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Originally published In Press as doi:10.1074/jbc.M412047200 on December 6, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7550-7561, March 4, 2005
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Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-peptidase I in Vitro and in Vivo*

Adam A. Golabek{ddagger}, Marius Walus, Krystyna E. Wisniewski, and Elizabeth Kida

From the New York State Institute for Basic Research in Developmental Disabilities, Department of Developmental Neurobiology, Staten Island, New York 10314

Received for publication, October 25, 2004 , and in revised form, December 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t1/2 of mature enzyme at pH 7.4 increased by ~8-fold in the presence of heparin) and in vivo (~2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tripeptidyl-peptidase I (TPP I,1 CLN2 protein) (EC 3.4.14.9 [EC] ) is the first mammalian representative of a growing family of serine carboxyl peptidases or sedolisins (1). However, unlike other sedolisins that show endopeptidase activity, TPP I acts as an exopeptidase that sequentially removes tripeptides from polypeptides with unsubstituted N terminus (2–4), demonstrating only minor endoprotease activity (5). TPP I is the only hydrolase with tripeptidyl-peptidase activity identified to date in lysosomes of mammalian cells. Cytoplasmic tripeptidyl-peptidase II, another enzyme with tripeptidyl-peptidase activity, shows no structural homology to TPP I and is functionally associated with the proteasome (6).

The increased interest in TPP I in recent years resulted from the fact that mutations in this protein lead to a fatal neurodegenerative lysosomal storage disorder of early childhood, classic late-infantile neuronal ceroid lipofuscinosis (CLN2, Jansky-Bielschowsky disease) (7, 8). This disease has onset at the age of 2–4 years with seizures followed by progressive blindness, dementia, and cerebellar, pyramidal, and extrapyramidal signs and is associated with widespread accumulation of storage material in lysosomes and severe neuronal loss and gliosis in brain tissue. Affected individuals usually die at 8–14 years of age (9, 10).

TPP I expression is developmentally regulated (11, 12). In adults, this enzyme is widely distributed in many tissues and types of cells showing strong immunoreactivity in cells with endocrine function (12). Natural substrates of the enzyme are unknown. However, TPP I degrades in vitro several neuropeptides and peptide hormones including glucagon, substance P, sulfated cholecystokinin-8, angiotensin II and III, and neuromedin B (4, 13, 14, 16). Synthetic amyloid-{beta} peptides 1–42 and 1–28 (14) and subunit c of mitochondrial ATP synthase (14, 17), a proteolipid that constitutes ~85% protein content of the CLN2 storage (18), also are degraded in vitro. Given that TPP I releases Gly-Pro-X triplets from synthetic collagen-like polymers and that an inhibitor of TPP I inhibits osteoclastic bone resorption in vitro, it was also proposed that TPP I is involved in the degradation of bone collagen (3). Recently, decreased expression of TPP I was found in fibroblasts from lung fibrosis (19), whereas increased TPP I immunoreactivity was disclosed in some neurodegenerative, inflammatory, and lysosomal storage disorders as well as several types of neoplasms (12). Interestingly, from 2- to 17-fold, higher TPP I activity was reported in breast cancer, which correlated positively with the levels of cathepsin D, a known breast cancer biomarker (20). Thus, it appears that TPP I is involved in numerous vital biological processes and its activity is critical for the proper function and survival of the neuronal cells in the central nervous system.

Human TPP I cDNA encodes a preproenzyme of 563 amino acid residues, which includes a 19-amino acid signal peptide cleaved off cotranslationally, a 176-amino acid prodomain removed during the maturation process, and a mature enzyme of 368 amino acid residues (7, 21–23). The enzyme utilizes in vivo all five potential N-glycosylation sites, and N-glycans ensure proper folding, stability, and transport of TPP I to lysosomes (24). The cleavage of TPP I prodomain in vivo takes place in lysosomes and appears to be facilitated by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride-sensitive serine protease (23). Similar to many other proteases, TPP I is also able to autoactivate in acidic pH in vitro (22), which as we have shown recently is accomplished through an intramolecular (in cis) mechanism (25). Autoprocessing of pro-TPP I in vitro and generation of the mature enzyme can be carried out in a broad range of pH values up to 6.2 (depending on the type of buffer used). However, the acquisition of enzymatic activity in vitro proceeds in a narrow pH range (usually 3.6–4.2) and the mature, inactive enzyme generated during pro-TPP I autoactivation at higher pH (4.5 and above) contains N-terminal extensions starting at 6 and 14 amino acid residues upstream of the prosegment/mature enzyme junction. All of these data suggested that a lysosomal milieu with pH values usually in the range of 4.5–5.5 (26–30) does not favor the generation of mature, active TPP I by autoactivation in cis of pro-TPP I, implying that proteolytic cleavage of pro-TPP I by another protease could predominate in vivo.

To further explore this issue, we conducted experiments to determine whether factors other than transproteolysis by another protease might facilitate the activation of human pro-TPP I. Here, we report that physiologically relevant factors, namely ionic strength and negatively charged compounds (glycosaminoglycans or GAGs), modulate the activation and activity of TPP I both in vitro and in vivo. We also demonstrate that binding to GAGs improves the thermal stability of TPP I and significantly protects the enzyme against alkaline pH-induced denaturation. These data show a potent influence of GAGs on the biology of TPP I and also suggest that human enzyme can utilize in vivo both in cis and in trans mechanisms of activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Heparin (sodium salt, Mr 6,000–30,000 and 3,000), dextran sulfate (Mr 8,000, 60,000, and 500,000), heparan sulfate (from bovine kidney), dermatan sulfate (chondroitin sulfate B from porcine intestinal mucosa), and poly-L-lysine (Mr 14,400) were purchased from Sigma. Cell culture components were from Invitrogen. Western blotting reagents were purchased from Bio-Rad and Amersham Biosciences. Protease inhibitor mixture (Complete) and FuGENE 6 transfection reagent were from Roche Applied Science. The endoproteolytic substrate peptide MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Arg-Leu-Lys-(Dnp)-r-NH2 (peptide 1) (5) was provided by Genemed Synthesis. Monoclonal antibodies (mAbs 8C4 and 2E12), which we raised to human recombinant TPP I expressed and purified from Escherichia coli, were described previously (12, 23). All other chemicals were from Sigma.

Purification of Human pro-TPP I—Human pro-TPP I was purified from serum-free secretions of CHO-DHFRneg cells (ATCC CRL-9096) stably transfected with plasmid encoding the full-length human pro-TPP I as described earlier (25).

SDS-PAGE and Western Blotting—Samples were solubilized in the reducing sample buffer, boiled for 5 min, and loaded onto 10% Tris/Tricine SDS-PAGE. Electrophoretically separated proteins were electrotransferred onto nitrocellulose membranes. Membranes were subsequently blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.05% Tween 20, incubated overnight with mAb anti-TPP I, washed extensively in PBS with 0.05% Tween 20, incubated with peroxidase-conjugated secondary antibodies diluted 1:5,000, and developed by using the ECL.

Time-course Analysis of Pro-TPP I Activation and Processing—Unless otherwise indicated, 2x concentrated stock solutions of purified pro-TPP I at the concentration specified in the figure legends (final concentrations) were prepared in 2 mM Tris, pH 8.0, 0.2% Triton X-100, mixed at 0 time with an equal volume of 200 mM sodium acetate (activation buffer), and maintained either at room temperature (25 °C) or 37 °C. At the indicated time, an aliquot of 10 µl was withdrawn and combined with either 10 µl of 0.5 M NaOH (for Western blot analysis) or 40 µl of 200 mM sodium acetate, pH 5.0 (for TPP I activity measurement).

TPP I Activity Measurement—Unless otherwise stated, TPP I activity was measured in the presence of 100 µM substrate Ala-Ala-Pheaminomethylcoumarin (AAF-AMC), 0.1% Triton X-100, 0.1 M sodium acetate, pH 5.0, in a total volume of 100 µl. TPP I activity measurement at pH 5.0 was selected to ensure that no further activation took place while TPP I activity was assessed (25). Reaction was carried out at 37 °C for 20 min and was terminated by the addition of 50 µl of 10% SDS. Released 7-amino-4-methylcoumarin was measured fluorometrically on CytoFluor 2000 (Applied Biosystems) (excitation wavelength 360 nm, emission wavelength 460 nm) after alkalizing the solution by adding 50 µl of 1 M Tris-HCl, pH 9.0.

