(Received for publication, October 20, 1995; and in revised form, January 5, 1996)
From the
Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8` or 8, respectively, into the SAP-B coding region of the transcribed mRNAs.
To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D).
In order to determine the biological function of the three
different SAP-B isoforms, SAP-precursor-deficient human
fibroblasts were loaded with recombinant SAP-precursor proteins with or
without 2- and 3-amino acid insertions, respectively, purified from the
medium of the baby hamster kidney cells. They were found to stimulate
at nanomolar concentrations the turnover of biosynthetically labeled
ceramide, glucosylceramide, and lactosylceramide. Since the
physiological function of SAP-B is to stimulate the degradation of
sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by
-galactosidase (EC 3.2.1.23) loading studies with the respective
exogenously labeled lipids on SAP-precursor-deficient fibroblasts were
performed. Addition of different purified SAP-precursors to the medium
of the lipid-loaded fibroblasts showed positive stimulation of the
lipid degradation by all three SAP-B isoforms derived from the
SAP-precursors. These findings establish that all three forms of the
SAP-B can function as sulfatide/globotriaosylceramide
activator.
The physiological degradation of glycosphingolipids with short
oligosaccharide chains is catalyzed by lysosomal exohydrolases in the
presence of small, heat-stable glycoproteins, so called sphingolipid
activator proteins (SAPs) ()or saposins(1) . Four of
the five known activator proteins (SAP-A to SAP-D or saposins
A-D) are derived from a common precursor polypeptide by
proteolytic processing(2, 3) . This precursor also
called prosaposin was identified as a protein of 68 kDa in human skin
fibroblasts by studying the synthesis and processing of
SAP-C(4) . After modification of its N-linked
oligosaccharides in the Golgi apparatus, a 73-kDa form is secreted into
the culture medium. Processing to the mature polypeptides of 8-13
kDa takes place after the transport of the precursor to the acidic
organelles of the cells.
Although different activating properties
for the SAPs (SAP-A to SAP-D) have been described, their physiological
relevance has been established only for SAP-B and SAP-C(1) .
The absence of SAP-C in human patients with Gaucher disease shows the
relevance of this activator protein for the degradation of
glucosylceramide by glucosylceramidase (EC 3.2.1.45) (1, 5) . While SAP-A is known to stimulate the
breakdown of several glycosphingolipids in
vitro(6, 7) , SAP-D has recently been
shown to be involved in ceramide degradation in
vivo(8) . This was established in an in vivo test
system, in which the breakdown of
[C]serine-labeled sphingolipids accumulating in
human fibroblasts derived from patients with SAP-precursor deficiency (9) was stimulated by addition of different SAPs (SAP-A to
SAP-D) to the culture medium.
SAP-B itself has capacity to bind
several sphingolipids, but its genetic defect leads to an accumulation
of sulfatides, globotriaosylceramide, digalactosylceramide, and
ganglioside G in a disorder classified as metachromatic
leukodystrophy(1, 10) .
The SAP-precursor gene localized on the long arm of human chromosome 10 (10q21-22) (11) consists of at least 15 exons, four of which (exon 6-9) code for SAP-B. Exon 8 consists of only 9 base pairs (12) and can be spliced differentially, generating three different cDNAs(13, 14) : one containing all 9 bases of exon 8, one containing the downstream 6 bases of exon 8 (also called exon 8`) and one completely lacking this exon (Fig. 1). At the protein level, only SAP-B without the amino acids derived from exon 8 has been detected so far(10, 15) . A recent report shows a differential tissue distribution of the mRNAs with or without exon 8 in different cell lines or tissues(16) . The multiple mRNA species were also detected in the mouse(17) . These findings prompt the question whether the different sequences within the SAP-B domain in the SAP-precursor have any biological significance. In the present report we describe the expression and processing of the three SAP-precursor forms in BHK cells stably transfected with the respective cDNAs. In addition, the stimulation of sulfatide and globotriaosylceramide degradation by the three SAP-B isoforms was tested in vivo in a cell culture system by adding purified recombinant precursor proteins (designated as SAP-precursor 0, 6, or 9) to the medium of lipid loaded human SAP-precursor-deficient fibroblasts.
