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J. Biol. Chem., Vol. 278, Issue 34, 31918-31923, August 22, 2003
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-Glucosidase to Proteolytic Degradation*
From the Division and Program in Human Genetics, Children's Hospital Research Foundation, Cincinnati, Ohio 45229-3039
Received for publication, March 18, 2003 , and in revised form, June 16, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-glucosidase
(GCase) requires saposin C for optimal in vitro and in vivo
hydrolysis of glucocerebroside. The deficiency of prosaposin/saposins
(PS–/–) in humans and mice leads to a decrease of GCase activity
in selected tissues. Concordant decreases (>50%) of GCase protein and
in vitro activity were detected in extracts of cultured fibroblasts
and hepatocytes from PS–/– mice and human prosaposin-deficient
fibroblasts. GCase RNA in the PS–/– cells was at wild-type levels.
Compared with that in wild-type cells (t
>24 h), the GCase
protein in the PS–/– cells had a faster disappearance rate
(t 
1 h in mouse and 8 h in human) as determined by
metabolic labeling and immunoprecipitation with anti-GCase antibodies.
Treatment of PS–/– cells with leupeptin, an inhibitor of cysteine
proteases, led to significant increases (2-fold) in GCase protein and
in vitro activity. Loading saposin C to human PS–/–
fibroblasts resulted in an enhancement of GCase protein and in vitro
activity. Saposin D loading had no effect. These data indicate that saposin C
is required for GCase resistance to proteolytic degradation in the cell. Thus,
diminished in vivo GCase activity would be greater than expected only
from the lack of GCase activation by saposin C. These results indicate a new
property for saposin C, an anti-proteolytic protective function toward
GCase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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80-amino
acid lysosomal glycoproteins that are encoded by a single gene, termed
prosaposin
(1–4).
The proteolytic processing of prosaposin to the individual saposins occurs
predominantly in acidified compartments including the lysosome
(5,
6). The physiological
importance of this locus has been demonstrated by the genetic deficiencies of
individual saposins or prosaposin that lead to various glycosphingolipid
storage diseases
(7–10).
For example, saposin B deficiency leads to sulfatide accumulation and a
metachromatic leukodystrophy-like disease
(11) that is similar to the
deficiency of arylsulfatase A, the cognate enzyme. Saposin C enhances acid
-glucosidase
(GCase)1 activity, and
its deficiency leads to a Gaucher-like disease with glucosylceramide
accumulation in cells (9,
12). Deficiencies of saposins
A and D have not been described in humans. A deficiency of saposin A in mice
leads to a late-onset, chronic form of globoid cell leukodystrophy
(13). A total deficiency of
human or mouse prosaposin results in the storage of multiple
glycosphingolipids in a variety of organs
(7,
8,
14).
Saposins enhance the activity of their respective cognate lysosomal enzymes
by different mechanisms to achieve optimal in vitro and in
vivo degradation of glycosphingolipids
(15–17).
Saposin B in a dimeric shell structure facilitates the partial extraction of
glycosphingolipids from membranes and the presentation of sulfatides or
globotriaosylceramide to arylsulfatase A or 
-galactosidase A,
respectively (18,
19). Saposin C promotes GCase
activity by inducing a conformation change in the enzyme at acidic pH
(20,
21). In vitro, both
saposin A and C enhance GCase activity, and negatively charged phospholipids
are required for their actions
(20,
22), but they have different
membrane interaction modes
(20). In vivo,
saposins A and C enhance galactosylcerebrosidase and GCase activities,
respectively (23). Saposin D
stimulates partially purified acid ceramidase for the degradation of ceramide
(24). Importantly, selected
nonphysiologic detergent-based assays can obviate the need for negatively
charged phospholipids and/or saposins in these GCase assays and can provide
estimates of its total catalytic power within cells
(25).
Although the involvement of saposins in the degradation of glycosphingolipids is well established, their in vivo mechanisms of action are not fully understood. Reductions of GCase, acid ceramidase, and galactosylcerebrosidase activities are present in extracts of tissues/cells from prosaposin knockout (PS–/–) mice and prosaposin-deficient humans (8, 14, 26). These data suggest that saposins might have roles, in addition to being activators, as protective or chaperone proteins for their cognate enzyme. The data presented here support such a role for saposin C in protecting GCase from proteolysis in lysosomes.
