Endocrinology Vol. 138, No. 9 3797-3803
Copyright © 1997 by The Endocrine Society
Proteolysis of Insulin-Like Growth Factors (IGF) and IGF Binding Proteins by Cathepsin D1
Max Claussen,
Bernd Kübler,
Martin Wendland,
Klaus Neifer,
Bernhard Schmidt,
Jürgen Zapf and
Thomas Braulke
Institute for Biochemistry II, University of Göttingen,
D-37073 Göttingen, Germany; and Metabolic Unit, Department of
Medicine, University Hospital (J.Z.), CH-8091 Zürich,
Switzerland
Address all correspondence and requests for reprints to: Thomas Braulke, Ph.D., Institute for Biochemistry II, University of Göttingen, Gosslerstrasse 12D, D 37073 Göttingen, Germany. E-mail: braulke{at}ukb2-00.uni-bc.gwdg.de
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Abstract
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Various proteinases have been postulated to function in limited
proteolysis of insulin-like growth factor binding proteins (IGFBPs)
contributing to the regulation of IGF bioavailability. In this study,
we report on the in vitro degradation of IGFs and IGFBPs
by the purified acidic aspartylprotease cathepsin D that has been shown
to proteolyze IGFBP-3. Recombinant human [125I] IGFBP-1
to -5 were processed by cathepsin D to fragments of defined sizes in a
concentration dependent manner, whereas IGFBP-6 was not degraded.
Ligand blotting revealed that none of the IGFBP-1 or -3 fragments
formed by cathepsin D retain their ability to bind IGF. By N-terminal
sequence analysis of nonglycosylated IGFBP-3 fragments produced by
cathepsin D, at least four different cleavage sites were identified.
Some of these cleavage sites were identical or differed by one amino
acid from sites used by other IGFBP proteases described. The
IGFBP-3 and -4 cleavage sites produced by cathepsin D are located
in the nonconserved central region. IGF-I and -II, but not the
unrelated platelet-derived growth factor BB, were degraded by cathepsin
D in a time and concentration-dependent manner. We speculate that the
major functional site of cathepsin D is intracellular and may be
involved 1) in the selected clearance either of IGFBP or IGFs via
different endocytic pathways or 2) in the general lysosomal
inactivation of the IGF system.
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Introduction
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SOLUBLE lysosomal enzymes such as the
aspartic protease cathepsin D are synthesized in the endoplasmic
reticulum as inactive prepro-forms of higher molecular weight. During
transport through the Golgi, the enzymes acquire phosphomannosyl
residues in their high-mannose type oligosaccharides. Following the
binding to mannose 6-phosphate specific receptors, the enzyme-receptor
complexes are segregated from the secretory pathway and exit the
trans-Golgi network in clathrin-coated vesicles. Upon fusion
with the acidic endosomal/prelysosomal compartment, the dissociation of
ligands occurs. The delivery of enzymes to lysosomes is accompanied by
a series of proteolytic cleavages into the mature enzyme forms (for
review see 1 . A variable fraction, depending on the cell type
studied, of newly synthesized lysosomal enzyme precursors escapes the
binding to receptors in the Golgi and is instead secreted (2). For
cancer cells, increased expression at the messenger RNA and protein
levels as well as increased activity and alterations in trafficking of
different classes of lysosomal proteases have been reported. Studies
using several approaches have indicated that the overexpression and
hypersecretion of cathepsins are responsible in part for the
degradation of basement membranes, invasive angiogenesis and metastasis
(3, 4). In addition, cathepsin D has been reported to be involved in
proteolytic processing of antigens, PTH and the insulin-like growth
factor binding protein (IGFBP)-3 (5, 6, 7).
Six distinct IGFBPs, differing in molecular mass, binding properties
for IGFs, and posttranslational modifications modulate the
bioavailability of IGFs (8, 9). Limited proteolysis of IGFBPs may be an
important mechanism in the release of bioactive IGFs both in
circulation and at cellular level, thereby increasing their
availability to cellular receptors. It appears that different proteases
are involved in IGFBP proteolysis in biological fluids and media of
cultured cells (10, 11, 12, 13).