Figure 1:
Relationship
of SAP-B to the human SAP-precursor gene (according to (14) ). a, structure of the human SAP-precursor gene, which occupies
about 20 kb of the long arm of chromosome 10. Open boxes correspond to exons 2-15 covering the cDNA sequence from the
codon Ala to the end. Black areas correspond to
untranslated regions. The putative exon 1 (in brackets) which
should cover the missing 5` end of the gene has not yet been
identified. b, the SAP-precursor cDNA. Exons and untranslated
regions are indicated as in a. c, the SAP-precursor
protein (527 amino acids). Domains A-D correspond to the mature
activator proteins A-D. N-Glycosylation sites are marked
by the closed circles. d, mature SAP-B. The mature
SAP-B is encoded on exons 6-9 of the prosaposin gene. Alternative
forms of SAP-B are encoded by different mRNA species generated by
splicing exon 7 either to exon 8 or to the last 6 bases of exon 8 (exon
8`) or directly to exon 9.
All other commercially available materials were obtained from the following suppliers: Sigma, Merck (Darmstadt, Federal Republic of Germany), Pharmacia LKB Biotechnologies (Freiburg, Federal Republic of Germany), and New England Biolabs.
[H]Sulfatide, which was
labeled in the sphingoid moiety, and
[
H]globotriaosylceramide labeled in the terminal
galactosyl residue were synthesized in our laboratory according to
published procedures(18, 19) .
The preparation of the goat anti-SAP-B and anti-SAP-D antisera has been described earlier(8) .
Stably transfected BHK cells were obtained by cotransfection of the expression plasmids and of the plasmids pSV2 pac and pSV2 neo conferring resistance to puromycin and G418 sulfate to the cells(27) , as described previously(28) . BHK cells transfected only with the expression plasmid pBHE were used as control.
Cell extracts were prepared with phosphate-buffered saline containing 1% Nonidet P-40, 10 mM EDTA, 2 mM phenylmethanesulfonyl fluoride, 1 mM pepstatin A and leupeptin each. Immunoprecipitations using the corresponding anti-SAP-antisera were performed as described previously(9) . Deglycosylation of the precipitated proteins with peptide-N-glycanase F and Endo H was performed according to the manufacturer's instructions. Immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis using gradient gels (7-17%). The gels were impregnated with Amplify for fluorography and exposed to the Kodak X-Omat AR film.
Figure 2:
Biosynthesis of the SAP-precursor isoforms
in transfected BHK cells. BHK cells transfected either with the
expression plasmids pBHE0, pBHE6, pBHE9, or only the vector pBHE
without a cDNA insertion as a control (Co) were pulsed with
[S]methionine for 1 h and chased for the times
indicated. SAP-precursors and SAP-C from the cell extracts and the
media were immunoprecipitated using a rabbit anti-SAP-C antiserum and
were analyzed by denaturing gel electrophoresis and fluoropgraphy. A
faint band at 46 kDa, detectable only in the immunoprecipitations of
the cell extracts and denoted by an asterisk, is caused by
unspecific contaminations of
immunoprecipitates.
This labeling experiment indicated formation of similar polypeptides in BHK cells transfected with the different SAP-precursor cDNAs as described for in human fibroblasts(4) . The different amounts of immunoprecipitable proteins in the transfected BHK cells were due to the fact that the totality of BHK cells resistant to the antibiotics were used for pulse-chase experiments and that the selection leaves varying numbers of non-resistant cells.