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EXPERMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-glucoside, and Triton
X-100 from Sigma; Immortomouse, containing the temperature-sensitive
TAg+ from Charles River Laboratories (Wilmington, MA); goat
anti-actin antibody and alkaline phosphatase-conjugated donkey anti-goat IgG
from Santa Cruz Biotechnology (Santa Cruz, CA); and Bradford reagent system
from Bio-Rad. Dr. Thierry Levade (INSERM, Toulouse, France) provided
SV40-transformed prosaposin-deficient and normal human fibroblasts
(26). Dr. Thomas Kolter
(Kekulé Institüt fur Organishe Chemie und Biochemie, Bonn,
Germany) provided fibroblasts from a human prosaposin-deficient patient. Cell Culture—Hepatocyte cultures were established from the wild type (WT);TAg+ and PS–/–;TAg+ mice. These mice were generated by breeding WT or PS–/– mice with the Immortomouse (TAg+), which contains the temperature-sensitive SV40 T antigen mutant driven by the mouse major histocompatibility complex H-2Kb promoter. PS–/–; TG mice were obtained by breeding PS+/– mice with mice containing the mouse prosaposin transgene (TG) and then back-crossing to obtain PS–/–;TG mice (27). TG expression was driven by the 3-phosphoglycerate kinase promoter. The fibroblast cultures were established as described (27). Mouse hepatocytes (32 °C) and fibroblasts (37 °C) and human fibroblasts were maintained in Dulbecco's modified Eagle's medium + 10% fetal bovine serum + 100 units/ml penicillin and 100 mg/ml streptomycin. For leupeptin treatment, the cells were incubated with media containing leupeptin (50 or 100 µM) for 48 h with one replacement of fresh medium and leupeptin at 24 h. For the saposin loading experiments, human prosaposin-deficient fibroblasts were incubated with medium containing human saposin C or D (75 µg/ml) for 4 days. The pure human saposins were produced in Escherichia coli using the pET 21a system (22).
Enzyme Assays—The cells or tissues were lysed in 1% sodium
taurocholate, 1% Triton X-100. GCase activities were determined
fluorometrically with 4-methylumbelliferyl--glucoside
(28). The activity is
expressed as nmol of 4-methylumbelliferone released/h/mg of protein. Protein
concentrations were estimated using the Bradford reagent system. Student's
t test was used to evaluate the data for Figs.
1,
2, and
4,
5,
6. Data are presented as means
± S.E.
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Immunoblots—The cell lysates were subjected to 12.5% SDS gel
and electroblotted onto polyvinylidene difluoride membranes. After blocking in
1% milk, GCase and -actin were detected by incubation with rabbit
anti-mouse GCase antiserum (1/1000) or goat anti-actin antibody (1/300)
diluted in 0.5% bovine serum albumin followed by treatment with alkaline
phosphatase conjugated to goat anti-rabbit (1/3000) or donkey anti-goat IgG
(1/500) diluted in TTBS (0.05% Tween 20, 20 mM Tris, 0.5
M NaCl, pH 7.4). GCase in human fibroblast cells were detected by
rabbit anti-human GCase antiserum (1/1500) in 1% milk, 1% bovine serum
albumin. The blots were quantified by Amersham Biosciences ImageQuant
software. The values obtained for GCase bands (Mr =
65,000) were normalized to the actin bands. Rabbit anti-mouse GCase was raised
against recombinant mouse GCase produced in E. coli using the pET 21a
system (22).
Pulse-Chase Studies—Proteins in mouse and human fibroblasts were radiolabeled for 1 h with 150 µCi of [35S]cysteine/methionine and chased with nonradioactive media for the indicated times. The radioactively labeled GCase protein in cell lysates were immunoprecipitated using anti-mouse or human GCase antiserum (27). The fibroblasts were matched for passage number and strain. The radioactive GCase bands were quantified by Amersham Biosciences ImageQuant software.