Determination of Kinetic Parameters—For each kinetic assay, seven serially diluted substrate concentrations were used. Both enzyme and substrate were prewarmed at 37 °C for 5 min, were mixed afterward, and incubated for 10 min. The reaction was terminated thereafter, as above. The linear rate of fluorescence change was measured to give the initial velocity (V). The Km and Vmax values were calculated by nonlinear regression fit of initial velocity versus substrate to Michaelis-Menten equation with the help of GraphPad Prism software. The kcat values were derived from the equation, Vmax = kcat [E], where [E] is the enzyme concentration.

Heparin-Agarose Affinity Chromatography—TPP I obtained by activation of pro-TPP I (at 2.5 µM) for 2 h at pH 3.5 and 37 °C was diluted 10-fold with the column equilibration buffer at the desired pH and analyzed on a 1-ml HiTrap (Amersham Biosciences) column custom-filled with heparin-agarose resin (Bio-Rad) and used at room temperature at a flow rate of 1 ml/min. Automated method scouting was used to achieve a desired buffer pH by using ÄKTA Explorer high pressure liquid chromatography, Unicorn software (both from Amersham Biosciences), and the following solutions: (a) 0.03 M Na2HPO4, 0.03 M sodium formate, and 0.03 M sodium acetate; (b) 0.1 M HCl; (c) H2O; and (d)2 M NaCl, each containing 0.05% Triton X-100. The pH of the buffer was verified after each run by manual measurement. After a wash, a linear NaCl gradient (0–1 M) over 20 min was used to elute the retained protein. Collected fractions (0.8 ml each) were monitored for TPP I enzymatic activity upon AAF-AMC substrate.

Cell Culture and Transfection—Control Chinese hamster ovary cells (CHO K1, ATCC CCL-61) and CHO cells deficient in xylosyltransferase 1 (pgsA-745, ATCC CRL-2242) were maintained in Ham's F-12 growth medium supplemented by 10% fetal calf serum, 2 mM glutamine, and antibiotics at 37 °C in a humidified atmosphere with 5% CO2. One day before transfection, cells were seeded on 35-mm culture dishes. Cells were transfected with the cDNA encoding wild-type human pro-TPP I cloned into the KpnI/NotI site of pcDNA3.1Hygro as described earlier (23) by using the FuGENE 6 transfection reagent according to the manufacturer's recommendation. 72 h after transfections, bafilomycin A1 was added to the culture media for 3 and 6 h (final concentration 100 nM) and then cells were lysed in 1% Triton X-100, 50 mM Tris, pH 7.4, and protease inhibitor mixture (Complete) supplemented with pepstatin A (lysis buffer) and TPP I was assessed by using Western blotting and enzymatic activity assay.

TPP I Uptake Experiments—For standard uptake assays, confluent CHO K1 and CHO pgsA-745 cells plated in 35-mm Petri dishes were rinsed twice with 2 ml of F-12 medium and then were maintained for 4 and 24 h at 37 °C in serum-free medium (Opti-MEM I) containing 10 µg/ml human pro-TPP I. Afterward, cells were washed in PBS, harvested with trypsin/EDTA solution to remove cell membrane-associated pro-TPP I, centrifuged twice at 2,000 x g for 2 min (first in full medium and then in PBS), and lysed as above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Effect of Ionic Strength on Autoactivation of Pro-TPP I—It was demonstrated that, during the activation of numerous zymogens including pepsinogen, procongopain, and yeast proteinase A, the reaction rate is a function of ionic strength (31–33). Although the theoretical isoelectric point (pI) of TPP I proenzyme (~5.91) is similar to the predicted pI of the prosegment (~6.12, excluding 19-amino acid signal peptide), the charge density of the prosegment polypeptide is distinctly higher than that of the mature TPP I. Thus, similarly to other lysosomal proenzymes, multiple salt bridges between the prosegment and the mature enzyme might form, stabilizing the prosegment-mature enzyme complex in its zymogen form. To study the role of ionic interactions in stabilization of the prosegment-mature enzyme complex in its zymogen form, we first analyzed the autoactivation of the pro-TPP I in the presence of NaCl at 0, 150, or 1,000 mM in the pH range of 3.5–6.0 for 15 and 120 min. As illustrated in Fig. 1, pro-TPP I was fully activated at pH 3.5 after only 15 min of activation in 50 mM sodium acetate buffer, independent of NaCl concentration. In the presence of 1 M NaCl (but not 150 mM NaCl), significant activation also was observed at pH 4.0 (Fig. 1, left panel). After 120 min of incubation, the activation of pro-TPP I in vitro at pH 4.0 was still very limited without the addition of salt to the reaction buffer (Fig. 1, right panel). However, at this pH, the enzyme was fully activated in the presence of 1 M NaCl and significantly activated in the presence of 150 mM NaCl. Under the experimental conditions employed, no pro-TPP I activation was observed at pH 4.5 and above, irrespective of NaCl concentration. These data, in agreement with our earlier observations (25), demonstrate that pro-TPP I activation in vitro takes place in a narrow pH range and show that, although a high ionic strength environment can enhance the activation at pH 4.0, it cannot initiate activation at pH higher than that.



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  		FIG. 1.
The effect of ionic strength on autoactivation of pro-TPP I in vitro. Pro-TPP I at 12.5 nM was activated at 37 °C in 0.1% Triton X-100, 100 mM sodium acetate at pH 3.5–6.0, and NaCl concentrations of 0, 150, or 1,000 mM for either 15 (left panel) or 120 min (right panel), and TPP I activity was measured at pH 5.0.

 
Activation of pro-TPP I in vitro is characterized by exponential kinetics at pH 3.5 and sigmoidal kinetics at pH 4.0 (25). To determine whether ionic strength can affect the kinetics of pro-TPP I activation, we analyzed in greater details the activation of pro-TPP I as a function of time and NaCl concentration by using a discontinuous method. As depicted in Fig. 2 (left panel), the kinetics of pro-TPP I autoactivation at pH 3.5 only slightly inversely correlated with salt concentration. Although increased NaCl concentrations somewhat reduced the rates of activation (which might have resulted from the direct effect of NaCl on TPP I activity (see below)), the overall yield of active TPP I was not dependent on ionic strength. In contrast, the rates of pro-TPP I activation at pH 4.0 were independent of the ionic strength of the buffer (Fig. 2, right panel); however, the yield of the active enzyme strongly correlated with salt concentration (Fig. 2, right panel, inset) and NaCl at 1 M led to an ~8-fold increase in the amount of active enzyme generated in comparison to control samples incubated in 100 mM acetate buffer without the addition of NaCl. Interestingly, the sigmoidal activation curve characteristic for pro-TPP I activated at pH 4.0 (25) was preserved even in the highest NaCl concentration. These data suggest that ionic strength affects the yields but not the rates or the mechanism of pro-TPP I activation in vitro.



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  		FIG. 2.
Kinetics of pro-TPP I activation in vitro at pH 3.5 and 4.0 as a function of NaCl concentration. Pro-TPP I was activated in 0.1% Triton X-100 and 50 mM sodium acetate and either pH 3.5 and concentration 12.5 nM at RT (left panel) or pH 4.0 and concentration 25 nM at 37 °C (right panel) in the presence of varying salt concentrations as labeled. At the time points indicated, an aliquot was withdrawn and analyzed for TPP I activity. Data points were fit to either two-phase exponential association equation (left panel) or sigmoidal equation (right panel). Inset shows maximal TPP I activity attained as a function of NaCl concentration. RT, room temperature.

 
The Effect of Ionic Strength on Activity of TPP I—The pH optimum of exopeptidase activity of TPP I toward a reporter substrate (AAF-AMC) is ~5.0 (3, 5). To evaluate whether the ionic strength might directly affect the activity of TPP I, the activity of mature TPP I was assayed in the presence of various NaCl concentrations at pH 3.5, 4.0, and 5.0. As illustrated in Fig. 3, NaCl slightly inhibited TPP I activity. In addition, at any given NaCl concentration, the inhibition of TPP I activity was the highest at pH 5.0 and then at 4.0 and 3.5. However, even at pH 5.0 and 2 M NaCl, there was only a ~22% decrease in TPP I activity in comparison with control samples assayed without NaCl.



