From the
Saposins A, B, C, and D are a group of homologous glycoproteins
derived from a single precursor, prosaposin, and apparently involved in
the stimulation of the enzymatic degradation of sphingolipids in
lysosomes. All saposins have six cysteine residues at similar
positions. In the present study we have investigated the disulfide
structure of saposins B and C using advanced mass spectrometric
procedures.
Electrospray analysis showed that deglycosylated
saposins B and C are mainly present as 79- and 80-residue monomeric
polypeptides, respectively.
Fast atom bombardment mass analysis of
peptide mixtures obtained by a combination of chemical and enzymatic
cleavages demonstrated that the pairings of the three disulfide bridges
present in each saposin are Cys
We have recently shown that saposin C interacts with
phosphatidylserine-containing vesicles inducing destabilization of the
lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R.,
Serafino, A., and Barca, A. (1994) FEBS Lett. 349,
181-186); this perturbation promotes the binding of the lysosomal
enzyme glucosylceramidase to the vesicles and the reconstitution of its
activity. It was presently found that the effects of saposin C on
phosphatidylserine liposomes and on glucosylceramidase activity are
markedly reduced when the three disulfide bonds are irreversibly
disrupted. These results stress the importance of the disulfide
structure for the functional properties of the saposin.
Saposins A, B, C, and D are a group of structurally similar
glycoproteins derived by proteolytic processing from a single precursor
protein, prosaposin
(1, 2, 3) . All saposins
contain about 80 amino acids and have 6 identically placed cysteine
residues.
The apparent function of the saposins is that of promoting
the enzymatic degradation of sphingolipids in lysosomes
(1, 2, 3) . It has been reported that saposin A
(Sap A)
The physiological significance of Sap B
and C has been unequivocally assessed by the discovery that a defect of
Sap B results in a variant form of metachromatic leukodistrophy, and a
defect of Sap C causes a variant form of Gaucher disease
(1, 2, 3) . The tissue accumulation of
sulfatides in the first case and of glucosylceramides in the second
indicates that in vivo Sap B has a fundamental role in the
regulation of arylsulfatase A activity and Sap C in that of
glucosylceramidase. The physiological role of Sap A and D is still
unclear.
The functional properties of Sap B and C have been
extensively investigated in vitro. The mechanism by which Sap
B promotes sphingolipid degradation has been well characterized by
showing that it forms water-soluble complexes with several
sphingolipids (sulfatide, globotriaosylceramide, ganglioside
G
Unlike Sap B, Sap C
was thought to stimulate glucosylceramidase by directly interacting
with the enzyme rather than with the sphingolipid substrate
(5, 9) . In contrast with this assumption, we have
recently demonstrated that the glucosylceramidase activation by Sap C
is a consequence of Sap C binding to acidic phospholipid-containing
liposomes. The association with the saposin affects the lipid
organization of the bilayer in such a way to promote the binding of
glucosylceramidase to the liposomes and the reconstitution of the
enzyme activity
(10, 11) .
A closer insight into the
function of Sap B and C requires the complete knowledge of their
structures. While the amino acid sequence
(12, 13, 14) and the nature and position of the carbohydrate moieties
(15, 16) have been firmly established, the location of
the disulfide bridges is still missing. This is a key point for the
assessment of saposin conformation, considering that the disulfide
cross-links limit the number of possible folded structures a protein
can assume.
In the present work, the disulfide structure of Sap B
and C has been elucidated. Moreover, the importance of the disulfide
bonds for the Sap C capacity to interact with phosphatidylserine (PS)
bilayers and to stimulate glucosylceramidase activity has been
investigated.
After CNBr treatment, Sap B and C were deglycosylated and then
submitted to different proteolytic digestions. Deglycosylation was
carried out by incubation with Peptide N-glycosidase F (1 unit
for 200 µg of saposin) at 37 °C for 16 h in 0.4% ammonium
bicarbonate buffer, pH 8.5, followed by lyophilization of the sample.
Pepsin hydrolysis of CNBr-treated Sap C was performed in 5% formic
acid at room temperature for 4 h (enzyme:substrate, 1:50 (w/w)). An
aliquot of the peptide mixture obtained after pepsin hydrolysis was
then subdigested with endoprotease Glu-C in 0.4% ammonium acetate, pH
4.0, at 40 °C for 6 h.
