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Journal of Lipid Research, Vol. 46, 2254-2264, October 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

Andrew J. Norris^1,*,, Julian P. Whitelegge^*, Arman Yaghoubian^*, Jean-Rene Alattia, Gilbert G. Privé, Tatsushi Toyokuni^**, Hubert Sun^*, Mai N. Brooks, Luigi Panza, Pamela Matto, Federica Compostella, Natascha Remmel^***, Ralf Klingenstein^***, Konrad Sandhoff^***, Claire Fluharty, Arvan Fluharty and Kym F. Faull^*

^* Pasarow Mass Spectrometry Laboratory, Departments of Psychiatry & Biobehavioral Sciences and Chemistry & Biochemistry, and Neuropsychiatric Institute, University of California Los Angeles, Los Angeles, CA 90024
Mental Retardation Research Center, University of California Los Angeles, Los Angeles, CA 90024
^** Department of Molecular & Medical Pharmacology, University of California Los Angeles, Los Angeles, CA 90024
Johnson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA 90024
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, M5G 2M9 Canada
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche, e Farmacologiche, Università del Piemonte Orientale, 28100 Novara, Italy
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, 20133 Milano, Italy
^*** Kekule Institut fur Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universitaet Bonn, 53121 Bonn, Germany

Published, JLR Papers in Press, August 1, 2005. DOI 10.1194/jlr.M500188-JLR200

¹ To whom correspondence should be addressed. e-mail: anorris{at}mednet.ucla.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the nonhydroxylated species of the sulfatide substrate over the corresponding hydroxylated species.

The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.

Abbreviations: ASA, arylsulfatase A; CSAct, cerebroside sulfate activator protein; ESI, electrospray ionization mass spectrometry; IS, internal standard; MALDI, matrix-assisted laser desorption ionization; MLD, metachromatic leukodystrophy; MRM, multiple reaction monitoring; MS/MS, tandem mass spectrometry; TOF, time-of-flight; U, unit of enzyme activity (the amount of enzyme activity that will catalyze the transformation of 1 µmol of the substrate per minute under standard conditions); UCLA, University of California Los Angeles

Supplementary key words saposins A, B, C, and D • electrospray ionization • tandem mass spectrometry

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Metachromatic leukodystrophy (MLD) is a neurodegenerative autosomal recessive disease in which affected individuals most commonly have a defect in arylsulfatase A (ASA) enzyme activity originating from 60 different known mutations (1). MLD can also arise as a result of a defective cerebroside sulfate activator protein (CSAct), also known as saposin B (2), as the sphingolipid activator protein 1 (3), or as the nonspecific activator protein (4). In this and other publications from the University of California Los Angeles (UCLA) group, this protein is referred to as CSAct to reflect the activity that is measured. The function of ASA is to metabolize intracellular sulfatide (sulfogalactosyl cerebroside) via enzymatic hydrolysis of the 3-O-galactosyl-sulfate group. However, ASA is apparently unable to perform this function as long as the lipid substrate is confined within the membrane. CSAct is thought to sequester sulfatide from membrane fragments incorporated into lysosomes by a poorly understood lipid-exchange mechanism (5), and in the resulting CSAct-sulfatide complex, the polar sugar sulfate moiety is sufficiently exposed for hydrolysis catalyzed by lysosomal ASA (Fig. 1) . During this process, the hydrophobic tail of the lipid is confined to a pocket within the dimeric protein shell and is shielded from the hydrophilic environment (6). Thus, the true in vivo substrate for ASA is the CSAct-sulfatide complex, and in this process CSAct serves as the lipid carrier.

View larger version (5K): [in this window] [in a new window]   Fig. 1. Scheme of the arylsulfatase A/cerebroside sulfate activator protein (ASA/CSAct) coupled reaction in which sulfatide (cerebroside sulfate) is converted to cerebroside with the release of inorganic sulfate. In this reaction, CSAct presents the sulfatide substrate to ASA, with subsequent sulfate hydrolysis.