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  		FIG. 3.
The effect of NaCl on TPP I activity in vitro. Pro-TPP I at a concentration 12.5 nM was preactivated in 50 mM sodium acetate, pH 3.5, for 2 h at 37 °C. Afterward, serially diluted NaCl at concentrations indicated were added to the reaction mixture for 5 min and TPP I activity was measured at pH 3.5, 4.0, and 5.0.

 
The Effect of GAGs on Pro-TPP I Maturation in Vitro—It was demonstrated that negatively charged compounds, such as dextran sulfate (DS), are able to accelerate the processing of several lysosomal enzymes including cathepsins B and L (34, 35). Hence, we first have tested the effect of the selected model polyanions (DS and heparin) on pro-TPP I maturation in vitro and compared it to the effect of a positively charged compound, poly-L-lysine. To this end, we studied the activation of pro-TPP I incubated for either 15 or 120 min at 37 °C in the absence or presence of tested compounds as a function of pH (3.5–6.0). As illustrated in Fig. 4A, the effect of polyanions on pro-TPP I strongly depended on the pH of the activation solution. At pH 3.5, both heparin and DS inhibited pro-TPP I activation after 15 min but not after 120 min of incubation. However, at pH 4.0 and 4.5, pro-TPP I activation occurred exclusively in the presence of DS and heparin under the experimental conditions used. During incubation of pro-TPP I with DS, an active TPP I was generated up to pH 5.5. The effect of heparin and DS on pro-TPP I activation was not dependent on the molecular size of GAGs tested (data not shown). Incubation with poly-L-lysine had no effect on pro-TPP I activation. Hence, both DS and heparin stimulated significantly the activation of pro-TPP I in vitro, and in their presence, autoactivation of pro-TPP I was possible in pH higher by approximately one unit than that of pro-TPP I activated alone (thus, up to pH 5.0 for TPP I incubated in the presence of heparin and even further away for DS).



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  		FIG. 4.
The effect of polyanions on activation and autoprocessing of pro-TPP I in vitro as a function of pH. Pro-TPP I at 12.5 nM was activated in 50 mM sodium acetate buffer, pH 3.5–6.0, for either 15 min (A and B, left panels) or 120 min (A and B, right panels,), either alone or in the presence of 12.5 µg/ml heparin, 12.5 µg/ml dextran sulfate, or 33 µg/ml poly-L-lysine, and both activity (A) and processing of TPP I (B) were analyzed. Western blot was developed by using mAb 8C4 and ECL. C, pro-TPP I was activated in 50 mM sodium acetate, 0.1% Triton X-100, pH 4.0 and 4.5, either alone or in the presence of heparin or dextran sulfate (both at 12.5 µg/ml) for 2 h at 37°C, and then samples were analyzed on silver nitrate-stained polyacrylamide gels. Bands migrating with an approximate molecular mass of ~12–20 kDa correspond to prosegment fragments (arrows) (25). Please note that the values of activity obtained for TPP I incubated alone and TPP I incubated in the presence of poly-L-lysine at pH 4.0–6.0 in A overlapped; therefore, the symbols for TPP I maintained in the presence of poly-L-lysine are not visible on the graph in A.

 
To see how polyanions affect the processing of pro-TPP I, we analyzed aliquots of samples incubated in the presence or absence of tested polyanions by SDS-PAGE and Western blotting. As illustrated in Fig. 4B, pro-TPP I incubated alone in the sodium acetate buffer was rapidly processed at pH 3.5. Autoprocessing at pH 4.0 was relatively slow, and there was very limited processing at pH 4.5 and none above. The presence of heparin and DS in the incubation buffer led to early generation of mature TPP I at less acidic pH values. In fact, pro-TPP I was fully processed after 15 min of incubation in samples containing DS at pH 5.0 and limited processing was present even up to pH 6.0 after 120 min of incubation. There was a clear disparity between the autoprocessing of pro-TPP I in the presence of DS and acquisition of TPP I activity (i.e. compare the amount of mature TPP I in the presence of DS at pH 5.0 at 15 and 120 min in Fig. 4B and the activity of TPP I at these time points in Fig. 4A). Also, there was little TPP I activity in pro-TPP I incubated for 120 min in the presence of heparin at pH 5.0 despite evident processing (compare Fig. 4, A and B). We have described previously the delayed acquisition or lack of TPP I activity, even though the proteolytic cleavage of the zymogen was taking place (25). Poly-L-lysine did not affect significantly the processing of pro-TPP I except for its slight stimulation after 15 min of incubation at pH 4.0. Interestingly, in the presence of GAGs in the activation mixture, degradation of the prosegment fragments of pro-TPP I generated during the autoprocessing was distinctly more effective in comparison with pro-TPP I activated alone (Fig. 4C).

To further explore the mechanism of the stimulatory effect of polyanions on pro-TPP I activation, we performed kinetic analyses (Fig. 5). Time-course analysis of pro-TPP I activation at pH 3.5 demonstrated that DS did enhance the earliest step of activation; however, after ~3 min of activation, DS inhibited the activation of pro-TPP I in a dose-specific manner, leading to a switch of the exponential to a linear curve (Fig. 5A). At pH 4.0, DS led to a robust activation of pro-TPP I (Fig. 5B). A similar effect was observed for heparin (data not shown). Poly-L-lysine as well as bovine serum albumin had no effect on activation rates or efficiency of pro-TPP I activation. Interestingly, the presence of polyanions not only led to a higher activation rate and yields of TPP I (~3-fold higher at 25 µg/ml DS) but also to a disappearance of the characteristic lag phase normally observed for TPP I activated at this pH (25). These results demonstrate that, similarly to other lysosomal proenzymes, increased negatively charged density afforded by DS and heparin can accelerate in vitro maturation of pro-TPP I.



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  		FIG. 5.
Time-course analysis of pro-TPP I activation in vitro in the presence of polyanions. Pro-TPP I was activated in 0.1% Triton X-100 and 50 mM sodium acetate, pH 3.5, and room temperature (RT)(A) and pH 4.0 and 37 °C (B) in the absence or presence of dextran sulfate, poly-L-lysine, and bovine serum albumin (BSA) at the concentrations indicated. At the time points labeled, an aliquot was withdrawn and analyzed for TPP I activity. Data points were fit to two-phase exponential association equation (A) or sigmoidal and one-phase exponential equation (B).

 
The Effect of Charged Compounds on TPP I Activity—The shape of the pro-TPP I activation curves in the presence of polyanions suggested that, once TPP I is generated, polyanions have a negative effect on its activity. To verify whether negatively charged compounds can directly affect TPP I activity, the activity of mature TPP I was measured in the presence of low concentration of heparin and DS at various pH values. As demonstrated in Fig. 6A, heparin and DS exerted a moderate inhibition of TPP I activity in almost the entire pH range tested (3.5–5.5) with the exception of pH 6.0 where TPP I alone did not display any activity, whereas there was some residual TPP I activity measured in the presence of DS. To quantitatively assess the inhibitory effect of polyanions on TPP I activity, we measured the enzymatic activity of TPP I in the presence of serially diluted DS and heparin. Unexpectedly, as depicted in Fig. 6B, the plot of TPP I activity versus DS and heparin concentration revealed a concave bell-shaped curve. Initially, as the GAG concentration progressed from 0 to ~100 ng/ml, the activity of TPP I was falling down rapidly to reach a plateau with saturable inhibition at ~100 ng–100 µg/ml and then it gradually increased with increased GAG concentration. The maximal percent inhibition was ~70% by DS and ~50% by heparin. A similarly shaped curve was obtained for endoproteolytic TPP I activity toward a respective substrate (peptide 1) in the presence of DS (data not shown). These data suggest that TPP I possesses two distinct binding sites for polyanions: 1) a high affinity inhibitory site and 2) a low-affinity stimulatory site. Nonlinear regression analysis of the activity data was used to calculate the 50% saturation of each site (IC50) and is presented in the Fig. 6B, inset.