In this study, we examined the ability of purified cathepsin D to
hydrolyze IGFBPs and IGF-II in vitro. The data presented
show, with the exception of IGFBP-6, that cathepsin D can efficiently
proteolyze all IGFBPs as well as IGF-I and -II. Furthermore, the
cleavage sites of IGFBP-3 and -4 were determined.
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Materials and Methods
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Recombinant IGF-I and -II were gifts from Dr. W. Märki
(Ciba Geigy, Basel, Switzerland). Recombinant IGFBP-1 and the
anti-IGFBP-3 antiserum were purchased from UBI (Lake Placid, NY), and
IGFBP-2 came from Austral Biological (San Ramon, CA). Glycosylated
(gly) and nonglycosylated (non-gly) IGFBP-3 were a generous gift of A.
Sommer and C. Maack (Celtrix, Santa Clara, CA). IGFBP-4, -5, and -6
were produced in yeast (14). IGFs and the IGFBPs were iodinated with
the aid of chloramine T and IODO-GEN (Pierce, Rockford, IL),
respectively, as described previously (15, 16). Na 125I,
[125I] platelet-derived growth factor BB, and rainbow
marker proteins were obtained from Amersham Corp. (Buchler, Germany).
Human cathepsin D was purchased from Sigma. The monoclonal antibody
against human IGF-II came from Amano Enzyme (Troy, VA).
Cathepsin D from mouse liver was purified to homogeneity by pepstatin
A-agarose affinity chromatography as described (17, 18). Briefly, liver
tissue was homogenized in 4 vol of 10 mM NaPi
buffer, pH 6.5, containing 150 mM NaCl, 5 mM
EDTA, and 1 mM mercaptoethanol. The postnuclear supernatant
was adjusted to 50 mM NaPi, pH 7.5, 50
mM NaCl and 0.1% Triton X-100, left for 45 min on ice, and
centrifuged at 100,000 x g for 1 h using a 45
Ti-rotor (Beckman). The supernatant was mixed with diethylaminoethyl
(DEAE) cellulose DE52 (Whatman, Maidstone, UK) and incubated for 3
h at 4 C under agitation. The nonbound material was loaded onto a
pepstatin A-agarose (Sigma) column and washed successively with 50
mM citrate buffer, pH 3.3, containing 500 mM
NaCl (buffer A) and buffer A containing 6 M urea. Cathepsin
D was specifically eluted with 50 mM Tris/HCl buffer, pH
8.5, containing 500 mM NaCl. When aliquots of eluted
fractions were analyzed by SDS-PAGE and silver staining, the purified
enzyme fractions exhibited a major 44-kDa band and a minor 30-kDa
polypeptide (Fig. 1
). These fractions
were pooled, concentrated and dialyzed against 10 mM
NaPi, pH 6.5, in ultrafiltration thimbles (Schleicher &
Schüll, Dassel, Germany, exclusion size 25,000). To evaluate
purity, the cathepsin D preparation was rechromatographed by RP-HPLC
(SMART, Pharmacia, Uppsala, Sweden) using a 220 x 2.1 mm C4
column (Aquapore, Applied Biosystem, Weiterstadt, Germany) equilibrated
with 0.1% trifluoroacetic acid in water. Proteins were eluted with an
increasing acetonitrile concentration at a flow rate of 0.3 ml per min.
SDS-PAGE and silver staining revealed no contamination by other
proteins. Both 44- and 30-kDa cathepsin D polypeptides were eluted at
an acetonitrile concentration between 54 and 71% (not shown).
Antiserum against cathepsin D was raised in rabbits. In Western
immunoblots, the antiserum recognized the precursor (52 kDa),
intermediate (44 kDa) and mature form (30 kDa) in extracts of embryonic
mouse fibroblasts but not in extracts of cathepsin D-deficient
mouse cells (19).
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Figure 1. Purification of mouse liver cathepsin D by
pepstatin A-agarose affinity chromatography: The cathepsin D containing
flow-through fraction (400 ml) from DEAE cellulose chromatography was
recycled over a pepstatin A-agarose column (5 ml) equilibrated with 50
mM NaPi, pH 7.5, 50 mM NaCl. After
washing, cathepsin D was eluted in 25 fractions of 10 ml each as
described in Materials and Methods. Aliquots of 0.1 ml
were analyzed by SDS-PAGE (10% acrylamide) and silver staining. The
positions of the molecular mass standard proteins (St) in kDa are
indicated. I, intermediate, and M, mature forms of cathepsin D.
