-1 was observed
Testican-1 inhibition of cathepsin L is competitive The thyropins thus far characterized have been found to act
as competitive inhibitors of cathepsin L [15,16,36], consis-tent with detection by X-ray crystallography of the p41 thyropin domain in the active site of cathepsin L [14] To confirm that testican-1 is a competitive inhibitor of cathep-sin L, kinetic assays were performed to measure the rate of cleavage of varying concentrations of substrate in the presence and absence of testican-1 The intersection of the Lineweaver–Burk plots on the y-axis above the origin indicates that cathepsin L is competitively inhibited by testican-1 at pH 5.5 (Fig 3A)
Testican-1 inhibition of cathepsin L at neutral pH Although lysosomal enzymes are assayed commonly at
pH 5.5, where the enzymes are most stable, we also assayed cathepsin L inhibition by testican-1 near neutral pH, as
Fig 3 Testican-1 is a competitive inhibitor of cathepsin L at pH 5.5 and pH 7.2 Testican-1 was added at the indicated concentrations to cathepsin L incubated at pH 5.5 or at pH 7.2 with concentrations of Z-Phe-Arg-NHMec between 154 n M and 7.7 l M The Lineweaver– Burk plots show the lines representing reactions at different testican-1 concentrations that all intercept at the y-axis, as expected for com-petitive inhibition The error bars represent the standard deviation of
at least three replicates Obvious outliers were discarded For each replicate, the reaction velocity was determined from the linear region of the curve as the rate of change of fluorescence over a period of at least
200 s Linear fits for the data at each testican-1 concentration were generated by linear regression, and all R2correlation coefficients were
>0.92.
Fig 2 Testican-1 with or without chondroitin sulfate inhibits
cathep-sin L proteolytic activity Cathepcathep-sin L was incubated with increacathep-sing
amounts of testican-1 in either its native form (m) or following
treat-ment with chondroitinase ABC (n) The inhibitory activity is
expressed as residual activity of the enzyme compared to the control
reaction without testican-1 set as 100% activity Residual activity is
graphed as a function of the molar ratio of testican-1 added to active
cathepsin L present Each point represents the mean of three
cates; error bars represent the standard deviation of each set of
repli-cates and the line represents the theoretical curve fit Testican-1
purified with chondroitin sulfate chains intact and testican-1 purified
after chondroitinase ABC digestion were used at the same protein
concentration in the enzymatic assays shown (Inset) Purification of
recombinant testican-1 A Coomassie-stained SDS/polyacrylamide gel
and a nitrocellulose blot of a parallel gel immunostained for
recom-binant testican-Myc-His 6 show its purification from the culture fluid of
transfected 293 cells The first lanes show the unfractionated culture
fluid The second lanes show testican-1 after treatment with
chond-roitinase ABC and purification by Ni-nitrilotriacetic acid affinity The
migration distances of Rainbow protein molecular mass markers in
these gels are indicated in kDa.
Trang 5testican-1 is a secreted proteoglycan As cathepsin L
denatures rapidly at neutral pH and above [37], data
collection was initiated immediately after addition of
enzyme Lineweaver–Burk plots of the data established that
testican-1 also inhibits cathepsin L competitively at pH 7.2
(Fig 3B) The Kmat pH 7.2 was 1.7 lM The Vmaxwas 385
and 72 fluorescence units RFU per second at pH 5.5 and
7.2, respectively
Ki was determined at both pH values from the
Line-weaver–Burk plots, as described in Experimental
proce-dures, but as such analysis is thought to produce a Kithat is
less accurate than nonlinear regression analysis for
tight-binding inhibitors [33], these values are only presented to
compare testican-1¢s inhibitory activity at pH 5.5 to that at
pH 7.2 The Kivalues derived by this method were 13 nMat
pH 5.5 and 8 nMat pH 7.2 The linear regression fits for
these data had R2values greater than 0.9 Thus, testican-1