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  		FIG. 6.
The effect of polyanions on TPP I activity in vitro. A, TPP I activity in the absence or presence of either heparin or dextran sulfate (both at 12.5 µg/ml) was measured in 100 mM sodium acetate, 100 mM NaCl, 0.1% Triton X-100, pH 3.5–6.0. B, TPP I activity was measured in the presence of serially diluted dextran sulfate and heparin in 100 mM sodium acetate, 100 mM NaCl, and 0.1% Triton X-100, pH 5.0. The resultant data points were fit to a bell-shaped curve equation allowing calculation of the GAG concentration, giving the 50% saturation of TPP I activity inhibition (IC50) and 50% saturation of the inhibition reversal (IC50R) (inset).

 
Because both NaCl and polyanions enhanced pro-TPP I activation, we next tested whether they acted through the same site on TPP I molecule or whether their effect might have been additive. To this end, pro-TPP I was preincubated either without or with 12.5 µg/ml heparin and NaCl at various concentrations and then was activated for 30 min at 37 °C and pH 4.0. As demonstrated in Fig. 7, both NaCl and heparin apparently bound to the same site on pro-TPP I because, at a very high NaCl concentration, pro-TPP I activation was not dependent on the presence of heparin; however, it took ~2 M NaCl to abolish the effect of ~1 µM heparin.



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  		FIG. 7.
The effect of ionic strength on pro-TPP I activation in vitro in the presence of heparin. Pro-TPP I was activated in the absence or presence of 12.5 µg/ml heparin in 100 mM sodium acetate, 0.1% Triton X-100, pH 4.0, at 37 °C for 30 min in serially diluted NaCl, and the resultant TPP I activity was measured.

 
To quantify kinetically the effect of polyanions on TPP I activity, we measured initial rates of enzymatic activity of TPP I at different substrate concentrations and varying concentrations of DS (Fig. 8). TPP I alone as well as in the presence of DS displayed the Michaelis-Menten characteristics when initial velocities were plotted against substrate concentration (data not shown). The calculated Km and Vmax values were displayed as a function of DS concentration and are presented in Fig. 8A. DS at ~1 µg/ml increased the Vmax of the reaction only slightly, whereas the Km was increased ~10-fold. Hence, at this concentration, DS behaved as an allosteric inhibitor. However, at higher DS concentrations, the Km was not affected significantly, whereas the Vmax of the reaction significantly decreased. Thus, the catalytic efficiency (kcat/Km) of TPP I was mostly restored (Fig. 8B). The presence of 1 M NaCl led to a small increase of the Km and a small decrease of Vmax of TPP I (data not shown).



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  		FIG. 8.
Kinetic analysis of polyanion effect on TPP I activity in vitro. Pro-TPP I at a concentration 12.5 nM was preactivated in 50 mM sodium acetate, pH 3.5, for 2 h at 37 °C. Initial velocity rates were measured for TPP I in the presence of serially diluted dextran sulfate, and the resultant Km and Vmax (A) or catalytic efficiency (kcat/Km)(B) was expressed as the function of DS concentration. C, serially diluted TPP I inhibitor (AAF-CMK) was preincubated for 5 min at room temperature with either TPP I alone or TPP I supplemented with 12.5 µg/ml dextran sulfate, and residual TPP I activity was measured and analyzed by a nonlinear regression equation to calculate IC50.

 
AAF-CMK is a specific TPP I inhibitor (4, 22). To determine whether binding to polyanions might affect the interaction of TPP I with AAF-CMK, we measured the inhibitory activity (IC50, the concentration of the inhibitor required to reach 50% inhibition of TPP I activity) of AAF-CMK in the absence and presence of DS. As illustrated in Fig. 8C, DS at 12.5 µg/ml increases the IC50 of AAF-CMK by ~40%. These data demonstrate that DS does not bind directly to the substrate-binding site of TPP I.

TPP I Binds Heparin-Agarose—To demonstrate directly the binding of TPP I to polyanions, we have used affinity chromatography on heparin-agarose. At pH 3.5, TPP I bound to heparin was eluted from heparin-agarose column at ~0.3 M NaCl (Fig. 9A). This interaction could be specifically disrupted by prior addition of 100 µg/ml heparin to TPP I solution. In addition, TPP I binding to heparin-agarose displayed a strong pH dependence with decreasing binding in increasing pH of the buffer (Fig. 9B), suggesting that histidines rather than arginines/lysines are involved in the interaction of TPP I with GAGs.



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  		FIG. 9.
Binding of TPP I to heparin-agarose. A, TPP I was preincubated either in the absence or presence of 100 µg/ml heparin, and its binding to heparin-agarose was analyzed as described under "Experimental Procedures." B, the pH-dependent affinity of TPP I to heparin-agarose resin was measured as the concentration of NaCl necessary to elute the enzyme from the column at a given pH.

 
The Effects of Glycosaminoglycans on Thermal and Alkaline pH Stability of TPP I—To analyze whether polyanions might structurally stabilize TPP I, we first assessed the effect of heparin on the thermal stability of TPP I. According to the data shown in Fig. 10A, heparin incubated with TPP I in vitro at the temperatures indicated afforded ~10 °C increase in thermal stability of the enzyme.



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  		FIG. 10.
The effect of GAGs on the stability of TPP I. A, the effect of heparin on thermal stability of TPP I. Activated TPP I was incubated either in the absence or the presence of 12.5 µg/ml heparin for 15 min at the indicated temperatures, and residual TPP I activity was measured. B, the effect of heparin on alkaline pH-induced TPP I inactivation. Activated TPP I at a concentration of 12.5 nM was incubated in 50 mM Tris, pH 7.4, 100 mM NaCl either in the absence or presence of 12.5 µg/ml heparin for the indicated periods, and residual TPP I activity was measured. C, CHO K1 and CHO pgsA-45 transiently transfected with wild-type human TPP I were exposed to 100 nM bafilomycin A1 for 0, 3, and 6 h and then lysed. Cell lysates were analyzed by Western blotting with mAb 8C4 (upper panel). Mock-transfected cells were run in parallel (20-µg protein of cell lysate/lane). Densitometry analysis of immunoblots quantifying the levels of mature TPP I in control and untreated cells and cells exposed to bafilomycin A1 is presented in the lower panel. Baf A, bafilomycin A1; M, mock transfectants.

 
It was documented that TPP I purified from bovine brain was losing over 90% activity when stored at pH 7.4 for 14 h at 4 °C and that no enzymatic activity remained when the enzyme was stored at pH >8.0 (14), indicating that mature TPP I is an acidic protease unstable at alkaline pH. Thus, in another set of experiments, we evaluated whether polyanions were able to protect mature TPP I against alkaline pH-induced inactivation. As demonstrated in Fig. 10B, heparin exerted a significant protection against TPP I inactivation in alkaline pH in vitro, extending the half-life of the enzyme incubated at pH 7.4 and 37 °C from ~2.5 to ~21.5 min. These data imply that, upon binding to polyanions, the resistance of TPP I to heat- and alkaline pH-induced denaturation greatly improves.

To test whether GAGs protect TPP I against inactivation in alkaline pH also in living cells, we analyzed the level and specific activity of human TPP I transiently expressed in CHO K1 cells and CHO pgsA-745 cells. Heparan sulfate and chondroitin sulfate are the two major GAG chain types in CHO cells, and wild-type CHO cells produce ~70% heparan sulfate and 30% chondroitin 4-sulfate (36). pgsA-745 CHO cells generated by means of chemical mutagenesis are defective in xylosyltransferase 1, an enzyme involved in the attachment of xylose to a specific serine residue of a core protein, which is the first step in the assembly of most GAGs with the exception of keratan sulfate and hyaluronan (37, 38). As a consequence of xylosyltransferase deficiency, pgsA-745 CHO cells fail to synthesize both chondroitin and heparan sulfate chains. To increase lysosomal pH in living CHO cells, we used bafilomycin A1. Bafilomycin A1, an inhibitor of vacuolar ATPase, leads to an increase in pH in acidic vacuolar compartments including lysosomes and late endosomes that causes degradation of preexisting lysosomal enzymes (39, 40).