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Growth factor and IGFBP-proteolysis
Proteolytic activity of cathepsin D against growth factors and
IGFBPs were measured by incubating 20,000–55,000 cpm of
[125I]IGFs, [125I]PDGF-BB, or
[125I]IGFBPs in a final volume of 50 µl in 0.1
M Na-acetate buffer pH 4.0 (or at the indicated pH) with
purified mouse or human cathepsin D (0.01–1.0 µg per assay) at 37 C
for 6 or 20 h. The samples were solubilized and separated by
SDS-PAGE (15 or 12.5% acrylamide) under nonreducing conditions. The
dried gels were exposed for autoradiography for 12–48 h (20).
Determination of cathepsin D cleavage sites in IGFBP-3 and -4
Ten micrograms of of gly- or non-gly IGFBP-3 or IGF-4 were
incubated with 3 µg cathepsin D in 50 µl 0.1 M
Na-acetate buffer, pH 4.0, at 37 C for 20 h. The samples were
solubilized, separated by SDS-PAGE (12.5% acrylamide) under reducing
or nonreducing conditions, and transferred to PVDF membranes
(Schleicher & Schuell, Germany). Coomassie blue stained polypeptides
were microsequenced using an Applied Biosystem model 477 A
fluid-phase protein sequenator (21).
Blot analysis
[125I] IGF-II ligand blotting and IGFBP-3
immunoblotting were carried out as described (22).
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Results
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Cathepsin D was purified from mouse liver by pepstatin-A affinity
chromatography. Other aspartyl proteinases, like renin and
cathepsin E, were removed by the initial adsorption on DEAE-cellulose
(17, 23). Analysis of purified cathepsin D by SDS-PAGE followed by
silver staining revealed the 44-kDa intermediate and the 30-kDa mature
heavy chain form (Fig. 1
), which are consistent with data in the
literature on cathepsin D purification from other species (2). Both the
44- and 30-kDa polypeptides represent enzymatic active forms. The
14-kDa light chain form was lost during the ultrafiltration step.
Cathepsin D has been reported to catalyze the proteolysis of IGFBP-3
(7). To determine the IGFBP specificity, recombinant human
[125I] labeled IGFBP-1 to -6 were incubated both in the
presence and in the absence of purified mouse cathepsin D (0.8 µg per
assay) for 20 h at various pH values (from 3.5 to 6.5), followed
by SDS-PAGE and autoradiography. Optimal activity to IGFBP-1 to -5 as
substrate was observed between pH 3.8 and 4.7 (not shown). No
fragmentation of the 26-kDa IGFBP-6 by cathepsin D was detected (Fig. 2). Further studies were carried out at
pH 4.0. When [125I] IGFBPs were incubated with cathepsin
D for 20 h, one major 10- to 12-kDa IGFBP-1 fragment, two 24- and
16-kDa IGFBP-2 fragments, one 14-kDa nonglycosylated (non-gly) IGFBP-3
fragment, and one 10-kDa IGFBP-4 fragment were found (Fig. 2). When
IGFBP-5 was assessed, several weak fragments of 14–23 kDa were
observed, whereas the greater part of the radioactivity migrated with
the dye front. Depending on the time period of storage at -80 C and
the IGFBP, the appearance of spontaneous degradation products were
observed even without incubation at 37 C and in the absence of
cathepsin D (e.g. IGFBP-1 and -3 in Fig. 2). However, these
radiolysis products differ in size from fragments produced by cathepsin
D.
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Figure 2. Effect of cathepsin D on IGFBP proteolysis:
[125I] labeled IGFBP-1 to -6 (each 40,000 cpm) were
incubated both in the presence (+) and absence (-) of 0.8 µg mouse
cathepsin D for 20 h at pH 4.0 at 37 C. The reaction products were
separated by nonreducing SDS-PAGE and visualized by autoradiography.
The position of the molecular mass marker proteins (in kDa) are
indicated.