In principle, both CSAct and ASA activity could be measured in vitro by measuring the temporal change in concentration of one or more of the participating reactants/products in the coupled reaction (Fig. 1). This would involve measuring the rate of either substrate (sulfatide) depletion or product (cerebroside and/or inorganic sulfate) formation. In practice, ASA activity can be measured using artificial substrates (p-nitrocatechol sulfate or 4-methylumbelliferyl sulfate) by following the formation of colored [p-nitrocatechol, _max = 550 nm (8)] or fluorescent [4-methylumbelliferol, _ex = 365 nm, _em = 450 nm (9)] products, respectively. These assays are performed without difficulty in a homogeneous milieu because all participants in the reaction are water-soluble. However, assays using natural sulfatide substrate are more complicated, because of the poor water solubility of monomeric sulfatide and its propensity to form aggregates. In this case, ASA activity can be measured using detergent-solubilized sulfatide (10), whereas CSAct activity can be measured using CSAct-solubilized sulfatide (11). As done previously in the UCLA laboratory, both of these procedures involve measuring the rate of inorganic sulfate formation. Because existing assays for inorganic sulfate were insufficiently sensitive, ³⁵S-radiolabeled sulfatide was used and the production of radioactive inorganic sulfate was measured. ³⁵S-radiolabeled sulfatide is prepared by in vivo administration of [³⁵S]sulfate to rat pups, followed by extraction and purification of the radiolabeled lipid (12). This radiolabeled lipid is not available commercially, and the tedious and time-consuming preparation requires relatively large amounts of radioactivity to yield a product with adequate specific activity. Furthermore, the short ³⁵S half-life of 87.32 days means that the effective shelf life of the [³⁵S]sulfatide is 6 months (unpublished observation), necessitating the periodic preparation of new material. More recently, ¹⁴C-radiolabeled sulfatide has been used as substrate and the production of [¹⁴C]cerebroside measured (6), but this substrate is expensive and until recently was not generally available.

There is a need for a more convenient assay with which the natural substrate system can be monitored. Mass spectrometry provides a valuable tool for the analysis of lipids and lipid-metabolizing enzymes (13). Previous work using mass spectrometry for the analysis of lipid-metabolizing enzymes has involved the use of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry (ESI) (14, 15). In the present case, the possibility of following sulfatide depletion by ESI has been tested by taking advantage of the strong signals that these lipids yield in the negative ion mode (16, 17); more importantly, the possibility of following cerebroside formation has been tested by taking advantage of the strong signals that these lipids yield as their lithiated adducts in the positive ion mode (18). These two approaches have been tried, and here we present the results of this comparison, which showed that monitoring cerebroside formation by ESI coupled to tandem mass spectrometry (MS/MS) provides a reliable, fast, specific, nonradioactive method for assays involving both CSAct- and detergent-solubilized sulfatide. The assays have been used to reveal hitherto obscure but potentially important details of the sulfatide specificity for binding by CSAct and the saposin specificity for the ASA-catalyzed reaction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Chemicals and reagents
Bovine brain sulfatide and cerebroside and semisynthetic [²H₃₅] stearoyl cerebroside ([²H₃₅]C₁₈:0 cerebroside) were obtained from Matreya, Inc. (State College, PA). Taurodeoxycholate, lithium chloride, and BSA were from Sigma Aldrich (St. Louis, MO). Quartz-distilled water (<16 m/cm) was produced in house, and all other reagents and solvents were of analytical grade or better.