Under standard conditions, the level of endogenous TPP I is very low in both CHO K1 and pgsA-745 cells with a weak band visualized on immunoblots only after their longer exposure (see Fig. 12). Therefore, for these experiments, we used cells transiently expressing human pro-TPP I. As shown in Fig. 10C (upper and lower panels), bafilomycin A1 treatment reduced the level of mature TPP I more markedly in pgsA-745 than in CHO K1 cells at both time points analyzed. According to densitometry analysis of immunoblots, the levels of mature TPP I in CHO K1 cells were lower by around 22% after 3 h and around 48% after 6 h of bafilomycin A1 treatment, whereas the reduction of mature TPP I levels was ~50 and ~90% after 3 and 6 h of bafilomycin A1 treatment, respectively, in pgsA-745 mutant cells. The amounts of pro-TPP I after bafilomycin A1 treatment were increased in both pgsA-745 and CHO K1 cells, a finding reflecting mostly altered intracellular transport of proenzymes to lysosomes and altered processing of proenzymes in alkalized endosomal/lysosomal compartments (40–42). These findings indicate that GAGs assist in the protection of mature TPP I against alkaline pH-induced inactivation and/or degradation not only in vitro but also in living cells.



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  		FIG. 12.
The effect of GAGs on TPP I activity in vivo. A, CHO K1 and CHO pgsA-745 cells were maintained in serum-free medium supplemented with 10 µg/ml pro-TPP I for 0, 3, and 24 h, trypsinized, lysed, and analyzed by Western blotting with mAb 8C4 and ECL (40-µg protein of cell lysate/lane) (upper panel). The specific TPP I activity was measured in each cell lysate, mock values were subtracted, and the values obtained were adjusted according to the TPP I band density and expressed as relative activity (lower panel). B, CHO K1 and CHO pgsA-745 cells transiently transfected with wild-type human TPP I and vector only were lysed 72 h posttransfection and analyzed by Western blotting with mAb 8C4 and ECL (20-µg protein of cell lysate/lane) (upper panel), or the specific TPP I activity was measured, mock values were subtracted, and the values obtained were adjusted according to the TPP I band density and expressed as relative activity (lower panel). K1, CHO K1 cells; 745, CHO pgsA-745 cells; wt hTPP I, wild-type human TPP I.

 
The Effect of Heparan Sulfate and Dermatan Sulfate on Activation and Activity of TPP I—Finally, we investigated the activation and activity of TPP I in the presence of heparan sulfate and dermatan sulfate (chondroitin sulfate B). In contrast to heparin, which is localized to storage granules of mast cells, heparan sulfate and dermatan sulfate are found in various types of cells and tissues both intracellularly (including endosomal/lysosomal compartments) at the plasma membrane and in the extracellular matrix (38, 43–45). As shown in Fig. 11, A–C, under in vitro conditions, both heparan sulfate and dermatan sulfate stimulated the activation of pro-TPP I (Fig. 11A) and inhibited the activity of the mature enzyme in a dose-dependent manner (Fig. 11B). The maximal percent inhibition was ~60% by dermatan sulfate and ~40% by heparan sulfate at the highest concentration of tested GAGs (100 µg/ml). Similar to what we observed for DS, both of the tested GAGs also allowed for effective pro-TPP I activation in less acidic pH in a concentration-dependent manner. Lower doses of GAGs (up to 25 µg/ml) allowed for pro-TPP I autoactivation at the pH up to 4.5 (data not shown). However, in the presence of 50 µg/ml heparan sulfate, effective autoactivation of pro-TPP I proceeded even at pH 6.0 (Fig. 11C).



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  		FIG. 11.
The effect of dermatan sulfate and heparan sulfate on autoactivation and activity of TPP I in vitro. A, dermatan sulfate and heparan sulfate at the concentrations indicated were incubated with pro-TPP I in 50 mM sodium acetate, 50 mM NaCl, 0.1% Triton X-100, pH 4.0, at 37 °C for 2 h, and the resultant TPP I activity was measured. B, preactivated TPP I was incubated for 10 min at room temperature with dermatan sulfate and heparan sulfate at the concentrations indicated, and residual TPP I activity was measured. C, 50 µg/ml dermatan sulfate and heparan sulfate were incubated with pro-TPP I in 100 mM sodium acetate, 0.1% Triton X-100 at 37 °C for 2 h at the pH indicated, and the resultant TPP I activity was measured.

 
To verify whether tested GAGs also affect TPP I activity in living cells, we compared TPP I activity in pgsA-745 cells that are deficient in heparan sulfate and chondroitin sulfate with that in wild-type CHO K1 cells. For these experiments, cells were either transiently transfected with human pro-TPP I and analyzed 72 h posttransfections or were maintained for 3 and 24 h in the culture medium supplemented with human pro-TPP I. Specific TPP I activity was measured afterward in cell lysates, and the values obtained were adjusted to the levels of TPP I polypeptide revealed by densitometry scanning of immunoblots and then presented as relative TPP I activity. As shown in Fig. 12, the relative TPP I activity was distinctly higher (26–54%) in cells deficient in GAGs than in control cells (Fig. 12, A and B, lower panels). These data demonstrate that, similar to what we found in vitro, GAGs also inhibit the activity of TPP I in vivo. Please note that the levels of endocytosed TPP I polypeptides are lower in pgsA-745 mutant cells than in CHO K1 cells (Fig. 12A, upper panel). This observation suggests that, although endocytosis and transport of TPP I to lysosomes depend on mannose 6-phosphate receptors (23, 46), GAGs in some way facilitate this process. This possibility, pointing to a potential new role of GAGs in the trafficking of even those lysosomal hydrolases that utilize mannose 6-phosphate receptors, must be verified by further experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Given that the values of the lysosomal pH most commonly reported are in the range of 4.5–5.5 (26–30), our previous studies suggested that lysosomal milieu could not favor effective autoactivation of pro-TPP I and its conversion to an active enzyme in vivo or that this process could be restricted to those cells that are able to acidify lysosomal milieu more prominently (47, 48). In this respect, the effect of ionic strength and GAGs on TPP I activation we demonstrate at present bears high physiological significance. Because of their high apparent stimulatory activity at a low physiologic concentration, especially GAGs may play an important role in the biology of TPP I. As we have evidenced, polyanionic compounds not only are able to markedly enhance the yields and rates of pro-TPP activation, but what may be especially important, they also are extending pro-TPP I activation toward less acidic pH, bringing the feasibility of pro-TPP I autoactivation within the physiological pH range of lysosomes. Similar stimulatory effect of GAGs also was reported for two lysosomal cysteine proteases, pro-cathepsins B and L (34, 35, 49). Interestingly, congopain, the major cysteine protease from Trypanosoma congolense, was activated in the presence of DS, even at a basic pH as high as 8.0 (33). These findings suggest that, in the presence of GAGs, molecules that are ubiquitous in various cell types and in the extracellular matrix, pro-TPP I could autoactivate in vivo without the need for any external proteolytic activity, implying that in vivo the enzyme can utilize both methods of maturation: autoactivation and proteolytic cleavage by another protease. That transproteolysis of TPP I takes place in vivo was demonstrated by our earlier inhibitory studies (23) and by the fact that human TPP I with active-site nucleophile mutated (Ser475) is processed both in primary cultured fibroblasts from a CLN2 patient (23) and when overexpressed in CHO cells (22).2

However, GAGs also exerted a potentially unfavorable biological effect on human TPP I by inhibiting its activity in vitro and in vivo. Inhibition of the activity of numerous lysosomal hydrolases by GAGs in vitro is well documented (50–56). For human leukocyte elastase and cathepsin G, the inhibition was classified as tight-binding, hyperbolic, and noncompetitive (51, 52, 54, 55). It required the presence of sulfated groups and inversely depended upon the chain length of oligosaccharides (51, 52, 54, 57, 58). The extent of inhibition of lysosomal hydrolases by GAGs varies, i.e. 4% control values was reported for human leukocyte elastase incubated in the presence of heparin (55), whereas arylsulfatase retained 80% control values when incubated with heparin or chondroitin sulfate (50). Moderate inhibition of TPP I by physiologically relevant concentrations of those GAGs that are present in mammalian tissues (~40, 50, and 60% maximal control values in the presence of dermatan sulfate, heparin, and heparan sulfate, respectively) appears to have a rather limited physiological relevance given that, under nonstressed conditions, usually only low activities of acid hydrolases are required (by many cells even as low as ~1%) (59). Nevertheless, this inhibition may bear a potential biological significance in situations when cellular requirements for TPP I are elevated to a great extent and the local levels of GAGs are sufficiently high.