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When [125I] IGFBP-1 to -4 were incubated with varying
concentrations of cathepsin D for 6 h at pH 4.0, several
intermediate IGFBP-fragments were observed depending on the IGFBP (Fig. 3). Thus, the more slowly migrating
IGFBP-1 form is preferentially affected more by cathepsin D-mediated
proteolysis than is the second form. With increasing concentration of
cathepsin D, the 12- to 16-kDa IGFBP-1 proteolysis products detected
first were reduced to a final fragment with an apparent molecular mass
of 10–12 kDa (see also Fig. 2). In the presence of various amounts of
cathepsin D, only the 24-kDa and 16-kDa doublet fragments of
[125I]-IGFBP-2 were formed.
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Figure 3. IGFBP proteolysis in dependency of cathepsin D
concentration: [125I] labeled IGFBP-1 to -4 and gly
IGFBP-3 (each 40,000 cpm) were incubated both in the presence and
absence (-) of the indicated amounts of mouse cathepsin D for 6 h
at pH 4.0 at 37 C. The reaction products were separated by nonreducing
SDS-PAGE and visualized by autoradiography. The positions of the
molecular mass marker protein (in kDa) are indicated.
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Depending on the cathepsin D concentration, proteolytic non-gly IGFBP-3
cleavage products of 25, 18, and 14–16 kDa were observed. During
proteolysis of 44/40-kDa glycosylated (gly) IGFBP-3 doublet with
cathepsin D, one 35/31 kDa doublet and one 22.5 kDa intermediate
fragment appeared transiently. The 16-kDa proteolytic gly
[125I]IGFBP-3 fragment was the final polypeptide observed
after prolonged incubation with cathepsin D. At similar enzyme-IGFBP
ratio, the proteolysis of non-gly IGFBP-3 was completed after 6 h,
whereas a higher percentage of glycosylated intermediate IGFBP-3
fragments remained (Fig. 3). Molecular mass analysis of IGFBP-4
fragments generated in dependence on cathepsin D concentration,
indicated major proteolytic cleavage products of 17- and 10-kDa and
minor fragments of 18, 16, and 12 kDa. Incubation of
[125I]IGFBP-5 with increasing concentrations of
cathepsin D resulted in the disappearance of stable radioactive bands
indicating cleavage at different sites producing various small
nondetectable labeled polypeptides (not shown). When commercially
available human cathepsin D was compared with mouse cathepsin D in
proteolysis assays with [125I]IGFBP-1, gly IGFBP-3 or
IGFBP-4, identical fragmentation patterns were observed (not shown; for
non-gly [125I] IGFBP-3 compare Figs. 3 and 5).
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Figure 5. Effect of cathepsin D on PDGF proteolysis:
[125I]PDGF (52,000 cpm) were incubated both in the
presence and absence (-) of the indicated amounts of human cathepsin D
for 6 h at pH 4.0 at 37 C. For comparison
[125I]IGFBP-3 (24,000 cpm) were incubated with 0.25 µg
cathepsin D for 6 h at 37 C. The reaction products were separated
by nonreducing SDS-PAGE and visualized by autoradiography (exposure
time of 12 h). The positions of the molecular mass marker protein
(in kDa) are indicated.
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Incubation of 60–100 ng nonlabeled IGFBP-1 or -3 with 0.5 µg
cathepsin D for 6 h followed by SDS-PAGE and ligand blotting,
revealed that none of the IGFBP-1 or -3 fragments formed by cathepsin D
showed [125I] IGF-II binding activity. When proteolyzed
IGFBP-samples were analyzed by Western immunoblotting, only the 12-kDa
fragment of IGFBP-1 and the 14-kDa fragment of IGFBP-3 were stained
weakly by the respective anti-IGFBP antiserum (not shown).
To determine the cleavage sites used by cathepsin D, large scale
proteolysis assays were performed with IGFBP-3 and -4 followed by
SDS-PAGE and electroblotting on PVDF membranes. The stained fragments
were subjected to Edman degradation. Non-gly IGFBP-3 were cleaved by
cathepsin D at tyrosine 126, lysine 187, glutamic acid 191, and
asparagine 228 (Table 1). Digestion of
gly IGFB P-3 with cathepsin D produced fragments N-terminally starting
with tyrosine 126 and asparagine 228. IGFBP-4 was cleaved by cathepsin
D at methionine 156.