In order to assess correct processing of all three different recombinant SAP-precursor to the four mature SAPs (SAP-A to SAP-D) and to analyze their carbohydrate structures, the BHK transfectants were metabolically labeled for 5 h, and the cell extracts were subjected to immunoprecipitation with polyclonal antisera against the corresponding mature SAP-A to SAP-D (Fig. 3). All three SAP-precursors resulted in formation of mature SAP-A, SAP-B, SAP-C, and SAP-D, differing with respect to their carbohydrate types as analyzed previously(34, 35) . In our hands, mature SAP-A never yielded a clear band in its glycosylated form, but only a broad patch ranging from 14.5 to 29 kDa. Upon deglycosylation, however, a clear band comparible in size with deglycosylated mature SAP-B, SAP-C, and SAP-D was obtained. The carbohydrate chains of SAP-A were Endo H-resistant, presumably being of the complex type, whereas the carbohydrate moieties of SAP-C and SAP-D were predominantly of the Endo H-sensitive high mannose or hybrid type. It is noteworthy that each SAP-precursor generated a mature SAP-B with an Endo H-resistant carbohydrate chain.
Figure 3:
Immunoprecipitation and N-glycosylation analysis of all mature SAPs (SAP-A to SAP-D)
from transfected BHK cells. The BHK cells transfected with the
expression plasmids pBHE0, pBHE6, and pBHE9 were labeled with
[S]methionine for 5 h. The mature SAPs (SAP-A to
SAP-D) were immunoprecipitated with the corresponding antisera as
indicated and treated either with peptide-N-glycanase F (PNGase F) or Endo H as described under ``Experimental
Procedures.''
Figure 4:
Endocytosis of SAP-precursor isoforms by
cultured human fibroblasts from a patient with SAP-precursor
deficiency. Medium from [S]methionine-labeled
BHK cells transfected with the SAP-precursor cDNA isoforms (pBHE0,
pBHE6, and pBHE9) or with the expression plasmid pBHE (Co)
were added to the culture medium of unlabeled fibroblasts derived from
a patient with SAP-precursor deficiency. After 24 h the fibroblasts
were harvested and analyzed for internalized immunoprecipitable SAP-B,
SAP-C, and SAP-D. Immunoprecipitations were carried out from the media
with an anti-SAP-C antiserum and from the cell extracts with the
antisera indicated. Asterisk, the faint bands visible in the
control precipitation with the anti-SAP-D antiserum presumable arose
from endogenous BHK cell SAP-precursor cross-reacting with this
antiserum.
Figure 5:
Concentration dependence of the turnover
of labeled sphingolipids in cultured fibroblasts on the complementation
of the culture medium with SAP-precursor9. Fibroblasts from a patient
with SAP-precursor deficiency and a normal control (NC) were
incubated for 24 h with medium supplemented with recombinant
SAP-precursor9 as indicated. Subsequently the cells were incubated with
[C]serine (1 µCi/ml) for 24 h. The medium
was changed and the cells were chased for 120 h with a medium
containing unlabeled serine and the same concentration of recombinant
SAP-precursor9 used during the preincubation period. After harvesting
of the cells, the sphingolipid fraction was isolated, equal amounts of
radioactivity were separated by TLC, and the radioactive spots
identified by autoradiography as described under ``Experimental
Procedures.'' SM, sphingomyelin.
In order to demonstrate the same effect on the lipid
turnover for all three purified prosaposins, the
[C]serine pulse-chase labeling experiment was
repeated, adding each SAP-precursor (0, 6, or 9, 0.5 µg/ml medium
of each) to the culture medium of the deficient fibroblasts during the
preincubation and chase periods (Fig. 6). To evaluate the
influence of the material copurified with recombinant SAP-precursors,
the control fractions from the media of untransfected BHK cells, as
described above, was also offered to labeled mutant fibroblasts.
Addition of all three SAP-precursors decreased the amount of
accumulated ceramide, lactosylceramide, and glucosylceramide in
quantitatively similar manner, while no significant influence on the
turnover of these lipids was detectable by the BHK control fraction
(Co).