GAGs are linear polysaccharides that contain repeating disaccharide units with an amino sugar (either N-acetylglucosamine or N-acetylgalactosamine) and a uronic acid (glucuronic acid and iduronic acid). A variety of combinations of monosaccharides, different type of linkages, and branch formation as well as secondary modifications such as sulfation, phosphorylation, methylation, and acetylation visualize the unusual complexity of these sugar chains, which when covalently attached to proteins in proteoglycans carry a large body of biological information (38, 60–62). The interactions between GAGs and lysosomal enzymes are electrostatic in nature (50, 51); however, the molecular mechanisms responsible for the effect of GAGs on activation and activity of lysosomal hydrolases are still not entirely clear.

Heparin, DS, heparan sulfate, and chondroitin sulfate B have a different carbohydrate backbone structure, but all contain sulfate groups providing a negative charge, which suggests that their effect on both activation and activity of TPP I results from anionic charge density rather than specific structure, similar to what was proposed for interactions of GAGs with other lysosomal enzymes (55). Further studies are necessary to characterize the polyanion-binding site(s) on TPP I molecule. Nevertheless, the effect of polyanions on both activation of pro-TPP I and activity of mature enzyme suggests that the binding site is localized in a mature portion of the enzyme. This contention is supported by the finding that polyanions only slightly protected the enzyme against inactivation by AAF-CMK, a specific TPP I inhibitor. Given that the steric hindrance to the access of the inhibitor to the enzyme active site imposed by the bound polyanions was limited, it appears that the binding site is not localized in direct proximity to the substrate-binding site. However, by kinetic analysis, the affinity of TPP I for the tripeptidyl substrate (Michaelis-Menten constant) in the presence of 10 ng–100 µg/ml of DS was diminished. Because the effect of GAGs on TPP I activation was absent in high salt concentration, the binding site could involve a cluster of positively charged residues on the enzyme surface away from the substrate-binding site, similar to what was proposed for cathepsin K (63) and dissimilar for cathepsin B and papain where the polyanion binding site is localized close to the active site of these proteases (56).

The bell-shaped curve of TPP I inhibition by both heparin and DS suggests that TPP I has at least two binding sites for these molecules with the opposite effects: a high affinity inhibitory site and a low-affinity stimulatory site. Because relatively high concentrations of heparin and DS are needed to saturate the stimulatory site, it can be assumed that, under most physiological conditions, only the inhibitory site would be occupied. Hence, the activity of TPP I would be moderately diminished. The similar effect of various tested GAGs reported for chymotrypsin- and elastase-like enzymes of the human granulocytes led to the suggestion that the interaction of neutral proteases with GAGs depends on their ratio (53). However, dermatan sulfate and heparan sulfate showed only an inhibitory effect on TPP I activity, nonsaturable even at the highest but still physiologically relevant concentrations we tested. These dissimilarities may stem from the higher negative charge density, resulting from the higher number of sulfate groups on disaccharide units of heparin and DS than of dermatan and heparan sulfate (64), but further studies are needed to elucidate this phenomenon.

The opposing effect of polyanions on the activation and activity of TPP I and their conformation-stabilizing properties revealed by increased thermal stability and reduced alkaline pH-induced degradation of the enzyme also suggest that the binding site(s) of polyanions on the TPP I molecule might partially overlap with the prodomain binding site. For cathepsin L, it was proposed that DS unfolds the tertiary structure of the propeptide at pH values below ~5.5, thereby increasing its susceptibility to proteolysis (65). However, no conformation changes of pro-cathepsin L during autoprocessing could be found by further studies (66), and similarly, we could not detect any major conformation changes during pro-TPP I autoactivation (25). Therefore, the mechanism proposed later on, implying that DS and related sulfated GAGs can substantially accelerate in vitro autoactivation of lysosomal proteases by weakening the interaction between the propeptide and the catalytic part (35) appears to be also relevant to the interactions of GAGs with TPP I. During the activation, the binding of polyanions to the mature portion of the pro-TPP I might interrupt the interaction of the prosegment with its cognate protease and displace the prodomain from the active site of the enzyme or misalign the interaction, rendering the junction site more vulnerable to the initial proteolytic scission. Our study also demonstrated that the prosegment fragments released during the autoprocessing of pro-TPP I in the presence of polyanions are degraded more efficiently than in pro-TPP I activated alone, suggesting that polyanions also enhance the susceptibility of the propeptides for proteolytic cleavage.

Both high salt concentration and polyanions enhanced the activation of pro-TPP I in vitro, either exclusively increasing the yields (NaCl) or improving the yields and rates (polyanions) of the activation and by shifting the pH of the activation (but not activity) to a less acidic range. Very high salt concentration stimulated the activation of pro-TPP I and inhibited the activity of mature enzyme almost as well as low concentrations of anionic polymers, which suggests a common mechanism of action. Because it took ~12.5% NaCl (2 M) to have the same stimulatory effect on pro-TPP I activation as 0.000125% heparin and 12.5% NaCl together, heparin appears to be 105 more efficient than NaCl in stimulating pro-TPP I activation. Even after taking into account the concentration of the heparin monomers in heparin solution, a strongly cooperative effect is evident in the binding of polyanions to TPP I in which many negative charges are held in juxtaposition versus the binding of chloride.

The physiological concentrations of GAGs in humans are in the range of nanograms/milliliters in serum, urine, and saliva, but concentrations in the range of 0.920–160 µg/ml for keratan sulfate in synovial fluid (67) or 15.2–65.4 µg/ml for sulfated GAGs in human wound fluids (68) also were reported. High local concentration of GAGs may be encountered also within intracellular compartments, i.e. a 30–40-fold increase of GAG content in liver from patients with mucopolysaccharidoses was noticed (69). At physiological concentrations, GAGs increase the yields and rates of TPP I activation, allow for effective autoactivation at less acidic pH only moderately inhibiting the activity of mature enzyme generated, and at the same time, protect the enzyme against alkaline pH-induced degradation. Thus, in total, GAGs facilitate the biological effects of TPP I. In this respect, the association with GAGs not only could protect the enzyme against potential intralysosomal fluctuations of pH (70) but also makes more plausible the involvement of TPP I in extracellular biological processes. It was proposed that TPP I may be implicated in bone resorption. In fact, osteoclastoma served as a source of one of the first highly purified preparations of TPP I and AAF-CMK at 2 µM inhibited bone resorption by osteoclasts by ~68% (3). That type I collagen constitutes 90–95% organic bone mass (71) would suggest that TPP I possesses collagenase activity. Bone and cartilage contain numerous sulfated GAGs. Thus, the binding of secreted pro-TPP I to them could enhance the activation of the proenzyme and stabilize the enzyme, providing support to the idea that TPP I could be involved in extracellular degradation of collagen. Only when complexed to specific GAGs does cathepsin K possess triple helical collagenase activity (72). It remains to be demonstrated directly whether TPP I is able to digest collagen and how polyanions contribute to this activity.

Other possible sites of extracellular action of TPP I are amyloid-{beta} plaques, a pathological hallmark of Alzheimer's disease brain. The presence of TPP I in a subset of amyloid-{beta} plaques (12), its activity in vitro toward amyloid-{beta} peptides (14) together with the occurrence of N-terminally truncated fragments of amyloid-{beta} peptide starting at the fourth amino acid in amyloid-{beta} plaques (73), and the widespread occurrence of GAGs in the form of various proteoglycans in amyloid plaques (74, 75) render this intriguing possibility feasible. Furthermore, cell surface heparan sulfate proteoglycans are in a constant turnover as a result of their continuous secretion and endocytosis (76–78) and proteins bound to heparan sulfate proteoglycans can be endocytosed together with proteoglycans (15, 79). Thus, due to the binding to GAGs, which significantly protects TPP I against alkaline pH-induced inactivation, TPP I could be shielded from inactivation in the extracellular matrix, be taken up by cells, and remain active in acidic compartments. These hypotheses must be elaborated by further experiments; however, the results of our study indicating that GAGs can significantly affect the activation and stability of TPP I elucidate a potent locally operating mechanism of TPP I regulation that is of high physiological relevance.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant NS047355 and the New York State Office for Mental Retardation and Developmental Disabilities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5208; Fax: 718-982-6346; E-mail: a.golabek{at}att.net.