Tryptic, chymotryptic, and endoprotease
Asp-N digestions of CNBr-treated Sap B were carried out in 0.4%
ammonium bicarbonate, pH 8.5, at 37 °C (enzyme:substrate, 1:50
(w/w)) for 4 h in the case of tryptic and chymotryptic hydrolyses and
overnight for endoproteinase Asp-N digestion.
All the digested
samples were lyophilized, resuspended in 5% acetic acid or 0.1
M HCl, and directly analyzed by mass spectrometry.
Fast atom bombardment (FAB) spectra were recorded
on a VG ZAB 2SE double focusing mass spectrometer fitted with a VG
cesium gun operating at 25 kV (2 µA). Samples were dissolved either
in 5% acetic acid or in 0.1 M HCl, and 2-µl aliquots
(about 30 pmol) were loaded onto a glycerol-coated probe tip;
thioglycerol was added just before inserting the probe into the ion
source. Spectra were recorded on ultraviolet-sensitive paper and
manually counted; signals were assigned on the basis of their molecular
masses with the aid of a suitable computer program
(19) .
The signals at m/z 1929 and 1858
were assigned to the peptide pairs
(25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) +
(44, 45, 46, 47, 48) and
(25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) +
(45, 46, 47, 48) ,
respectively, which originated from enzymatic hydrolyses of the peptide
pair
(20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) +
(44, 45, 46, 47, 48) , thus
confirming the occurrence of a disulfide bridge between Cys
The conservation of the cysteine residues along the sequence
of saposins (A, B, C, and D) indicates the importance of the disulfide
arrangement for the function of this group of proteins. In the present
study we have investigated the location of disulfide bridges in Sap B
and C. Since native saposins are resistant to proteolytic attack
(28, 29) , we have obtained fragments suitable for an
unambiguous determination of their disulfide structure by combining
CNBr cleavage of native Sap B and C with proteolytic digestion. Using
advanced mass spectrometric procedures, it was established that in both
saposins the first two half-cystine residues are disulfide-linked to
the last two, and two half-cystine residues centrally placed are linked
to each other, the actual pairing being as follows:
Cys
Recently, we showed that Sap C
binds with high affinity to PS-containing vesicles
(10) . Upon
association with Sap C, the lipid surface is perturbed and, under
appropriate conditions, the vesicles can also fuse
(11) . These
effects occur at low but not at neutral pH, suggesting that Sap C may
participate in some membrane related function associated with acidic
cell compartments, such as lysosomes, where mature saposins are
localized
(1, 2, 3) . So far, information on the
importance of the disulfide bonds for the Sap C interaction with
membranes has not been reported. By comparing the destabilizing power
of native and S-carboxymethylated saposin, we have found that
the capacity of Sap C to perturb PS membranes is greatly reduced when
the three disulfide bonds are irreversibly disrupted. Most likely,
disulfide bonds contribute to the maintenance of functional integrity
of Sap C and changes in the gross conformation of the molecule might
result in a decreased affinity toward lipid surfaces and/or in a
reduced penetration into the bilayer. S-Carboxymethylation
also causes a reduction of the Sap C capacity to stimulate
glucosylceramidase. This result further supports our previous findings
showing that the glucosylceramidase activation by Sap C mainly depends
on the saposin-induced perturbation of PS-containing membranes
(10, 11) .
In vivo, the failure to form
normal disulfide bonds in the Sap C molecule may have negative
consequences for the glucosylceramidase action. In fact, mutations in
the coding region of Sap C that result in the substitution of
Cys
It has
been noted previously that saposins have a remarkable homology with the
surfactant-associated protein B (SP-B)
(34) , a protein that
presumably enhances the adsorption of surfactant lipids to the
air-water interface of the lung
(35, 36, 37, 38) . All these proteins
are similar in size and in the placement of several amino acids,
including the six cysteine residues. The present results show that also
their disulfide structure, with one central big loop surrounded by two
smaller ones, is identical
(35, 36) . This further
similarity strongly suggests a relatedness between saposins and SP-B.
Despite their different roles and localization, a similarity in the
mode of action of SP-B, Sap B, and Sap C can be pointed out. In fact,
experimental data indicate that to express their functional properties
all these proteins interact with lipids, SP-B with the major
phospholipids of the pulmonary surfactant
(37, 38) , Sap
C with acidic phospholipids
(10) , and Sap B with sphingolipids
(3) . Moreover, SP-B and Sap C show strikingly similar effects
on model membranes such as liposomes, since both proteins are able to
increase the permeability of acidic phospholipid-containing bilayers
and, under appropriate conditions, to induce liposome fusion
(11, 37, 38) .