Figure 6:
Effect of the different SAP-precursor
isoforms on the turnover of labeled sphingolipids in cultured
fibroblasts. Fibroblasts from a patient with SAP-precursor deficiency
and a normal control (NC) were incubated for 24 h with
recombinant SAP-precursor isoforms purified from the medium of
transfected BHK cells (0.5 µg/ml medium SAP-precursor 0, 6, or 9)
and with an equal volume of control protein fraction isolated from BHK
cells transfected with the expression plasmid pBHE (Co).
Subsequently the fibroblasts were incubated for 24 h with
[C]serine (1 µCi/ml). Then the medium was
changed, and the cells chased for 120 h with medium supplemented with
unlabeled serine and with recombinant SAP-precursor 0, 6, or 9 as used
in the preincubation period. After the cell harvest the sphingolipid
fraction was isolated, equal amounts of radioactivities were separated
on TLC and identified as described under ``Experimental
Procedures.'' SM, sphingomyelin.
One known physiological function of SAP-B is to stimulate the
degradation of sulfatide by arylsulfatase A. Since the metabolic
labeling of sphingolipids with [C]serine in
cultured fibroblasts resulted only in the pronounced labeling of those
sphingolipids which are endogenously synthesized in fibroblasts, the
effect of SAP-B isoforms on the turnover of sulfatide was determined by
loading experiments. [
H]Sulfatide was added to
the medium of the cultured fibroblasts and during the preincubation and
chase periods the culture medium was supplemented with 0.5 µg/ml of
each purified SAP-precursor isoform. The metabolic fate of the
[
H]sulfatide taken up by the cells was examined
by TLC (Fig. 7). While in untreated SAP-precursor-deficient
fibroblasts about 97.6 ± 6.0% of the total radioactivity
remained in the accumulated sulfatide, its proportion decreased to 89.2
± 4.1% in the cells treated with each of the recombinant
SAP-precursor isoforms. In contrast, treatment with the control protein
fractions (Co) had no effect on sulfatide turnover. In normal
fibroblasts up to 45.2 ± 15.7% of the radioactivity was
detectable in the sulfatide fraction.
Figure 7:
Effect of the SAP-precursor isoforms on
the metabolism of endocytosed [H]sulfatide.
Fibroblasts derived from a patient with SAP-precursor deficiency and a
normal control (NC) were incubated with SAP-precursor 0, 6, or
9 purified from transfected BHK cells (0.5 µg/ml medium) and an
equal volume of BHK cell control protein fraction (Co) for 24
h. Then the fibroblasts were incubated for 48 h with
[
H]sulfatide (0.288 µCi/ml). Subsequently the
cells were chased for 120 h with fresh medium supplemented with the
same amount of SAP-precursor 0, 6, or 9 as used in the preincubation
period. The cells were harvested and the sphingolipid fractions
isolated. Radioactivity corresponding to equal amounts of cell protein
was separated on TLC and the spots identified as described under
``Experimental Procedures.''
The presence of the radiolabeled ceramide and sphingomyelin, arising from degradation of sulfatide and reutilization of ceramide in all SAP-deficient fibroblasts cultured in present of the purified proteins as well as in the normal control clearly pointed out that all SAP-B isoforms have been able to stimulate the sulfatide degradation in vivo (Fig. 7).
An analogous feeding experiment was performed
with [H]globotriaosylceramide in order to examine
the function of all three SAP-B isoforms on the degradation of
globotriaosylceramide (Fig. 8). Due to the fact that the label
was located in the terminal galactose residue, no metabolic products
were detectable. In the normal control cells, the radioactivity of the
isolated globotriaosylceramide constituted 32.2 ± 17.5% of total
radioactivity. In the mutant fibroblasts, untreated and treated with
the control protein fractions (Co), globotriaosylceramide
bound radioactivity remained 78.9 ± 21.8% and 81.9 ±
18.1% of the total radioactivity, respectively. In
SAP-precursor-deficient fibroblasts cultured in the presence
of purified SAP-precursor 0, 6, or 9, a decrease of the accumulated
globotriaosylceramide was generally detectable (62.9 ± 11.1% of
the total radioactivity), indicating that all three SAP-B
isoforms stimulate degradation of globotriaosylceramide to a similar
degree.