is similarly effective at inhibiting cathepsin L at pH 5.5 and
at pH 7.2
Testican-1 enhances cathepsin L stability at neutral pH
At pH 7.2, cathepsin L proteolytic activity in the absence of
testican-1 begins to decline before 10 min (Fig 4),
consis-tent with the measurements of others [38] This is not merely
due to depletion of substrate, as indicated by the progress
curve of the control reaction at pH 5.5 in the absence of
testican-1 (A) When cathepsin L activity was assayed near
neutral pH in the presence of testican-1, the loss of activity
was noticeably slower (B) This increase in enzyme stability
was observed at testican-1 concentrations as low as 5 nM, a
25 : 1 inhibitor-to-enzyme ratio Thus at pH values similar
to those outside cells, the presence of testican-1 allows the
enzyme to remain active longer, at the cost of a reduced rate
of proteolytic activity
Cathepsin L could potentially cleave testican within the
thyropin domain that would thus act as a competitive
substrate, but no change in enzyme velocity was detected
over 20 min, as might be expected were the protease
degrading the inhibitor (Fig 4A) This is unlikely to have
affected our Kidetermination (Fig 2) as these experiments
utilized lMconcentrations of substrate and nM
concentra-tions of testican
Discussion
Testican-1, a secreted proteoglycan with a thyropin domain,
was determined to be a potent competitive inhibitor of the
lysosomal cysteine protease, cathepsin L At pH 5.5, the
physiological pH for a lysosomal enzyme, the proteoglycan
inhibited the enzyme with a Ki of 0.7nM Using an
alternative method, we also demonstrated that testican-1
was similarly effective as an inhibitor of cathepsin L at
pH 5.5 and at pH 7.2 The affinity of testican-1 for
cathepsin L is similar to that observed for another
physio-logical inhibitor, cystatin B [39], but is significantly lower
than the affinity of the isolated thyropin domain of the p41
form of the MHC invariant chain for cathepsin L, which
has a Kiof 1.7· 10)3nM[36] Proteins containing thyropin
domains have been found to inhibit a variety of
papain-related cysteine proteases with Ki values in the low
picomolar to low nanomolar range [15]
Testican-1 is unusual in having multiple specific protease inhibitor activities within a single polypeptide We show that testican-1 inhibits the cysteine protease cathepsin L, while the N-terminal domain unique to testicans 1–3 has been shown to inhibit pro-matrix metalloproteinase-2 activation
by membrane-type 1 and 3 matrix metalloproteinases [7] In addition, the protein contains a domain homologous to inhibitors of a third family of proteases, the serine proteases Another human gene family with recognizable homologies
to multiple specific protease inhibitors in a single protein has been recognized recently by data bank homology searches and one of the domains similar to Kunitz-type protease inhibitors has been shown to inhibit trypsin [40–42] Cathepsin L inhibition is a novel activity for the protein core of proteoglycans and thus expands our appreciation of the regulatory role of these molecules The multidomain structure characteristic of proteoglycans enables them to interact with various molecules including growth factors, cell adhesion proteins, and other extracellular matrix components
Fig 4 Testican-1 increases the stability of cathepsin L at neutral pH Cathepsin L at 0.2 n M was incubated at 30 C with 350 n M Z-Phe-Arg-NHMec at pH 5.5 (A) and at 7.2 (B) without testican-1 (d) and with testican-1 at the indicated concentrations (h, e) Each progress curve is representative of four replicate curves at the given conditions.
As fluorescence was measured immediately after the enzyme was added to the dithiothreitol-containing reaction mixture on ice, the initial lag period represents the time required for the enzyme to react with dithiothreitol and the reaction mixture to reach 30 C The arrow indicates the time by which human cathepsin L was previously shown
to be inactivated when incubated at pH 7at 30 C with the same substrate [38].