Preparation of synthetic sulfatides
Nonhydroxylated sulfatides with fatty acyl chain (R moiety; Fig. 1) compositions of C₁₆:0 (palmitic), C₁₈:0 (stearic), C₂₂:0 (behenic), and C₂₄:1¹⁵ (nervonic) were synthesized as described (19). Hydroxylated sulfatides with fatty acyl chain (R moiety; Fig. 1) compositions of -hydroxylated C₁₈:0 (hC₁₈:0, -hydroxy-stearic) and hC₂₂:0 (-hydroxy-behenic) were synthesized from racemic mixtures of the corresponding hydroxylated fatty acids according to the following procedure. First, ß-D-galactosylsphingosine was prepared through stereoselective glycosylation of 3-O-benzoyl azidosphingosine (20) with 2,3,4,6-tetra-O-pivaloyl--D-galactopyranosyl trichloroacetimidate, followed by removal of the protecting groups and azide reduction (19). Then, the corresponding fatty acid (1.5 eq.), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.0 eq.) and N-hydroxybenzotriazole (1.5. eq.), was added to a solution of the ß-D-galactosylsphingosine (0.03 g) in dichloromethane (2 ml). The mixture was refluxed (1 h) and diluted with methanol, and then the solvent was removed under vacuum. After purification by flash chromatography with dichloromethane-methanol (85:15, v/v), the galactosylceramide was selectively sulfated at position 3 as described previously (19) and the 3-O-sulfated products were recovered in 50% yields.

Negative ion ESI of the final products (17; see below) from solutions in chloroform-methanol-triethylamine-water (4:5:1:0.05, v/v) showed intense signals for each negatively charged molecule (mass spectra not shown) corresponding to the (M-H)^– anions: C₁₆:0, observed m/z 778.2, calculated 778.51 Da; C₁₈:0, observed m/z 806.4, calculated 806.54 Da; C₂₂:0, observed m/z 862.8, calculated 862.61 Da; C₂₄:1¹⁵, observed m/z 888.9, calculated 888.62 Da; hC₁₈:0, observed m/z 822.6, calculated 822.54 Da; hC₂₂:0, observed m/z 878.7, calculated 878.60 Da. Based on the negative ion ESI spectra, the purity of each sample was estimated to be in excess of 90%.

Aqueous sulfatide suspensions were prepared as described previously (21). Briefly, aliquots of stock solutions in chloroform-methanol-water (63:32:5, v/v) were dried under nitrogen. The dried residue was redissolved in 40 µl of the same solvent. Sufficient boiling water was then added to bring the mixture to 1 mg/ml, and it was then vigorously mixed, sonicated briefly, and vigorously stirred for 2 h, during which time the temperature of the mixture slowly returned to room temperature. The result was a faintly turbid suspension that was stored frozen at –80°C. Before use, it was brought to room temperature and vigorously stirred for 30 min.

Detergent-solubilized sulfatide was prepared from bovine sulfatide [125 µl, 12 mg/ml in chloroform-methanol (2:1, v/v)] and dried to near dryness under a stream of nitrogen gas in a 2 ml conical glass vial. The white precipitate was then redissolved in 250 µl of taurodeoxycholate (5 mg/ml in 4 mM sodium sulfate containing 0.73% sodium chloride) by vigorous stirring for 1 h at room temperature.

ASA
ASA was purified from human liver as described previously (22) and stored at –80°C in 25 mM Tris-Cl at pH 7.5 with a specific activity of 3,000 units (U) of enzyme activity (the amount of enzyme activity that will catalyze the transformation of 1 µmol of the substrate per minute under standard conditions)/mg protein as determined by a modified Baum, Dodgson, and Spencer (8) assay using p-nitrocatechol sulfate as substrate. When needed for the CSAct and ASA assays, the stock was diluted in 25 mM Tris-HCl, pH 7.5, containing 1 mg/ml BSA to an activity of 250 U/ml.

CSAct and saposins A, C, and D
Native CSAct was purified from pig kidney as described previously (11). The preparation contained predominantly a glycosylated 79 amino acid residue protein and has been characterized extensively by chromatographic and mass spectrometric analysis (23), with biological activity demonstrated by in vitro and in vivo assays (21, 24, 25). The material was stored as a 27 mg protein/ml solution in 25 mM Tris-HCl, pH 7.4, at –80°C and diluted as indicated for the various assays.