Figure 8:
Effect of the SAP-precursor isoforms on
the metabolism of endocytosed
[H]globotriaosylceramide. Fibroblasts from a
patient with SAP-precursor deficiency and a normal control (NC) were preincubated with 0.5 µg/ml SAP-precursor 0, 6,
or 9 or BHK control protein (Co) for 24 h. Then the cells were
incubated with [
H]globotriaosylceramide (GTC) (0.231 µCi/ml) for 48 h. Subsequently the cells were
chased for 120 h with fresh medium supplemented with the same
SAP-precursor 0, 6, or 9 or control protein as used during the
preincubation period. After cell harvest the sphingolipid fractions
were isolated, radioactivity corresponding to same amounts of cell
proteins separated on TLC and the radioactive spots identified as
described under ``Experimental
Procedures.''
That the SAP-precursor gene is alternatively spliced producing three different cDNAs, with or without inclusion of 9 or 6 base pairs of exon 8 has been known for several years(14) , but no protein product encoded by the longer cDNAs has been detected in tissues so far. This can be due to several reasons: the translation products of the longer cDNAs may be processed differently and generate different sets of final products, the longer SAP-B forms may be unstable, or expression pattern of the sources from which SAP-B was usually purified favors the protein without the insertion(15) . The results presented in this report indicate that all three polypeptides encoded by the different SAP-precursor cDNAs are transported and processed in the same manner as described for SAP-precursor in human fibroblasts and that all three SAP-B isoforms are stable and have the same sphingolipid activator function(4) .
Recently, the relative abundance of the
alternatively spliced mRNA with or without 9 bases was determined by
reverse transcriptase-polymerase chain reaction in various human
tissues and cell lines(16) , but it was difficult to correlate
the presence of the mRNA forms with the occurrence of mature SAP-B at
the protein level, especially as there are no antibodies available to
differentiate between the three protein forms. Since we have shown that
the maturation of SAP-B proceeds in a comparable way in BHK cells and
in human fibroblasts, the question arises if there is a significance
for the alternative spliced mRNAs and their protein products. The
detection of immunoprecipitable, stable mature SAP-B derived from the
different SAP-precursors allowed the conclusion that the stability of
mature SAP-B is not affected dramatically by these insertions.
Examination of several patients suffering from genetic SAP-B deficiency
demonstrated that disruption of the correct protein structure of SAP-B
led to lack of cross-reacting material in the tissues or fibroblasts of
these patients followed by an accumulation of sulfatides and
globotriaosylceramide(1) . One of these patients had a
G
C mutation leading to substitution of a cysteine
residue for a serine (14) and no mature SAP-B was detectable in
patient's tissue and fibroblasts by immunological
methods(36) . A deletion at the N terminus of the SAP-B domain
due to a splice site mutation in the prosaposin gene had the same
effect(37) . In a third case, the insertion of 33 additional
bases by a point mutation introducing a new splice site at the 5` end
of exon 8 resulted in the insertion of 11 amino acids into the
SAP-B region of the SAP-precursor. No stable SAP-B was
released from this precursor either (38, 39) .
The expression studies in BHK cells revealed that the major part (more than 60%) of the expressed SAP-precursors was secreted into the culture medium and that a minor amount was targeted to the acidic organelles of the BHK cells where proteolytic cleavage to the mature SAPs (SAP-A to SAP-D) took place. An analogous phenomenon was reported for the expression of cathepsin D precursor in BHK cells and the authors concluded that if synthesized at a higher rate a particular group of lysosomal proteins was secreted into the medium without concomitantly compromising the targeting of other lysosomal enzymes (40) .