Interestingly, another
disulfide-rich protein of 77 amino acids, the pore-forming amoeba
peptide, has a cysteine pattern identical with that of saposins and
SP-B
(39) and preferentially inserts into negatively charged
phospholipid bilayers causing perturbation of the membrane
(40) . This observation suggests that the conserved six cysteine
residues might form a common motif for stabilizing a favorable
structure to affect the biophysical properties of membranes.
The
availability of detailed information about structural similarities
between saposins and homologous proteins will help to reach a deeper
understanding of saposin functions.
Sap C was treated with CNBr, deglycosylated, and
then digested with pepsin. An aliquot of the peptide mixture obtained
after pepsin hydrolysis was subdigested with endoprotease Glu-C.
Sap B was treated with CNBr, deglycosylated, and
then digested with trypsin. An aliquot of the peptide mixture obtained
after trypsin hydrolysis was subdigested with endoprotease Asp-N and
chymotrypsin.
This paper is
dedicated to the memory of our much missed colleague and friend
Gianpaolo Nitti.
We thank Prof. D. Barra for helpful discussions, E.
Raia for technical assistance, and G. M. Spinelli for reading the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Cys
,
Cys
-Cys
, Cys
-Cys
for
saposin B and Cys
-Cys
,
Cys
-Cys
, Cys
-Cys
for
saposin C.
(
)
and saposin C (Sap C) stimulate the
hydrolysis of glucosylceramide and galactosylceramide
(2, 4, 5, 6) , saposin B (Sap B) that of
several sphingolipids including sulfatide and G
ganglioside
(3, 7) , and saposin D (Sap D) that of
sphingomyelin
(8) .
, etc.) thus making them accessible to the respective
sphingolipid hydrolases
(3, 7) .
Materials
PS from bovine brain was from Avanti
Polar Lipids.
1,2-Dioleoyl-3- sn-phosphatidyl-L-[3-C]serine
(54 mCi/mmol) was from Amersham International. Calcein was from
Molecular Probes. 4-Methylumbelliferyl-
-D-glucopyranoside
was obtained from Koch-Light Laboratories. Peptide
N-glycosidase F, endoproteinase Glu-C, and endoproteinase
Asp-N were from Boehringer Mannheim. Trypsin, chymotrypsin, pepsin,
cyanogen bromide (CNBr), glycerol, and thioglycerol were purchased from
Sigma. Phenylisothiocyanate was obtained from Pierce. The PLRP-S
reverse-phase column was from Polymer Laboratories, Amherst, MA. All
other chemicals were of the purest available grade.
Glucosylceramidase Preparation
Glucosylceramidase
was purified from human placenta following the procedure described by
Murray et al. (17) .
Sap B and C Preparation
Sap B and C were purified
from spleens of patients with Type 1 Gaucher's disease following
a previously reported procedure
(18) ; it consisted of heat and
acid treatment of a water homogenate, ion exchange chromatography on
DEAE-Sephacel, gel filtration on Sephadex G-75, and reverse-phase HPLC
on a C4 column (Vydac). The purity and identity of Sap B and C
preparations were verified by SDS-polyacrylamide gel electrophoresis,
Western blotting, and N-terminal sequence analysis.
Deglycosylation of Sap B and C
In a final volume
of 50 µl, 200 µg of either Sap B or C were incubated in 10
mM sodium phosphate buffer, pH 8.6, containing 0.1 mM
EDTA and 0.6% (w/v) Triton X-100, with 10 units of Peptide
N-glycosidase F at 37 °C for 24 h. The deglycosylated
proteins were purified by reverse-phase HPLC on a PLRP-S reverse-phase
column (300 Å, 8 µm, 150 4.6 mm). The column was
equilibrated with buffer A (2.5% acetonitrile, 2.5% isopropyl alcohol,
95% 50 mM ammonium bicarbonate, pH 8.5) and eluted with a
linear gradient of 0-100% buffer B (40% acetonitrile, 40%
isopropyl alcohol, 20% 50 mM ammonium bicarbonate, pH 8.5)
over 90 min at a flow rate of 0.8 ml/min. Deglycosylated Sap B and C
eluted later than the corresponding native proteins. The HPLC-purified
samples were lyophilized, redissolved in water, and submitted to
accurate molecular mass determination by electrospray mass
spectrometry.