Incubation of [125I]labeled IGF-I (not shown) and IGF-II
(Fig. 4) with purified cathepsin D
resulted in three radiolabeled IGF-II fragments with faster mobility in
SDS-PAGE than intact IGF-II. In Western blots, the fragments were not
detected by a monoclonal anti-IGF-II antibody (not shown). With
increasing enzyme concentration or incubation time, the intensity of
radiolabeled polypeptides decreased indicating cleavage into smaller
fragments not detectable under the conditions used. When the unrelated
28-kDa [125I] platelet-derived growth factor (PDGF) BB
was incubated with increasing amounts of human cathepsin D (0.01–1
µg per assay) for 6 h under identical conditions as used for
[125I] IGFBP-3, no PDGF-proteolysis products were
detected (Fig. 5).
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Figure 4. Proteolysis of IGF-II by cathepsin D:
[125I]labeled IGF-II (50,000 cpm) was incubated in the
presence or absence of mouse cathepsin D for 6 h at pH 4.0 at 37
C. The reaction products were separated by SDS-PAGE (15% acrylamide)
and visualized by autoradiography.
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Discussion
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Our experiments demonstrate that the affinity purified aspartic
protease cathepsin D efficiently cleaves IGFBP-1 to -5. Conover and
colleagues were the first to describe the capacity of cathepsin D to
generate under acidic conditions IGFBP-3 fragments resembling those
detected after incubation of IGFBP-3 in acid-activated conditioned
media from a variety of human cell lines (7, 24). In addition, upon
acidification of conditioned media from human embryonic tissue
explants, proteolytic activity against IGFBP-1, -3, and -4 was observed
(22). Both the inhibition by pepstatin and the immunodepletion of
cathepsin D from conditioned media indicate that cathepsin D may play
an important role in IGFBP-3 proteolysis (7). However, these
experiments cannot rule out that cathepsin D may be required for
activation of specific IGFBP proteases. Furthermore, whether the
acid-activated IGFBP proteolysis in conditioned media significantly
contributes to IGFBP regulation in vivo remains unclear.
Less than 20% of newly synthesized cathepsin D is secreted in most
types of cultured cells as inactive precursor form into the culture
medium (1), which might be primarily responsible for acid-activated
IGFBP proteolysis. In contrast, in breast cancer cell lines,
overexpression and specific hypersecretion of procathepsin D (up to
66% of the newly synthesized protease) have been reported (25).
Evaluation of procathepsin D secreted by the breast cancer cell line
MCF7 by quantitative Western blotting revealed up to 10
µg procathepsin D/ml and mg protein in 48 h, which is in the
range of cathepsin D amounts used in the present in vitro
study (T. Braulke, unpublished results). The elevated extracellular
level of procathepsin D is thought to be correlated with malignancy and
metastatic potency. Cathepsin D might participate in these processes
not only by degrading the extracellular matrix (26), but also by
proteolysis of IGFBPs, required for increased IGF bioavailability and
stimulated cell growth. Furthermore, extracellular cathepsin D may
arise from several cells of the hemopoietic lineage that respond to
external stimuli by secretion of lysosomal enzymes from these cells
(27). Alternatively, cathepsin D might play a role in IGFBP proteolysis
in intracellular compartments, e.g. in recycling endosomes.
These tubular elements are part of the early endosome characterized by
an acidified internal pH and a rapid export of internalized membrane
components, lipids and fluid-phase markers (28). In certain cell types,
proteolytic processes occur during endocytic recycling. Thus, in B
lymphoblasts, endocytosed antigens are partially degraded in a
specialized, cathepsin D-containing compartment (29). MHC class II
molecules in this compartment acquire antigenic peptides and are
transported to the cell surface. It remains to be elucidated whether,
in certain cell types cathepsin D-mediated proteolysis of IGFBPs occurs
in intracellular organelles followed by recycling and release of IGFBP
fragments into the extracellular space.