Two different sources of recombinant human saposins were used. The human prosaposin sequence numbering will be used throughout (Swiss-Prot primary accession number P07602 at http://us.expasy.org). One set of proteins was prepared as nonglycosylated hexahistidine-tagged molecules in a yeast (Pichia pastoris) expression system according to standard Invitrogen^TM protocols and purified by ion exchange and affinity chromatography (26). Saposins A and C were stored as 0.66 and 0.60 mg protein/ml solutions, respectively, in 50 mM sodium phosphate buffer containing 300 mM sodium chloride, pH 4.0, at –80°C. Saposin D was stored as a 1.0 mg protein/ml solution in 10 mM citrate buffer, pH 4.0, at –80°C. MALDI-time-of-flight (TOF) mass spectrometry (Applied Biosystems DE STR; linear mode with time-lagged focusing using internal calibration with bovine myoglobin) showed agreement within experimental error between the measured and calculated masses of the principal components in each preparation after consideration for some N- and C-terminal proteolytic trimming, which presumably occurred during sample purification: saposin A (Fig. 2A) , measured m/z 10,719; calculated 10,716.3 Da for MH⁺ [EAEAYV-human saposin A (S₆₀–K₁₄₃)-HHHHHH; three disulfide bonds]; saposin C (Fig. 2B), measured m/z 9,204, calculated 9,200.7 Da for MH⁺ [V-human saposin C (S₃₁₁–G₃₉₀)-R; three disulfide bonds]; and saposin D (Fig. 2C), measured m/z 9,962 (10%), 9,825 (50%), 9,688 (100%), and 9,553 (60%), calculated 9,963.5, 9,826.4, 9,689.3, and 9,552.2 Da for MH⁺ [EAEA-human saposin D (G₄₀₇–S₄₈₄)-nH; three disulfide bonds] for six, five, four, and three C-terminal histidine residues, respectively]. The differences between measured and calculated molecular masses (0.8–3.3 Da) are within the accuracy typically obtained with MALDI-TOF of proteins (±10 Da at 10 kDa) (27). The purified materials were biologically active, as demonstrated by their support of the efficient hydrolysis of lactosylceramide by activator protein-deficient cultured human fibroblasts (26). The stock solutions were diluted in 25 mM Tris-HCl buffer (pH 7.5) for use in the CSAct assay.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectra of recombinant human saposins A (A), C (B), and D (C) produced by the P. pastoris expression system. The measurement errors are within the accuracy typically obtained with MALDI-TOF of proteins (±10 Da at 10 kDa). See Materials and Methods for details.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Electrospray ionization mass spectrometry (ESI) spectra of recombinant human saposins A (A) and C (B) produced by the E. coli expression system. The insets represent the computer-generated molecular mass profiles of the two samples. The molecular mass measurement errors are within the accuracy typically obtained with ESI of proteins on a standard quadrupole mass spectrometer (±1 Da at 10 kDa). See Materials and Methods for details.

Taurodeoxycholate-potentiated ASA assay
A microtized variation of the established procedure for this detergent-based assay (10) used 0.2 ml polypropylene tubes and a total volume adjusted to 50 µl with 25 mM Tris-HCl buffer, pH 7: sodium acetate buffer (5 µl, 1 M, pH 4.5); 10 µl of taurodeoxycholate-solubilized bovine sulfatide substrate (60 µg of bovine sulfatide; see above); and 25 µl of 17 mM Tris-HCl (pH 7.5; to substitute for the saposin protein used in the CSAct assay). The reactions were started by the addition of 5 µl of ASA enzyme (250 U/ml); blank reactions with no added ASA used 5 µl of 1 mg/ml BSA in 25 mM Tris-HCl (pH 7.5). During incubation (37°C), 5 µl aliquots were removed at 0.5, 1, 1.5, and 2 h and placed into new 1 ml tubes containing 330 µl of chloroform-methanol (2:1, v/v) and 70 µl of 4 mM sodium sulfate containing 0.12 M sodium chloride. These mixtures were vortexed (30 s) and then centrifuged (3,000 g, 5 min). An aliquot of the lower phase (200 µl) was then removed with a 1 ml glass gas-tight syringe fitted with a Chaney adaptor, placed into a new tube, and dried with a stream of dry nitrogen gas. For injection into the mass spectrometer, the dried samples were redissolved in 100 µl of chloroform-methanol (1:4, v/v) containing 250 pmol of [²H₃₅]stearoyl cerebroside IS and 100 mM lithium chloride.