Reduction and Alkylation of Sap C
In a final
volume of 150 µl, Sap C (300 µg) was reduced by incubation with
dithioerythritol (0.8 mg) in 0.5 M Tris/HCl buffer, pH 8.1,
containing 2 mM EDTA and 6 M guanidine HCl, for 4 h
at 50 °C under nitrogen. The solution was then cooled to room
temperature and iodoacetamide (1.7 mg) was added. After 30 min in the
dark, the protein sample was freed from salts and the excess of
reagents by passing the reaction mixture through a PD-10 prepacked
column (Pharmacia) equilibrated and eluted with 0.4% ammonium
bicarbonate, pH 8.5. The S-carboxymethylated Sap C solution
was then lyophilized.
Chemical and Enzymatic Hydrolyses of Sap B and
C
Cleavage of peptide bonds adjacent to methionine residues of
Sap B or C was carried out by CNBr treatment. Each saposin, dissolved
in 70% trifluoroacetic acid, was incubated with CNBr (15-fold weight
excess over protein) overnight at room temperature under an inert
atmosphere in the dark. The sample was then diluted 10-fold with water
and evaporated to dryness in a Speed Vac concentrator (Savant).
Mass Spectrometry
Intact protein samples were
analyzed by electrospray mass spectrometry (ES-MS) using a VG BIO-Q
triple quadrupole mass spectrometer equipped with an electrospray ion
source. Samples were injected into the ion source via a loop injection
at a flow rate of 2 µl/min; spectra were recorded at 3.4 kV cone
voltage by scanning the first quadrupole from 500 to 1600 m/z at 10 s/scan; data were acquired and elaborated using the LAB-BASE
program (VG analytical). Mass calibration was performed by means of the
multiply charged ions from a separate injection of horse heart
myoglobin (average molecular mass 16950.5 Da); all masses are reported
as average mass.
Vesicle Preparation
Large unilamellar PS vesicles
were prepared by filter exclusion using a high pressure extrusion
apparatus (Lipex Biomembranes, Vancouver, Canada) as described
previously
(20) .
Leakage Assay
The leakage of liposome content was
monitored by the release of calcein trapped inside the vesicles
(21) . Vesicles for leakage experiments were prepared by
hydrating dried films of PS in 60 mM calcein, pH 7.4, followed
by 10 cycles of freeze-thawing. The resulting multilamellar vesicles
were extruded 10-15 times through two 0.1-µm diameter pore
polycarbonate filters (Nucleopore Corp., Pleasanton, CA). Free calcein
was separated from the dye-containing vesicles by chromatography on a
Sephadex G-75 column. Upon addition of Sap C, leakage of calcein to the
external medium was followed by the increase in fluorescence caused by
calcein dilution and the consequent relief of self-quenching
(excitation 470 nm, emission 520 nm). 100% leakage was established by
lysing the vesicles with 0.3% (w/v) Triton X-100. Leakage experiments
were carried out at 37 °C and monitored with a Fluoromax
spectrofluorometer equipped with a constant temperature cell holder and
stirrer (Spex Industries Inc., Edison, NJ).
Stimulation of Glucosylceramidase Activity by Sap C and
Large PS Vesicles
The glucosylceramidase stimulation by Sap C
and large PS vesicles was evaluated as described previously
(10, 11) . Different amounts of Sap C were added to an
assay mixture containing, in a final volume of 0.2 ml, 10 mM
acetate buffer, pH 5.0, 150 mM NaCl, 1 mM EDTA
(buffer A), 2.5 mM
4-methylumbelliferyl--D-glucopyranoside, 2 µg of
large PS vesicles, and 3 ng of purified placental glucosylceramidase.
The assay mixtures were incubated for 30 min at 37 °C. The extent
of reaction was estimated fluorometrically. All assays were carried out
in duplicate and the results agreed within 5%.