Observations that the SAP-precursor occurs in different human fluids such as milk, cerebrospinal fluid, and seminal plasma (41, 42) and the fact that all three isoforms were secreted into the medium of the transfected BHK cells call for a detection system for the individual SAP-precursor isoforms. Such a method would allow determination of the existence and amounts of the different protein forms in different body fluids. In addition to findings that demonstrated binding and transport of gangliosides by the SAP-precursor(43) , this protein has been suggested to act as a neuroprotective or regenerative agent in vivo(44) . More recently, the SAP-precursor has been suggested to be a neurotrophic factor(45) , because it elicited differentiation of neuroblastoma cells when applied in the nanomolar range. The neurotrophic function is said to be localized on the SAP-C domain(46) , indicating that alternative splicing does not modulate this function.
For our studies on the stimulatory effect of SAP-B on the lysosomal degradation of sphingolipids we purified the prosaposin isoforms from the media of transfected BHK cells. In all preparations obtained by the purification procedure, partially processed intermediates copurified with 73-kDa precursor form. A similar phenomenon was observed during the first purification steps of SAP-precursor from the medium of infected Sf9 cells, seminal plasma, and milk(29) . The intermediates were identified to be tri- and disaposins, and a possible pathway of proteolysis of SAP-precursor was discussed(29) . However, our endocytosis studies demonstrate clearly that all endocytosed higher protein forms were processed within 24 h to the mature SAPs (SAP-A to SAP-D). Therefore all effects shown on lipid degradation (in the time period between 24 and 168 h after feeding of SAP-precursor 0, 6, or 9, see Fig. 4and Fig. 5) were caused by mature SAP forms rather than by the SAP-precursor or partially processed intermediates.
It has been
shown previously that alternative splicing causing a polymorphism at
the protein level may alter the binding specificity and/or function of
the expressed proteins, including hormone precursors, DNA-binding
proteins and structural proteins(47, 48) . A recent
report indicated that synthetic peptides derived from the
SAP-B amino acid sequence (from Ser to
Glu
with or without the Gln-Asp-Gln insertion) have
different binding affinities for G
ganglioside, sulfatide,
and sphingomyelin(16) . From these results the authors
suggested that alternative splicing of the SAP-precursor gene may
change binding specificity in the encoded SAP-B presumably to adapt to
the variable sphingolipid composition of tissues. However, our
observations indicate that all SAP-B isoforms are able to stimulate the
degradation of sulfatide and globotriaosylceramide in cultured
fibroblasts without significant differences among them.
Two models
have been proposed for the structure of SAP-B. Potier based his model
on sequence homology of SAP-B with influenza virus
neuraminidase(49) . The model predicts a high proportion of
-sheet and one
-helix at the C-terminal end of SAP-B, which
should contain the additional two or three amino acids inserted by
alternative splicing(16) . Based on the disulfide bonding
pattern the second model suggests a four-helix bundle
structure(11, 50) . In this model the additional amino
acids would also be placed into a
-helical structure with the
majority of the hydrophobic residues forming a potential lipid binding
core in the interior of the molecule. A mechanism has been proposed for
the interaction of SAP-B with membrane surfaces and for the binding of
single lipid molecules(11) . In this model the helices are
oriented parallel to the lipid head group. After extraction of the
lipid from the membrane the SAP-B helices should create a hydrophobic
environment for the lipid hydrocarbon tail.
Recently, a structural
model for a family of proteins called saposin-like proteins (SAPLIP)
was discussed basing on the four -helical bundle first proposed by
Stevens et al.(50) and using crystal structural data
derived from hemerythrin as a template for modeling SAP-B(51) .
In this model the inserted 3 amino acids are located between helices
III and IV and might affect lipid binding directly or indirectly.
All these different models point to the fact that only structural work on SAP-B and its different isoforms can test the validity of the above models and may indicate the possible functional significance of the amino acids insertions.