1 The abbreviations used are: TPP I, tripeptidyl-peptidase I; AAF-CMK, Ala-Ala-Phe-chloromethylketone; CHO, Chinese hamster ovary; Ala-Ala-Phe-AMC, Ala-Ala-Phe-aminomethylcoumarin; DS, dextran sulfate; GAG, glycosaminoglycan; mAb, monoclonal antibody; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PBS, phosphate-buffered saline. Back

2 A. A. Golabek, M. Walus, K. E. Wisniewski, and E. Kida, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Maureen Stoddard-Marlow for copy-editing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wlodawer, A., Li, M., Gustchina, A., Oyama, H., Dunn, B. M., and Oda, K. (2003) Acta Biochim. Pol. 50, 81–102[Medline] [Order article via Infotrieve]
  2. McDonald, J. K., Hoisington, A. R., and Eisenhauer, D. A. (1985) Biochem. Biophys. Res. Commun. 126, 63–71[CrossRef][Medline] [Order article via Infotrieve]
  3. Page, A. E., Fuller, K., Chambers, T. J., and Warburton, M. J. (1993) Arch. Biochem. Biophys. 307, 354–359
  4. Vines, D., and Warburton, M. J. (1998) Biochim. Biophys. Acta 1384, 233–242[CrossRef][Medline] [Order article via Infotrieve]
  5. Ezaki, J., Takeda-Ezaki, M., Oda, K., and Kominami, E. (2000) Biochem. Biophys. Res. Commun. 268, 904–908[CrossRef][Medline] [Order article via Infotrieve]
  6. Tomkinson, B. (1999) Trends Biochem. Sci. 24, 355–359[CrossRef][Medline] [Order article via Infotrieve]
  7. Sleat, D. E., Donnelly, R. J., Lackland H., Liu, C. G., Sohar, I., Pullarkat, R. K., and Lobel, P. (1997) Science 277, 1802–1805[Abstract/Free Full Text]
  8. Sleat, D. E., Gin, R. M., Sohar, I., Wisniewski, K. E., Sklower Brooks, S., Pullarkat, R., Palmer, D. N., Lerner, T. J., Boustany, R. M., Uldall, P., Siakotos, A. N., Donnelly, R. J., and Lobel, P. (1999) Am. J. Hum. Genet. 64, 1511–1523[CrossRef][Medline] [Order article via Infotrieve]
  9. Wisniewski, K. E., Kida, E., Golabek, A. A., Kaczmarski, W., Connell, F., and Zhong, N. (2001) Adv. Genet. 45, 1–34[Medline] [Order article via Infotrieve]
  10. Kida, E., Golabek, A. A., and Wisniewski, K. E. (2001) Adv. Genet. 45, 35–68[Medline] [Order article via Infotrieve]
  11. Suopanki, J., Partanen, S., Ezaki, J., Baumann, M., Kominami, E., and Tyynelä, J. (2000) Mol. Genet. Metab. 71, 190–194[CrossRef][Medline] [Order article via Infotrieve]
  12. Kida, E., Golabek, A. A., Walus, M., Wujek, P., Kaczmarski, W., and Wisniewski, K. E. (2001) J. Neuropathol. Exp. Neurol. 60, 280–292[Medline] [Order article via Infotrieve]
  13. Du, P.-G., Kato, S., Li, Y.-H., Maeda, T., Yamane, T., Yamamoto, S., Fujiwara, M., Yamamoto, Y., Nishi, K., and Ohkubo, I. (2001) Biol. Chem. 382, 1715–1725[CrossRef][Medline] [Order article via Infotrieve]
  14. Junaid, M. A., Wu, G., and Pullarkat, R. K. (2000) J. Neurochem. 74, 287–294[CrossRef][Medline] [Order article via Infotrieve]
  15. Mikhailenko, I., Kounnas, M. Z., and Strickland, D. K. (1995) J. Biol. Chem. 270, 9543–9549[Abstract/Free Full Text]
  16. Warburton, M. J., and Bernardini, F. (2002) Neurosci Lett. 331, 99–102[CrossRef][Medline] [Order article via Infotrieve]
  17. Ezaki, J., Tanida, I., Kanehagi, N., and Kominami, E. (1999) J. Neurochem. 72, 2573–2582[CrossRef][Medline] [Order article via Infotrieve]
  18. Palmer, D. N., Jolly, R. D., van Mil, H. C., Tyynelä, J., and Westlake, V. J. (1997) Neuropediatrics 28, 45–48[Medline] [Order article via Infotrieve]
  19. Waldburg, N., Kahne, T., Reisenauer, A., Rocken, C., Welte, T., and Buhling, F. (2004) Pathol. Res. Pract. 200, 147–154[CrossRef][Medline] [Order article via Infotrieve]
  20. Junaid, M. A., Clark, G. M., and Pullarkat, R. K. (2000) Int. J. Biol. Markers. 15, 129–134[Medline] [Order article via Infotrieve]
  21. Liu, C.-G., Sleat, D. E., Donnelly, R. J., and Lobel, P. (1998) Genomics 50, 206–212[CrossRef][Medline] [Order article via Infotrieve]
  22. Lin, L., Sohar, I., Lackland, H., and Lobel, P. (2001) J. Biol. Chem. 276, 2249–2255[Abstract/Free Full Text]
  23. Golabek, A. A., Kida, E., Walus, M., Wujek, P., Mehta, P., and Wisniewski, K. E. (2003) J. Biol. Chem. 278, 7135–7145[Abstract/Free Full Text]
  24. Wujek, P., Kida, E., Walus, M., Wisniewski, K. E., and Golabek, A. A. (2004) J. Biol. Chem. 279, 12827–12839[Abstract/Free Full Text]
  25. Golabek, A. A, Wujek, P., Walus, M., Bieler, S., Soto, C., Wisniewski, K. E., and Kida, E. (2004) J. Biol. Chem. 279, 31058–31067[Abstract/Free Full Text]
  26. Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3327–3331[Abstract/Free Full Text]
  27. Mellman, I., Fuchs, R., and Helenius, A. (1986) Annu. Rev. Biochem. 55, 663–700[CrossRef][Medline] [Order article via Infotrieve]
  28. Vergne, I., Constant, P., and Laneelle, G. (1998) Anal. Biochem. 255, 127–132[CrossRef][Medline] [Order article via Infotrieve]
  29. Bach, G., Chen, C. S., and Pagano, R. E. (1999) Clin. Chim. Acta 280, 173–179[CrossRef][Medline] [Order article via Infotrieve]
  30. Golabek, A. A., Kida, E., Walus, M., Kaczmarski, W., Michalewski, M., and Wisniewski, K. E. (2000) Mol. Genet. Metab. 70, 203–213[CrossRef][Medline] [Order article via Infotrieve]
  31. McPhie, P. (1972) J. Biol. Chem. 247, 4277–4281[Abstract/Free Full Text]
  32. Van Den Hazel, H., Wolff, A. M., Kielland-Brandt, M. C., and Winther, J. R. (1997) Biochem. J. 326, 339–344[Medline] [Order article via Infotrieve]
  33. Serveau, C., Boulange, A., Lecaille, F., Gauthier, F., Authie, E., and Lalmanach, G. (2003) Biol. Chem. 384, 921–927[CrossRef][Medline] [Order article via Infotrieve]
  34. Mason, R. W., and Massey, S. D. (1992) Biochem. Biophys. Res. Commun. 189, 1659–1666[CrossRef][Medline] [Order article via Infotrieve]
  35. Rozman, J., Stojan, J., Kuhelj, R., Turk, V., and Turk, B. (1999) FEBS Lett. 459, 358–362[CrossRef][Medline] [Order article via Infotrieve]
  36. Lindholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2267–2271[Abstract/Free Full Text]
  37. Esko, J. D., Stewart, T. E., and Taylor, W. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3179–3201