Analysis of Sap C and Sap B Size by Electrospray Mass
Spectrometry
An amino acid sequence of either 80
(13) or
82
(1) amino acid residues has been previously reported for
human Sap C. The presence of a truncated form, two amino acid residues
shorter at the N-terminal end, has also been observed
(13) . In
order to confirm the existence of the various forms and their chemical
structure, a preparation of Sap C was deglycosylated and then submitted
to ES-MS analysis. Fig. 1( upper panel) shows the
spectrum transformed on a real mass scale. Three components could be
recognized; the major component ( A) showed a molecular mass of
8945.01 ± 1.24 Da which agrees with the predicted mass value for
the 80-amino acid monomeric form of Sap C (theoretical mass value
8944.2 Da). The molecular mass measured for component B ( B)
was 8741.38 ± 0.44 Da; on the basis of this mass value,
component B was tentatively identified as the truncated form of Sap C
in which the N-terminal dipeptide Ser-Asp is missing
(13) (theoretical mass value 8742.0 Da). Finally, component C
( C) (observed mass value 9147.55 ± 1.42 Da) was
interpreted as intact Sap C with a single N-acetylglucosamine
residue bound to the glycosylation site (theoretical mass value 9147.2
Da). This component, also present in the ES mass spectrum of the native
form of Sap C (data not shown), was apparently not attacked by Peptide
N-glycosidase F. Human deglycosylated Sap C is thus a
monomeric polypeptide composed of 80 amino acids. The presence of
components B and C confirms its previously reported microheterogeneity
(1, 2, 3, 13) .
Figure 1:
Transformed
electrospray mass spectra of deglycosylated Sap C ( upper
panel) and Sap B ( lower panel). The multiply charged ion
spectra are transformed on a real mass scale. The molecular mass of the
components of the two protein preparations are reported as
daltons.
Sap B was also
submitted to ES-MS analysis. It was previously reported that it
consisted of either 79
(14) , 80
(22) , or 81
(1, 2) amino acid residues. Fig. 1( lower panel)
shows the ES mass spectrum of deglycosylated Sap B. The major component
exhibited a molecular mass of 8869.67 ± 0.94 which well agrees
with the mass value predicted on the basis of the 79-amino acid
sequence (8869.2 Da). Minor components in the spectrum were likely due
to oxidized forms of Sap B produced during sample manipulations. The
ES-MS analysis of Sap B did not reveal any structural
microheterogeneity at the protein level. The previously considered
possibility that Sap B might exist as a dimer stabilized by
intermolecular disulfide bridges
(3) can be excluded on the
basis of the ES-MS analysis.
Location of Disulfide Bridges in Sap C
All of the
six cysteine residues in Sap C have been shown to participate in
disulfide bridges
(18, 23) . The complete pattern of S-S
bridges in Sap C was derived from FAB-MS analysis of the peptides
obtained by combining chemical and enzymatic digestions of the protein
(24) . As Sap C is resistant to most proteases
(1, 2, 3) , it was first cleaved with CNBr at
the level of the two methionine residues in order to disrupt the tight
native conformation. The resulting fragments were then deglycosylated,
digested with pepsin, and examined by FAB-MS. Fig. 2 A shows the high mass region of the FAB spectrum. Two intense mass
signals were detected at m/ z 2612 and 2320. The first
signal occurred 2-Da units lower than the expected mass value for
peptide 36-59 which contains two cysteine residues, thus
suggesting the occurrence of an intramolecular S-S bridge joining
Cysand Cys
(24) . This assignment was
confirmed by the presence of a signal at m/z 2614 due to the
partial reduction of the intramolecular disulfide bridge occurring
under FAB-MS conditions
(25) . Total reduction by dithiothreitol
led to the occurrence of a single mass signal at m/z 2614
(data not shown), confirming the presence of an intramolecular bridge
between Cys
and Cys
.
Figure 2:
Partial FAB-MS spectra of Sap C showing
the mass signals relative to disulfide-bridged peptides. A,
high mass region of the spectrum of Sap C treated with CNBr,
deglycosylated, and digested with pepsin. B, peptide mixture
resulting from subdigestion with endoprotease Glu-C of an aliquot of
the peptic digest.