ESI
A Perkin-Elmer Sciex API III⁺ triple quadrupole mass spectrometer equipped with an Ion Spray^TM source was tuned and calibrated in the positive ion mode as described previously (30). Under these standard resolution conditions, the polypropylene glycol/NH₄⁺ singly charged ion clusters were resolved with a 40% valley. Flow injection was used for sample introduction (20 µl per injection), with water-acetonitrile-formic acid (50:50:0.1, v/v) as solvent for proteins, and methanol-chloroform (4:1, v/v) as solvent for lipids, both constantly infused into the source (15 µl/min). For proteins, an m/z 400–2,000 range was scanned (positive mode, orifice 90 V, 0.3 Da step size, 5.61 s/scan). For lipids, an m/z 100–1,000 mass range was scanned (0.3 Da step size, 6.66 s/scan). For cerebrosides, the positive ion mode was used with the orifice set to +120 V. For sulfatides, the instrument polarity was reversed and the orifice was set to –90 V. Fragment ion spectra of Q1 preselected cerebroside parent ions [(M+Li)⁺, 99.999% argon collision gas with a collision gas thickness instrument setting of 200 and a rod offset (R₀–R₂) of 40 V] were recorded by scanning Q3 from m/z 50 to 1,000 (0.3 Da step size, 7.43 s/scan). Up to eight of the following cerebroside transitions were recorded for multiple reaction monitoring (MRM) measurements: C₁₆:0, 706.4544.4; C₁₈:1, 732.6570.6; C₁₈:0, 734.6572.6; hC₁₈:0, 750.6588.6; [²H₃₅]C₁₈:0, 769.7607.7; C₂₂:0, 790.7628.6; hC₂₂:0, 806.7644.6; C₂₄:1¹⁵, 816.7654.6; C₂₄:0, 818.7656.6; hC₂₄:0, 834.7672.6; C₂₆:1, 844.7682.6; C₂₆:0, 846.7684.7.

Data processing
Representative spectra were computed as the average of all spectra accrued from each injection using instrument-supplied software (MacSpec^TM, version 3.3; Perkin-Elmer Sciex). MRM profiles were smoothed and peak areas determined using the IGOR Pro^TM software package (version 3; WaveMetrics, Inc., Lake Oswego, OR) and converted to moles of cerebroside using the response from the IS. Reaction rates were expressed as moles of product per hour per milligram of saposin.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
As reported previously, the negative ion ESI spectra of brain sulfatide reveal a series of ions from m/z 806 to 950, reflecting the relative abundance of the various species of this lipid in the brain (Fig. 4A) (16, 17). The positive ion spectra of brain cerebrosides in the presence of lithium (18) reveal a corresponding series of ions from m/z 734 to 860, reflecting a similar pattern of relative abundance to that of the sulfatides (Fig. 4B). The 72 Da difference between the signals from corresponding sulfatide and cerebrosides is accounted for by the mass difference attributable to sulfation (80 Da), the loss of a proton in the negative ion sulfatide spectra, and lithium adduction (7 Da) in the positive ion cerebroside spectra. Surprisingly, the intensity of the lithiated adducted cerebroside signals did not diminish in the presence of up to 100 mM sodium chloride (Fig. 5) .

View larger version (22K): [in this window] [in a new window]   Fig. 4. ESI spectra of bovine brain sulfatide (A; negative ion mode) and bovine brain cerebroside (B; positive ion mode, lithium adducted) and the tandem mass spectrometry spectrum (C) of the m/z 816.5 parent ion from the spectrum in B.