RNA Analyses—Total RNA was extracted using Trizol reagent. Northern blots were performed as described (27) using the mouse GCase cDNA as probe. Real time RT-PCR reactions were performed to quantify mouse prosaposin and human GCase mRNA levels in the ABI Prism 7000 sequence detection system (29). 18 S RNA was used as internal control. The primers for RT-PCR reactions were as follows: mouse prosaposin, 5'-GCGACATATGCAAAACTGTTGTC-3' (forward) and 5'-CACAGGTCTTCTCCAGGTAATGAA-3' (reverse); human GCase, 5'-GCAGTGTCGTGGGCATCA-3' (forward) and 5'-AGACACACACCACCGAGCTGTA-3' (reverse); 18S, 5'-GCATGGCCGTTCTTAGTTGG-3' (forward) and 5'-TGAACGCCACTTGTCCCTCT-3' (reverse).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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5–7% relative to the endogenous prosaposin in the corresponding WT
samples. This low level of prosaposin TG expression was sufficient to increase
the GCase activity by more than 2-fold relative to PS–/– mice
(Fig. 1B). These
results established a relationship between PS expression, the presence of
saposin/prosaposin protein, and the in vitro level of GCase activity.
Clearly, small amounts of PS expression were required to re-establish GCase
activity. Importantly for these studies, the detergent assays used for
estimating GCase in vitro activity do not require negatively charged
phospholipids or saposin C. Indeed, the detergents (taurocholate and Triton
X-100) replace the function of these two more natural activators and provide
an assessment of the maximal catalytic power of GCase in cells
(25). Thus, the activities are
not related to the in vivo levels but rather reflect the level of
catalytically active GCase within cells. Such reductions could be due to
decreased synthesis of GCase, its folding, or its stability within the
cells.
Reduction of GCase Protein in PS–/–
cells—To address the mechanism for GCase activity reductions,
fibroblast and hepatocyte cultures were established from the wild type and
PS–/– mice and used for immunoblot analysis. To eliminate the
potential for diminished GCase gene expression, GCase RNA levels were
determined by Northern analyses and were found to be the same as in WT and
PS–/– samples (Fig.
2A). Importantly, GCase activities in the
PS–/– cell extracts were reduced by 
50% relative to WT
(Fig. 2B). Immunoblots
using anti-mouse GCase antiserum showed a concordant decrease in GCase protein
(Fig. 2C). Essentially
identical results were obtained with prosaposin-deficient cultured human
fibroblasts using rabbit anti-human GCase antibodies
(Fig. 5). These results show
that the reduction of GCase activity in PS–/– cells is due to a
decrease of GCase protein and not because of misfolding of GCase into an
inactive conformer. However, this does not exclude the potential for
diminished translation of the normal amounts of GCase mRNA.
Turnover of GCase Protein in PS–/– and WT
cells—Pulsechase studies were conducted to evaluate the synthetic
levels of GCase protein and its stability in both mouse and human fibroblasts.
Following metabolic labeling and chase, GCase in the cell lysates were
immunoprecipitated using anti-mouse or anti-human GCase antibodies.
Quantitative immunoprecipitation of the GCase in cultured fibroblasts showed
essentially the same levels of GCase signal from WT and PS–/–
cells at 0 h of chase (Fig. 3).
This excludes the possibility of a grossly abnormal translation of the amount
of GCase mRNA in PS–/– cells. However, in WT cultured fibroblasts
GCase protein was detectable for up to 72 h of chase. In contrast, GCase in
PS–/– cells was at very low levels after 24 h of chase
(Fig. 3). The GCases in WT
cells have a t of >24 h and in PS–/– cells a
t of 1 h in mouse or 8 h in human. These results
show that essentially normal amounts of GCase are synthesized in
PS–/– cells and that it was degraded at a much greater rate than
in WT cells. Such enhanced degradation could occur by proteolysis in different
compartments including the endoplasmic reticulum, Golgi, or
endosome/lysosomes. To establish the mechanism of saposin/prosaposin effect on
GCase degradation, studies were conducted with protease inhibitors that exert
their major effects in the lysosome.