The signal at m/z 2320 was tentatively assigned to a three-peptide cluster
consisting of fragments 1-11, 71-74, and 75-80 held
together by two disulfide bridges involving the remaining four
cysteines at positions 5, 8, 72, and 78 (). This
interpretation was derived from the unique mass value and the
disappearance of the signal following dithiothreitol treatment. A
single step of manual Edman degradation confirmed the assignment
producing a shift of the signal to a lower mass value (2021) due to the
removal of Ser, Val
, and Leu
(24, 26) . The direct identification of the
correct cysteine pairings within the peptide cluster was related to the
possible cleaving of the polypeptide chain between Cys
and
Cys
both occurring in fragment 1-11. The presence of
a glutamic acid residue at position 6 suggested an endoprotease Glu-C
subdigestion experiment. Fig. 2 B shows the partial FAB
spectrum of the resulting peptide mixture. The enzymatic hydrolysis at
the level of Glu-6 splits the three-peptide cluster into two peptide
pairs giving origin to the mass signals at m/z 1341 and 998.
These signals were assigned to the peptide pairs
(1, 2, 3, 4, 5, 6) +
(75, 76, 77, 78, 79, 80) linked by the S-S bridge between Cys
-Cys
and
(7, 8, 9, 10, 11) +
(71, 72, 73, 74) linked by a disulfide bridge between Cys
and
Cys
(). The S-S bridge arrangement is further
confirmed by the signal at m/z 1337 corresponding to the
peptide 7-11 linked to 68-74 via the same S-S bridge.
Location of Disulfide Bridges in Sap B
The
disulfide bridge pattern of Sap B was elucidated following the same
strategy used for Sap C. Saposin B was first hydrolyzed with CNBr and
then deglycosylated. The resulting peptide mixture was incubated with
trypsin, and the unfractionated digest was directly analyzed by FAB-MS
producing the mass spectrum shown in Fig. 3 A. Three mass
signals at m/z 2751, 2274, and 1197 were associated with
disulfide-containing peptides as indicated by their disappearance
following dithiothreitol incubation. The peak at m/z 2751 was
assigned to peptides
(20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) +
(44, 45, 46, 47, 48) and that at m/z 1197 to peptides
(34, 35, 36, 37, 38) +
(44, 45, 46, 47, 48) linked by
an S-S bridge involving Cysand Cys
, the
former being due to an incomplete cleavage at the level of Lys
(). This interpretation was confirmed by the shift
to the expected lower mass values (2579 and 997, respectively)
following a single step of manual Edman degradation
(24, 26) .
Figure 3:
Partial FAB-MS spectra of Sap B showing
the mass signals relative to disulfide-bridged peptides. A,
high mass region of the spectrum of Sap B treated with CNBr,
deglycosylated, and digested with trypsin. B, peptide mixture
resulting from subdigestion with endoprotease Asp-N and chymotrypsin of
an aliquot of the tryptic digest.
The signal at m/z 2274 was
assigned to the peptide pair
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) +
(69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79) linked by two S-S bridges involving the four cysteine
residues, Cys, Cys
, Cys
, and
Cys
, two located in fragment 1-10 and two in
fragment 69-79 (). The presence of this signal
excluded the occurrence of disulfide bonds linking
Cys
-Cys
and Cys
-Cys
,
leaving the possibility of either Cys
-Cys
and
Cys
-Cys
or Cys
-Cys
and Cys
-Cys
pairings. The identification
of the correct S-S pairings depended on the possible cleavage of the
polypeptide chain between these cysteines residues. The peptide mixture
was then submitted to a double enzymatic hydrolysis using first
endoprotease Asp-N and then chymotrypsin. The FAB-MS spectrum of the
resulting peptide mixture is shown in Fig. 3 B. The
signal at m/z 1124 corresponds to peptides 6-10 and
69-73 linked via an S-S bridge involving Cys
and
Cys
(). The formation of these peptides
demonstrated that the enzymatic hydrolyses had produced the expected
cleavages of fragment 1-10 at the level of the N-terminal side of
Asp
and of fragment 69-79 at Leu
. This
was further confirmed by the presence of a mass signal at m/z 943 which was associated with the peptides 1-5 and
74-77 linked by the S-S bridge Cys
-Cys
().
and Cys
().
Importance of the Sap C Disulfide Bonds for the Saposin
Functional Properties
We have previously found that at pH 5.0,
presumably the pH of the lysosome environment, Sap C induces
destabilization of PS-containing vesicles
(11) and promotes the
binding of glucosylceramidase to the bilayers reconstituting the enzyme
activity
(10) . PS large liposomes which by themselves are
unable to fully stimulate glucosylceramidase, upon interaction with Sap
C, become powerful enzyme activators
(10) . In order to assess
the importance of the Sap C disulfide bridges for its ability to
perturb vesicles, we have compared the effect of either native or
reduced and alkylated Sap C on the permeability of PS large liposomes.