  38. Kolset, S. O., Prydz, K., and Pejler, G. (2004) Biochem. J. 379, 217–227[CrossRef][Medline] [Order article via Infotrieve]
  39. Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M., and Tashiro, Y. (1991) J. Biol. Chem. 266, 17707–17712[Abstract/Free Full Text]
  40. Ishidoh, K., Takeda-Ezaki, M., Watanabe, S., Sato, N., Aihara, M., Imagawa, K., Kikuchi, M., and Kominami, E. (1999) J. Biochem. 125, 770–779[Abstract/Free Full Text]
  41. van Deurs, B., Holm, P. K., and Sandvig, K. (1996) Eur. J. Cell Biol. 69, 343–350[Medline] [Order article via Infotrieve]
  42. Lecaiile, F., Muno, D., Kominami, E., and Ishidoh, K. (2004) Biol. Chem. 385, 511–516[CrossRef][Medline] [Order article via Infotrieve]
  43. Trowbridge, J. M., and Gallo, R. L. (2002) Glycobiology 12, 117R–125R[Abstract/Free Full Text]
  44. Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., and Kitagawa, H. (2003) Curr. Opin. Struct. Biol. 13, 612–620[CrossRef][Medline] [Order article via Infotrieve]
  45. Dennissen, M. A., Jenniskens, G. J., Pieffers, M., Versteeg, E. M., Petitou, M., Veerkamp, J. H., and van Kuppevelt, T. H. (2002) J. Biol. Chem. 277, 10982–10986[Abstract/Free Full Text]
  46. Lin, L., and Lobel, P. (2001) Biochem. J. 357, 49–55[CrossRef][Medline] [Order article via Infotrieve]
  47. Van Dyke, R., Root, K. V., Schreiber, J. H., and Wilson, J. M. (1992) Biochem. Biophys. Res. Commun. 184, 300–305[CrossRef][Medline] [Order article via Infotrieve]
  48. Montcourrier, P., Mangeat, P. H., Valembois, C., Salazar, G., Sahuquet, A., Duperray, C., and Rochefort, H. (1994) J. Cell Sci. 107, 2381–2391[Abstract]
  49. Ishidoh, K., and Kominami, E. (1995) Biochem. Biophys. Res. Commun. 217, 624–631[CrossRef][Medline] [Order article via Infotrieve]
  50. Avila, J. L., and Convit, J. (1975) Biochem. J. 152, 57–64[Medline] [Order article via Infotrieve]
  51. Baici, A., Salgam, P., Fehr, K., and Böni, A. (1980) Biochem. Pharmacol. 29, 1723–1727[CrossRef][Medline] [Order article via Infotrieve]
  52. Baici, A., Diczházi, C., Neszmélyi, A., Móczár, E., and Hornebeck, W. (1993) Biochem. Pharmacol. 46, 1545–1549[CrossRef][Medline] [Order article via Infotrieve]
  53. Marossy, K. (1981) Biochim. Biophys. Acta 659, 351–361[Medline] [Order article via Infotrieve]
  54. Redini, F., Tixier, J.-M., Petitou, M., Choay, J., Robert, L., and Hornebeck, W. (1988) Biochem. J. 252, 515–519[Medline] [Order article via Infotrieve]
  55. Volpi, N. (1996) Biochim. Biophys. Acta 1290, 299–307[Medline] [Order article via Infotrieve]
  56. Almeida, P. C., Nantes, I. L., Chagas, J. R., Rizzi, C. C. A., Faljoni-Alario, A., Carmona, E., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (2001) J. Biol. Chem. 276, 944–951[Abstract/Free Full Text]
  57. Volpi, N. (1997) Chem. Biol. Interact. 105, 157–167[CrossRef][Medline] [Order article via Infotrieve]
  58. Frommherz, K. J., Faller, B., and Bieth, J. G. (1991) J. Biol. Chem. 266, 15356–15362[Abstract/Free Full Text]
  59. Grabowski, G. A., and Hopkin, R. J. (2003) Annu. Rev. Genomics Hum. Genet. 4, 403–436[CrossRef][Medline] [Order article via Infotrieve]
  60. Iozzo, R. V. (1998) Annu. Rev. Biochem. 67, 609–652[CrossRef][Medline] [Order article via Infotrieve]
  61. Bernfield, M., Götte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729–777[CrossRef][Medline] [Order article via Infotrieve]
  62. Honke, K., and Taniguchi, N. (2002) Med. Res. Rev. 22, 637–654[CrossRef][Medline] [Order article via Infotrieve]
  63. Li, Z., Hou, W. S., Escalante-Torres, C. R., Gelb, B. D., and Brömme, D. (2002) J. Biol. Chem. 277, 28669–28676[Abstract/Free Full Text]
  64. Hileman, R. E., Fromm, J. R., Weiler, J. M., and Linhardt, R. J. (1998) BioEssays 20, 156–167[CrossRef][Medline] [Order article via Infotrieve]
  65. Jerala, R., Zerovnik, E., Kidriè, J., and Turk, V. (1998) J. Biol. Chem. 273, 11498–11504[Abstract/Free Full Text]
  66. Ménard, R., Carmona, E., Takebe, S., Dufour, E., Plouffe, C., Mason, P., and Mort, J. S. (1998) J. Biol. Chem. 273, 4478–4484[Abstract/Free Full Text]
  67. Møller, H. J., Larsen, F. S., Ingemann-Hansen, T., and Poulsen, J. H. (1994) Clin. Chim. Acta 225, 43–45[CrossRef][Medline] [Order article via Infotrieve]
  68. Penc, S. F., Pomahac, B., Wikler, T., Dorschner, R. A., Eriksson, E., Herndon, M., and Gallo, R. L. (1998) J. Biol. Chem. 273, 28116–28121[Abstract/Free Full Text]
  69. Minami, R., Abo, K., Tsugawa, S., Oyanagi, K., and Nakao, T. (1981) Tohoku J. Exp. Med. 134, 215–220[Medline] [Order article via Infotrieve]
  70. Butor, C., Griffiths, G., Aronson, N. N., and Varki, A. (1995) J. Cell Sci. 108, 2213–2219[Abstract]
  71. Krane, S. M., and Simon, L. (1994) in Scientific American Medicine (Rubenstein, E., and Federman, D. D., Eds) Vol. 3, pp. 1–26, Scientific American, Inc., New York
  72. Li, Z., Yasuda, Y., Li, W., Bogyo, M., Katz, N., Gordon, R. E., Fields, G. B., and Brömme, D. (2004) J. Biol. Chem. 279, 5470–5479[Abstract/Free Full Text]
  73. Sergeant, N., Bombois, S., Ghestem, A., Drobecq, H., Kostanjevecki, V., Missiaen, C., Wattez, A., David, J.-P., Vanmechelen, E., Sergeraert, C., and Delacoutre, A. (2003) J. Neurochem. 85, 1581–1591[CrossRef][Medline] [Order article via Infotrieve]
  74. Snow, A. D., Mar, H., Nochlin, D., Kimata, K., Suzuki, S., Hassell, J., and Wight, T. N. (1988) Am. J. Pathol. 133, 456–463[Abstract]
  75. van Horssen, J., Kleinnjenhuis, J., Maass, C. N., Rensink, A. A. M., Otte-Holler, I., David, G., van den Heuvel, L. P. W. J., Wesseling, P., de Waal, R. M. W., and Verbeek, M. M. (2002) Neurobiol. Aging 23, 537–545[CrossRef][Medline] [Order article via Infotrieve]
  76. Kramer, P. M. (1971) Biochemistry 10, 1437–1445[CrossRef][Medline] [Order article via Infotrieve]
  77. Bienkowski, M. J., and Conrad, H. E. (1984) J. Biol. Chem. 259, 12989–12996[Abstract/Free Full Text]
  78. Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10260–10269[Abstract/Free Full Text]
  79. Reiland, J., and Rapraeger, A. C. (1993) J. Cell Sci. 105, 1085–1093[Abstract]

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