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GCase Activity and Protein in PS–/– Cells
Treated with Protease Inhibitors—PS–/– cultured mouse
fibroblasts and hepatocytes were incubated with leupeptin, a cysteine protease
inhibitor. Leupeptin inhibits protein degradation in the lysosome by 85% and
in non-lysosome compartments by 15%
(30). After 48 h of leupeptin
exposure, both GCase activity and protein levels increased by
1.7–2-fold (Fig. 4).
The final GCase activity and protein levels were comparable with those in WT
fibroblasts (Fig. 4). Leupeptin
treatment had little or no effect on WT GCase activities or protein. Treatment
with pepstatin A, an aspartic protease (including cathepsins D and G)
inhibitor, produced an 1.4-fold increase in GCase activity and protein in
both PS–/– cell types (data not shown).
In prosaposin-deficient human fibroblast cells, GCase RNA is at the same
level as in WT control (Fig.
5A). Consistent with mouse samples, GCase activity and
protein levels showed increases of 2- and 1.7-fold, respectively, after
incubation with leupeptin-containing medium for 48 h
(Fig. 5). Cathepsin inhibitor
II, a selective lysosomal cysteine protease inhibitor, increased GCase
activity and protein by 1.3-fold in human PS–/– fibroblasts
(data not shown). Leupeptin (Fig.
5) and cathepsin inhibitor II treatment had no significant effect
on GCase activity and protein in WT controls. The same results were obtained
from primary human fibroblast cells. These results implicate the
cysteine/aspartic protease participation in the degradation of GCase in the
lysosome. In these cells, leupeptin, cathepsin inhibitor II, or pepstatin had
no effect on -glucuronidase activity, a soluble lysosomal enzyme that is
not involved in glycosphingolipid metabolism.
Saposin C Led to Recovery of GCase Activity and Protein in PS–/– Cells—PS–/– cultured human fibroblasts were incubated with human saposin C or saposin D in the medium. The cells loaded with saposin C showed significant increase of GCase activity and protein (Fig. 6). Saposin D had no effect on GCase in PS–/– cells (Fig. 6). The effects of saposins A and B on GCase activity were studied using the fibroblast from saposin A-deficient mice and a human saposin B-deficient patient. No alteration of GCase activity was observed in those cells.
The results from our present study support saposin C as the protector for GCase. Saposins A and C enhance in vitro GCase activity (22, 23), but only isolated saposin C deficiency leads to glucosylceramide storage and a "Gaucher disease-like" phenotype (9, 12). In saposin A mutant mice, no accumulation of glucosylcerebroside was found (13), and GCase activity was not altered in fibroblasts from such mutant mice. Thus, saposin A is not a physiologically important activator or protector for GCase. Furthermore, saposins B and D do not have any effect on GCase activity (31). GCase activity was not reduced in human saposin B-deficient fibroblast. Loading saposin D to PS–/– cells had no effect on GCase. These results show that saposin C has a novel role as the anti-proteolytic protector for GCase in addition to being enzymatic activator.
These studies indicate that the enhanced degradation of GCase in
prosaposin-deficient mice and humans occurs largely in the lysosome. This
finding has two implications. 1) GCase is normally folded and trafficked to
the lysosome in PS–/– cells. The protection by the lysosomally
directed protease inhibitors suggests that endoplasmic reticulum-localized
proteolysis of GCase is unlikely or limited. Also, the presence of several
isoforms of GCase in human prosaposin-deficient fibroblast indicates passage
through the Golgi and consequent oligosaccharide remodeling
(32). 2) The
"anti-proteolytic protective effect" of saposin/prosaposin results
from interaction of GCase and mature saposins and not prosaposin. Prosaposin
is processed proteolytically to the individual saposin in a late endosomal or
lysosomal compartment (6,
33). Thus, these findings
support a mechanism of GCase protection whereby GCase is synthesized normally
and trafficked to the lysosome in PS–/– cells. Upon arrival in the
late endosome/lysosome compartments, GCase interacts with mature saposin C in
such a manner as to inhibit its degradation by lysosomal proteases. The
"protector protein" needed for -galactosidase/neuraminidase
stability and assembly represents a similar example, except that
macromolecular assembly occurs in the Golgi
(34,
35).