Several in vitro studies have demonstrated the presence of
additional proteases [divalent cation-dependent serine proteases,
matrix metalloproteases (MMP), prostate specific antigen (PSA)]
degrading IGFBP-3, -4, and -5 in biological fluids and conditioned
media of various cell types (11, 12, 30, 31, 32). Sequence analysis of
IGFBP-3 fragments formed by MMP-1, -2 and -3 revealed a major cleavage
site between tyrosine 126 and leucine 127 (33). By N-terminal sequence
analysis, five separate cleavage sites were identified for PSA in
IGFBP-3 comprising the major cleavage sites prior alanine 125, lysine
187 and serine 201 (32). The 14 kDa IGFBP-4 fragment, formed by an
IGF-dependent IGFBP-4 protease in conditioned media of human
fibroblasts, begins with lysine 157 (34). Furthermore, rat IGFBP-4 was
cleaved by a protease from the rat B 104 neuronal cell line into two
fragments. The first 120-amino acid-long fragment begins at the
N-terminus of the mature IGFBP-4, whereas the second peptide begins
with a lysine homologous to lysine 157 of the human IGFBP-4 (35).
N-terminal sequence analysis in the present study demonstrated the
presence of at least four separate cleavage sites for cathepsin D in
IGFBP-3. Because small oligopeptides can be lost during the separation
by SDS-PAGE, the total number of cleavage sites used by cathepsin D
cannot be determined, and therefore no fragment length predictions can
be made. Distinct cleavage sites for both IGFBP-3 and IGFBP-4 were
localized one amino acid proximal or distal to those generated by
matrix metalloproteinase or PSA in IGFBP-3, respectively, or by an
IGFBP-4 protease from conditioned media (Fig. 6, 32–34). In our proteolysis assays
using [125I]labeled IGFBPs, only those fragments that
contain iodinated tyrosins are observed. Due to limitation in amounts
of recombinant IGFBPs available, a more extended investigation to
detect other non-labeled IGFBP fragments, e.g. by
immunoblotting, was not possible. Preliminary experiments showed that
cathepsin D hydrolyzes non-gly IGFBP-3 faster than gly IGFBP-3. Because
the identified cleavage sites are located in the non-conserved central
region that contains the three N-glycosylation sites asparagine 116,
136, and 199 (36), it is likely that the oligosaccharide chains
interfere with the cathepsin D recognition sites.
It has been suggested that proteolysis of IGFBPs may function in
release of IGFs by lowering their affinity for IGFs (11, 37). Indeed,
the proteolytic fragments formed by cathepsin D mediated cleavage of
IGFBP-1 or -3 showed no IGF binding activity. However, because at the
acidic pH required for cathepsin D activity the ability also of intact
IGFBP to bind IGFs is reduced or lost, it is rather unlikely that
cathepsin D catalyzed proteolysis of IGFBP may represent a mechanism
for modulation of free IGF levels. Moreover, in this study the
selective cleavage of IGFs performed by cathepsin D in vitro
has been demonstrated. It remains to be determined whether the
proteolysis of IGFs by cathepsin D results in loss of biological
activity to assess the role of the protease in a more general catabolic
process of inactivation of both IGFBPs and IGFs. Alternatively, IGFBPs
and IGFs are accessible for cathepsin D in different subcellular
compartments, i.e. IGFBPs in the endosomal recycling pathway
resulting in the release of binding incompetent fragments and IGFs in
the lysosomal degradative pathway after IGF receptor-mediated
internalization. However, more experimental data are needed to
discriminate between these possibilities and to understand the
functional significance of IGFBP proteases in modulating IGF
activity.
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Acknowledgments
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The excellent technical assistance of Nicole Jacksch is
gratefully acknowledged. We thank Cyrilla Cole-Maelicke and Angelika
Thiel for their help with the manuscript and Fritz Ropeter and Angelika
Misgaiski for processing the photographic material.
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Footnotes
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1 This study was supported by the Deutsche Forschungsgemeinschaft
(Sonderforschungsbereich 402/A6), the Fonds der Chemischen Industrie,
and the Swiss National Science Foundation, Grant 32–31281.91 (J.Z.).
Parts of this work were presented at the Third International Symposium
on Insulin-Like Growth Factor Binding Proteins, Tübingen,
Germany, October 6–8, 1995. 
Received March 7, 1997.
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Top
Abstract
Introduction
) and in both gly and non-gly
IGFBP-3 (
) are shown below. IGFBP-3 cleavage sites generated by
matrix metalloproteases (
, Ref. 33) and PSA (
; Ref. 32) are
indicated above.