View larger version (15K): [in this window] [in a new window]   Fig. 5. ESI spectra of bovine brain cerebrosides (positive ion mode, lithium adducted) in the presence of increasing concentrations of sodium chloride, demonstrating the absence of ion suppression by salt. Bovine cerebrosides, 100 pmol/µl in methanol-chloroform (4:1, v/v) containing 100 mM lithium chloride, were analyzed in the presence or absence of the indicated concentrations of sodium chloride or the lithiated solvent alone (bottom spectrum). Intensity (in arbitrary units) refers to the maximal signal recorded during the collection of each spectrum; current (µA) refers to the maximal observed read-back of the electrospray needle current during the collection of each spectrum. The cerebroside masses are identified by their aminoacyl-linked fatty acid (R) chains. The calculated m/z values for the various lithiated species are as follows: C18:0, 734.6; hC18:0, 750.6; C22:0, 790.7; hC22:0, 806.7; C24:1, 816.7; hC24:0, 834.7; and C26:0, 846.7.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Multiple reaction monitoring analysis of cerebroside product formation in the coupled ASA/CSAct reaction. At the indicated amounts of CSAct (abscissa) in the reaction mixture, the following transitions were measured: C18:0, 734.6572.6 (trace 1); hC18:0, 750.6588.6 (trace 2); C22:0, 790.7628.6 (trace 3); [2H35]C18:0, 769.7607.7 (trace 4); C24:1, 816.7654.6 (trace 5); C24:0, 818.7656.6 (trace 6); hC24:0, 834.7672.6 (trace 7); and C26:1, 844.7682.6 (trace 8). The data show increasing signal intensity for each of the seven product transitions recorded (traces 1–3 and 5–8) in the presence of a relatively constant response from the internal standard (IS; trace 4).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effect of the concentration of native (pig kidney) CSAct on the rate of formation of seven different cerebrosides in the coupled ASA/CSAct reaction in which bovine sulfatides were used as substrate. The fatty acyl compositions of the seven cerebroside products are indicated.

View larger version (10K): [in this window] [in a new window]   Fig. 8. Comparison of the rate of cerebroside formation from a near-equimolar mixture of the indicated synthetic sulfatide substrates. Ordinate values were obtained from activity measurements as shown in Fig. 7 (slopes) after normalization by the corresponding sulfatide substrate abundance. Each data point represents the mean of three experiments with the error bars indicating standard deviation within each respective data set.

To address the question of the saposin specificity in the ASA-catalyzed reaction, two different sets of recombinant saposins were compared. The results show that cerebroside formation occurred only with recombinant CSAct (saposin B), and there was no detectable cerebroside formation when recombinant saposins A, C, or D from the P. pastoris expression system (Fig. 9) , or when recombinant saposins A and C from the E. coli expression system (data not shown), were used. The earlier observations that saposins C and D have low but measurable activity in this assay were made with proteins purified from natural sources (37). We conclude that this result is a consequence of incomplete separation of the various proteins using the conventional chromatography that was available at the time, resulting in a low level of CSAct contamination in the saposin C and D samples. The in vitro data presented here clearly indicate that saposins A, C, and D cannot substitute for CSAct. However, caution should be exercised when extrapolating to the in vivo situation, because the in vitro assay does not exactly reflect the catabolic situation of sulfatides within the lysosomal compartment. The assay presents the sulfatide substrates in a micellar form in the absence of any other membrane lipids. In the lysosomal compartment, sulfatide substrates are presumably presented as components of inner lysosomal membranes rich in bis(monoacylglycero)phosphate. This lipid is specific for the inner membranes and stimulates sphingolipid hydrolysis. The topology of lysosomal digestion (38) may affect the specificity of CSAct and ASA, and especially their variants present in patients, in a different way than in the micellar situation used in the in vitro assay.