The FAB-MS analysis of the tryptic digest of the
S-carboxymethylated saposin indicated that all six cysteines
were alkylated
(27) (data not shown). The destabilization of
the bilayer was measured by the release of an encapsulated fluorescent
dye, calcein, from PS large vesicles. Native Sap C, at concentrations
as low as 12 nM, was able to induce a fast release of calcein
while the modified saposin, at the same concentration, had almost no
effect on the membranes (Fig. 4). A significant release of the
fluorescent dye could only be observed raising the amount of modified
Sap C in the assay, i.e. liposomes released about 25% calcein
after a 3-min incubation with 100 nM S-carboxymethylated saposin.
Figure 4:
Leakage of PS large liposomes induced by
either native or S-carboxymethylated Sap C. Time course of the
Sap C-induced release of calcein entrapped in PS large liposomes.
Different amounts of either native (--) or
S-carboxymethylated (- - -) Sap C were
injected into a stirred cuvette thermostated at 37 °C, containing a
1-ml solution of liposomes (50 µM in lipid) in buffer A,
pH 5.0. The final concentration of Sap C in the mixtures, expressed as
nanomolar, is indicated in the figure.
It was expected that the
reduction and alkylation that impair the Sap C capacity to perturb
membranes would equally affect the saposin ability to promote
glucosylceramidase stimulation by PS large liposomes. It was found that
S-carboxymethylated Sap C is a much less effective enzyme
activator than the native saposin, especially at low concentrations
(Fig. 5).
Figure 5:
Effect of either native or
S-carboxymethylated Sap C on the stimulation of
glucosylceramidase activity by PS large liposomes. Different amounts of
either native (--
) or
S-carboxymethylated (
- - -
) Sap C
were added to the glucosylceramidase assay mixture, and the enzyme
activity was measured as reported under ``Materials and
Methods.'' The concentration of PS liposomes in the assays was 10
µg/ml; in their absence, neither native nor
S-carboxymethylated Sap C stimulated glucosylceramidase
activity.
-Cys
, Cys
-Cys
,
Cys
-Cys
for Sap B and
Cys
-Cys
, Cys
-Cys
,
Cys
-Cys
for Sap C (Fig. 6). The
disulfide bonds, that establish interactions between distant parts of
the saposin molecules, most likely play a major role in stabilizing
their conformation. The observation that in Sap B and C similarly
positioned cysteines are paired in an identical manner suggests that
the same arrangement is present also in Sap A and D, where the six
cysteine residues are present at strictly conserved positions.
Figure 6:
Schematic disulfide structure of Sap B and
Sap C. Black lines linking cysteines indicate disulfide bonds.
The numbers in circles indicate the N-glycosylation
sites.
This
is the first demonstration of the actual location of disulfide bridges
in saposins. It reaches firm conclusions incompatible with the
hypothetical models so far developed
(1, 12, 30) . A folding structure for Sap B,
having Cyslinked with Cys
and Cys
with Cys
, was previously proposed by comparison with
a homologous region of influenza virus neuraminidase
(30) . A
three-helical wheel model was also put forward for Sap B, with cysteine
residues properly positioned to form internal disulfide bridges;
according to this model, Cys
faced Cys
and
Cys
faced Cys
(12) . A similar
structure with regard to potential arrangement of disulfide bridges was
proposed for Sap A and C
(1) .
with either phenylalanine or glycine have been found
in two patients with an atypical form of Gaucher disease
(31, 32) . These patients accumulate glucosylceramide in
tissues, although their glucosylceramidase is normal. Moreover, two
recombinant Sap C molecules, created to contain the same mutations
found in the patients (Cys
Gly or Cys
Phe), were unable to stimulate glucosylceramidase in
vitro (33) . These findings stress the essentiality of the
cysteine residues and thus of the disulfide bonds for maintaining the
appropriate conformation of the functional site(s) of Sap C.
Table:
FAB-MS
characterization of peptides obtained by chemical and enzymatic
hydrolysis of Sap C
Table:
FAB-MS
characterization of peptides obtained by chemical and enzymatic
hydrolysis of Sap B
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.