Trang 6Testican-1 had no significant inhibitory effect on the
endopeptidase activity of a related lysosomal cysteine
protease, cathepsin B This is consistent with the finding
that the structurally similar p41 thyropin domain inhibits
cathepsin L but does not inhibit cathepsin B [36] Equistatin
[16] and saxiphilin [15] both inhibit cathepsin B, but they
bind with lower affinity than to cathepsin L Cathepsin B
differs from cathepsin L in that it has an additional loop of
approximately 20 amino acids which partially occludes the
active site and thus affects interactions with competitive
inhibitors such as stefins [43]
Our experiments establish that addition of the full-length
testican-1 polypeptide results in inhibition of cathepsin L
activity Specific fragments of the polypeptide can be found
in cerebral spinal fluid [44], blood [45] and human semen [1],
indicating that testican-1 undergoes maturation or
process-ing which might expose, free, or destabilize the thyropin
domain We observed that a preparation containing
primarily proteolytic fragments of recombinant testican-1
also inhibited cathepsin L activity (data not shown) This is
consistent with isolation of only the thyropin domain of p41
with cathepsin L purified from kidney [13] This p41
domain is a competitive inhibitor of cathepsin L after it is
cleaved from the invariant chain by endosomal proteases
[36] The identification in seminal plasma of testican-1
fragments cleaved within the thyropin domain [1] suggests
that this cathepsin L inhibitor can also eventually be
degraded by proteases that may be present in blood [45]
Testican interaction with proteases could be mediated by
the polypeptide backbone of a proteoglycan, by its
glycos-aminoglycans, or by both Two glycosaminoglycan
attach-ment sites are localized near the C-terminus of testican-1,
at Ser residues 383 and 388 [2] Significantly, the two
preparations of testican-1, with and without chondroitin
sulfate, were equally efficient inhibitors of the protease,
suggesting inhibition was mediated by the protein core
and not affected by the large glycosaminoglycan moieties
The high homology of the testican-1 thyropin domain to the
cathepsin L-inhibitory domain of the p41 variant of the
MHC invariant chain is consistent with the conclusion that
cathepsin L inhibition is primarily mediated by protein–
protein interactions
While cathepsin L is an intracellular protease localized
within lysosomes under normal conditions, the protease is
secreted when expression levels are elevated by cell
trans-formation [19,46,47], in response to signaling [19–21], or
during specific developmental stages [48,49] The thyropin
domain in testican-1 could serve merely to reduce the
potentially destructive activity of this secreted cysteine
protease Alternatively, a thyropin domain presented in the
context of a proteoglycan could alter cathepsin L-mediated
proteolysis There are ample reports of extracellular
pro-teolysis ascribed to cathepsin L, however, it has not been
clear how a protease unstable at neutral pH mediates
extracellular proteolysis pH-induced unfolding has been
reported to cause rapid inactivation of mature cathepsin L
[38] While the presence of testican-1 reduced the rate of
enzymatic cleavage of substrate, it also significantly slowed
the expected loss of cathepsin L activity due to denaturation
at neutral pH Thus, our data suggest that testican-1 may
actually stabilize the mature cathepsin L protease, so that its
half-life is increased, although its velocity is reduced
A role for testican-1 in inhibiting, yet also stabilizing, protease activity is completely consistent with the recent findings that the p41 alternatively spliced variant of the MHC invariant chain is not merely an inhibitor of cathepsin L activity but also serves as a chaperone that helps to maintain a pool of active protease in late-endocytic compartments of antigen presenting cells [50] Precedent for this role comes from the observation that coexpression
of p41 with p31 modifies endosomal proteolysis of p31 [51] Cathepsin L activity is also stabilized extracellularly when this p41–enzyme complex is secreted by activated macro-phages [52] Heparin-like glycosaminoglycans have recently been reported to protect human cathepsin B from pH-induced inactivation in vitro [53], while heparan sulfate
on ectodomains of cell membrane proteoglycans shed to wound fluids are known to protect serine proteases from interaction with their endogenous inhibitors, thus modify-ing the proteolytic balance of the fluid [54] This physio-logical modulation of proteolysis primarily depends on protease interactions with the glycosaminoglycans of proteoglycans and does not require specific protein–protein interaction as occurs between testican-1 and cathepsin L Through regulation of testican-1 expression levels, the more specific protein–protein interaction may spatially and temporally control the activity of secreted cathepsin L, allowing the enzyme to serve multiple, specific roles in different tissues
Acknowledgements
We thank Dr Tom Traut for his expert advice on enzyme kinetics, Dr Mike Caplow for helpful suggestions, Susan Jones for assistance with the fluorescence microplate reader, and Dr Mohammad BaSalamah for stimulating the initiation of this study This work was supported in part
by National Institutes of Health RO1 HL55452 to C.-J E and by a University of North Carolina Medical Faculty Award to A E.
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