View larger version (10K): [in this window] [in a new window]   Fig. 9. Effect of the concentration of recombinant CSAct and saposins (SAP) A, C, and D on the rate of cerebroside formation from bovine sulfatide substrate in the in vitro assay. The ordinate represents the summed formation of the C18:1 + hC18:0 + C22:0 + C24:1 + C24:0 + hC24:0 + C26:1 cerebrosides.

Finally, it should be noted that earlier efforts focused on using the strong negative ESI signals obtained with sulfatide for the development of an MRM assay for the ASA/CSAct coupled reaction (39). The procedure was based on measuring the temporal decline in sulfatide (substrate) concentration. With the conventional Ion Spray^TM source, concentrations of nonvolatile inorganic salt in excess of 1 mM typically result in significant reductions in ion current from organic analytes. Although this is true for the detection of negative sulfatide ions, it is paradoxically not true for the detection of positively charged lithiated cerebroside ions, as already mentioned. To avoid changing the reaction conditions, it was necessary to develop a method for the effective removal of inorganic salt while maintaining reasonable sulfatide recovery. Several methods were tried, including dialysis against water (Spectra/Por^TM membrane), hexane/2-proponal extraction (40), and size-exclusion chromatography (Toso Haas Toyopearl^TM 40 HW HW 40S resin and elution with hexane/2-proponal). None of these methods adequately removed the inorganic salt without a significant loss of sulfatide. The final method of repeated aqueous 0.1 M ammonium acetate (pH 6.5) extractions gave a recovery of 65–70% of radiolabeled sulfatide through the entire sample preparation procedure, and the resulting extracts gave sulfatide MRM signals that were not suppressed. This procedure yielded comparable data to those reported above for the positive ion MRM assay in which the lithiated cerebroside ions were monitored. However, in addition to being a simpler and more comprehensive procedure, the measurement of product (cerebroside) formation is preferred over the measurement of substrate (sulfatide) depletion, particularly in the ASA/CSAct coupled assay that is used in the range of 0–10% substrate hydrolysis (12).

We have developed a mass spectrometric method for assaying the coupled ASA/CSAct reaction that provides information on the rate of formation of specific cerebroside products, thus allowing a more complete understanding of the kinetic properties of the reaction than is possible with the conventional radioactive method. The procedure can be used in CSAct-potentiated and detergent-potentiated reactions. No modifications to the standard assay protocols are needed. Future studies will include application of this method for the characterization of CSAct mutants as a way of better understanding the molecular basis of MLD.

	ACKNOWLEDGMENTS

 
This work was supported in part by grants from UCLA, the National Institutes of Health (NS-31271), Principal Investigator, Arvan L. Fluharty, the W. M. Keck Foundation, the Pasarow Family Foundation, the Ministero dell'Istruzione, dell'Università e della Ricerca (Programmi di Ricerca Scientifica di Rilevante Interesse Nazional 2004-Fondo per gli Investimenti della Ricerca di Base 2001), and the Deutsch Forschungsgemeinschaft (SFB 645). Participants in the UCLA Developmental Disabilities Immersion Program (DDIP), the Student Research Project (SRP), and the Neuroscience Interdepartmental Program (IDP), Anup Patel (DDIP), Farzin Feizbahchk (SRP), and Gregory Wong (IDP), helped with the early stages of this work.

Manuscript received May 10, 2005 and in revised form July 7, 2005 and in re-revised form July 19, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

1. von Figura, K., V. Gieselmann, and J. Jaeken. 2001. Metachromatic leukodystrophy. In The Metabolic and Molecular Bases of Inherited Disease, 8th edition, C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, editors. McGraw Hill, New York. 3695–3724.

2. O'Brien, J. S., and Y. Kishimoto. 1991. Saposin proteins: structure, function, and role in human lysosomal storage disorders. FASEB J. 5: 301–308.[Abstract]

3. Inui, K., and D. A. Wenger. 1984. Biochemical, immunological, and structural studies on a sphingolipid activator protein (SAP-1). Arch. Biochem. Biophys. 233: 556–564.[CrossRef][Medline]

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