©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Analysis of Saposin C and B
COMPLETE LOCALIZATION OF DISULFIDE BRIDGES (*)

Anna Maria Vaccaro (1), Rosa Salvioli (1), Alessandra Barca (1), Massimo Tatti (1), Fiorella Ciaffoni (1), Bruno Maras (2), Rosa Siciliano (3), Francesca Zappacosta (3), Angela Amoresano (3), Piero Pucci (3) (4)

From the (1) Laboratorio Metabolismo e Biochimica Patologica, Istituto Superiore di Sanit, 00161 Roma, Italy, the (2) Dipartimento di Scienze Biochimiche e Centro di Biologia Molecolare, Consiglio Nazionale delle Ricerche, 00185 Roma, Italy, (3) Servizio di Spettrometria di Massa, Consiglio Nazionale delle Ricerche, 80131 Napoli, Italy, and the (4) Dipartimento di Chimica Organica e Biologica, Universitá di Napoli, 80134 Napoli, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-Cys, Cys-Cys, Cys-Cysfor saposin B and Cys-Cys, Cys-Cys, Cys-Cysfor saposin C.

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.


INTRODUCTION

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)() 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 Gganglioside (3, 7) , and saposin D (Sap D) that of sphingomyelin (8) .

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, etc.) thus making them accessible to the respective sphingolipid hydrolases (3, 7) .

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.


EXPERIMENTAL PROCEDURES

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).

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.

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.

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) .

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%.


RESULTS

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 Cysand 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 Cysand Cysboth 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-Cysand (7, 8, 9, 10, 11) + (71, 72, 73, 74) linked by a disulfide bridge between Cysand 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-Cysand Cys-Cys, leaving the possibility of either Cys-Cysand Cys-Cysor Cys-Cysand Cys-Cyspairings. 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 Cysand 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 Aspand 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().

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 Cysand 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.




DISCUSSION

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-Cys, Cys-Cys, Cys-Cysfor Sap B and Cys-Cys, Cys-Cys, Cys-Cysfor 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 Cysand Cyswith 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, Cysfaced Cysand Cysfaced Cys(12) . A similar structure with regard to potential arrangement of disulfide bridges was proposed for Sap A and C (1) .

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 Cyswith 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.

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.

  
Table: FAB-MS characterization of peptides obtained by chemical and enzymatic hydrolysis of Sap C

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.


  
Table: FAB-MS characterization of peptides obtained by chemical and enzymatic hydrolysis of Sap B

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.



FOOTNOTES

*
This work was partly supported by CNR (Progetto Finalizzato Ingegneria Genetica). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of our much missed colleague and friend Gianpaolo Nitti.

The abbreviations used are: Sap B, saposin B; Sap C, saposin C; SP-B, surfactant protein B; PS, phosphatidylserine; HPLC, high performance liquid chromatography; FAB-MS, fast atom bombardment mass spectrometry; ES-MS, electrospray mass spectrometry.


ACKNOWLEDGEMENTS

We thank Prof. D. Barra for helpful discussions, E. Raia for technical assistance, and G. M. Spinelli for reading the manuscript.


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