The decrease of GCase activity shown in selected tissues of PS–/– mice suggests that saposin C may have a tissue-specific protective effect for GCase. For example, GCase in vitro activities are reduced in liver and kidney tissues but are not significantly altered in the brain of PS–/– mice. GCase is a lysosomal membrane-associated protein, and negatively charged phospholipids are required for the activation effect. In prosaposin-deficient cells, the disrupted sphingolipid metabolism appears to lead to a modification of lysobisphosphatidic acid organization.2 Because this lipid may be the physiologic negatively charged phospholipid for GCase activation, saposin C deficiency may alter the interaction of GCase with this lipid and lead to proteolytic degradation. The degree of such phospholipid reorganization may be different in various tissues, particularly in the brain. This degree of lipid alteration also might contribute to the tissue-specific reduction of GCase activity in PS–/– mice. In addition, the group of proteases participating in degradation of GCase could be differentially expressed in the brain. The underlying mechanism of such tissue specificity needs to be investigated.
It is unlikely that all of the glycosphingolipid activators would have
protective functions for their cognate enzyme. -Hexosaminidase A, the
cognate enzyme for GM2 activator, maintained nearly normal activity
in GM2 activator-deficient mice
(36) and humans
(37). Arylsulfatase A, which
requires saposin B for optimal activation, showed normal levels in both human
and mice with either saposin B or prosaposin deficiency
(14,
38,
39). The level of
arylsulfatase A protein was normal in PS–/–
fibroblasts.2 In
comparison, saposin A is essential for galactosylcerebrosidase to achieve
maximal activity (40). The
mouse models with deficient saposin A or prosaposin show decreased in
vitro galactosylcerebrosidase activity
(13,
14). Decreased acid ceramidase
activity also was observed in prosaposin-deficient patient cells
(26,
41) and prosaposin-deficient
mouse tissues. However, the acid ceramidase protein level was not altered in
PS–/–
samples.2 Whether
galactosylcerebrosidase was protected by saposin A and the possibility of
saposin D being the chaperone for acid ceramidase are under investigation.
In vitro and ex vivo turnover studies shows that GCase interacts with the inner lysosomal membrane (21, 42, 43). Using artificial membrane systems of negatively charged phospholipids, saposin C facilitates the association of GCase with the membranes to favor the degradation of glucosylcerebroside (44). The membrane attachment is essential for GCase stability and catalytic function. In addition to its activator functions, saposin C, as a protector, might play the role of enhancing the stability and efficiency of GCase in vivo by facilitating the association of enzyme to membrane. These functions of saposin C have implications for understanding the molecular pathogenesis of Gaucher disease and the development of more efficient therapies. Most of the GCase mutations that cause Gaucher disease disrupt the inherent catalytic function and/or the proteolytic stability of the mutant enzyme (45, 46). The latter results primarily from misfolding of GCase, but one could envision specific mutations that preclude or diminish interaction of GCase and saposin C. This could lead to an unstable enzyme, which could have nearly normal in vitro intrinsic residual activity as assessed with detergents. Certainly, mutations that affect the saposin C/GCase interaction site would lead to greater decreases of in vivo activity than expected from the GCase mutation alone. Similarly, disruption of the normal stoichiometry between GCase and saposin C would lead to proteolytic susceptibility of GCase. For example, GCase can be supplied by delivery to macrophages as purified enzyme or by over-expression in selected cells, e.g. a gene therapy approach. The delivery of large excesses of GCase to these cells could overwhelm the saposin C available for interaction. This excess enzyme would be very susceptible to proteolytic degradation and diminish the expected therapeutic benefit. Thus, preservation of the appropriate stoichiometry of saposin C and GCase could significantly enhance the efficacy/cost benefit of such therapeutic approaches.
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FOOTNOTES |
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To whom correspondence should be addressed: Division and Program in Human
Genetics, Children's Hospital Medical Center, 3333 Burnet Ave., Pavilion
E5.253, Cincinnati, OH 45229-3039. Tel.: 513-636-7290; Fax: 513-636-2261;
E-mail:
greg.grabowski{at}chmcc.org.
1 The abbreviations used are: GCase, acid -glucosidase; WT, wild type;
TG, transgene; PS, prosaposin/saposin; RT-PCR, reverse transcriptase PCR.
2 X. Qi, Y. Sun, and G. A. Grabowski, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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