Kidney Sulfatides in Mouse Models of Inherited GlycosphingolipidDisordersDETERMINATION BY NANO-ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY*

Received for publication, November 6, 2001, and in revised form, March 14, 2002Published, JBC Papers in Press, March 27, 2002, DOI 10.1074/jbc.M110641200

Roger Sandhoff‡§, Stefan T. Hepbildikler¶, Richard Jennemann‡, Rudolf Geyer�,Volkmar Gieselmann**, Richard L. Proia‡‡, Herbert Wiegandt‡, and Hermann-Josef Grone‡

From the ‡Deutsches Krebsforschungszentrum Heidelberg, Abteilung fur Zellulare und Molekulare Pathologie,INF 280, 69120 Heidelberg, Germany, the ¶Kekule-Institut fur Organische Chemie und Biochemie, Universitat Bonn,Gerhard-Domagk-Str. 1, 53121 Bonn, Germany, the �Biochemisches Institut am Klinikum der Justus-Liebig-UniversitatGiessen, 35392 Giessen, Germany, the **Physiologisch Chemisches Institut, Rheinische Friedrich Wilhelms Universitat,53115 Bonn, Germany, and the ‡‡Genetics of Development and Disease Branch, National Institute of Diabetes andDigestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Sulfatides show structural, and possibly physiologicalsimilarities to gangliosides. Kidney dysfunction mightbe correlated with changes in sulfatides, the majoracidic glycosphingolipids in this organ. To elucidatetheir in vivo metabolic pathway these compounds wereanalyzed in mice afflicted with inherited glycosphingo-lipid disorders. The mice under study lacked the genesencoding either �-hexosaminidase �-subunit (Hexa�/�),the �-hexosaminidase �-subunit (Hexb�/�), both �-hex-osaminidase � and �-subunits (Hexa�/� and Hexb�/�),GD3 synthase (GD3S�/�), GD3 synthase and GalNActransferase (GD3S�/� and GalNAcT�/�), GM2 activatorprotein (Gm2a�/�), or arylsulfatase A (ASA�/�). Quan-tification of the sulfatides, I3SO3

�-GalCer (SM4s), II3SO3�-

LacCer (SM3), II3SO3�-Gg3Cer (SM2a), and IV3, II3-(SO3

�)2-Gg4Cer (SB1a), was performed by nano-electrospraytandem mass spectrometry. We conclude for the in vivosituation in mouse kidneys that: 1) a single enzyme (Gal-NAc transferase) is responsible for the synthesis ofSM2a and GM2 from SM3 and GM3, respectively. 2) Inanalogy to GD1a, SB1a is degraded via SM2a. 3) SM2a ishydrolyzed to SM3 by �-hexosaminidase S (Hex S) andHex A, but not Hex B. Both enzymes are supported byGM2-activator protein. 4) Arylsulfatase A is required todegrade SB1a. It is probably the sole sphingolipid-sulfa-tase cleaving the galactosyl-3-sulfate bond. In addition,a human Tay-Sachs patient’s liver was investigated,which showed accumulation of SM2a along with GM2storage. The different ceramide compositions of bothcompounds indicated they were probably derived fromdifferent cell types. These data demonstrate that in vivothe sulfatides of the ganglio-series follow the same met-abolic pathways as the gangliosides with the replace-ment of sulfotransferases and sulfatases by sialyltrans-ferases and sialidases. Furthermore, a novel neutralGSL, IV6GlcNAc�-Gb4Cer, was found to accumulate onlyin Hexa�/� and Hexb�/� mouse kidneys. From this weconclude that Hex S also efficiently cleaves terminal�1–6-linked HexNAc residues from neutral GSLs in vivo.

Sulfatides, (designating all sulfated glycosphingolipids) suchas galactosylceramide I3-sulfate, occur enriched in the myelinsheets of the central and peripheral nervous system and inglandular epithelial tissues of mammals. Sulfatides of morecomplex structure have been found in the kidney (1). Inthe human renal cell carcinoma cell line SMKT-R3 high lev-els of sulfatides including gangliotriaosylceramide-II3 sulfate(SM2a)1 were observed (2) that they may modulate the meta-static potential of these cells (3). In addition, complex sulfatideshave been recognized to rank among the strongest ligands forNKR-P1. This membrane protein, with an extracellular Ca2�-dependent lectin domain, is expressed on natural killer cellsthat display innate immunity (4, 5). Other proteins involved ininnate immunity, properdin and factor H, have also been re-ported to bind specifically to the sulfatides (6). More recently ithas been shown that intracellular sulfation of lactosylceramidesuppresses the expression of integrins (7).

In mice, complex kidney sulfatides belong to the ganglio-series glycosphingolipids (GSL) and thus show structural sim-ilarity to “brain type”-gangliosides (Fig. 1). This relationship isfurther suggested by largely identical carbohydrate substitu-tion positions for sulfate and sialic acid. These mouse kidneysulfatides include lactosylceramide-II3 sulfate (SM3), SM2a,and gangliotetraosylceramide-II3, IV3 bis-sulfate (SB1a) (8). Ingeneral, GSLs are synthesized from a ceramide core by modi-fication with glycosyl- and sulfotransferases in the endoplasmicreticulum and Golgi. Degradation of GSL takes place at thesurface of intra-lysosomal vesicles by the action of exoglycosi-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

§ To whom correspondence should be addressed. Tel.: 49-6221-424358; Fax: 49-6221-424352; E-mail: r.sandhoff@dkfz-heidelberg.de.

1 The abbreviations used are: SM2a, gangliotriaosylceramide II3-sulfate; ASA, arylsulfatase A (EC 3.1.6.8); C/A, chloroform/acetone;C/M/W, chloroform/methanol/water; GalNAc-T, GalNAc-transferase(�1,4-N-acetylgalactosaminyltransferase, UDP-N-acetyl-D-galactosa-mine:GM3/GD3 �1,4-N-acetyl-D-galactosaminyltransferase (EC2.4.1.92)); GM2, II3-N-acetyl(or N-glycolyl)-neuraminyl gangliotriaosyl-ceramide; GM1a, II3-N-acetyl(or N-glycolyl)-neuraminyl gangliotetrao-sylceramide; GD1a, II3,IV3-bis-N-acetyl(or N-glycolyl)-neuraminyl gan-gliotetraosylceramide; GD3 synthase, CMP-sialic acid:GM3 �-2,8-sialyltransferase (EC 2.4.99.8); GM1-�-galactosidase (EC 3.2.1.23);GM2AP, GM2-activator protein; GSL(s), glycosphingolipid(s); Hex(A, B,or S), �-hexosaminidase (A, B, or S) (EC 3.2.1.52); MS, mass spectrom-etry; nano-ESI-MS/MS, nano-electrospray ionization tandem massspectrometry; SAP B, sphingolipid activator protein B (saposin B);sialidase, neuraminidase (EC 3.2.1.18); SM, sphingomyelin; sulfatideswere abbreviated according to Ishizuka (1), i.e. SM4s, galactosylceram-ide sulfate, GalCer I3 –sulfate; SM4g, seminolipid, galactosyl-1-alkyl-2-acyl-sn-glycerol I3-sulfate; SM3, lactosylceramide sulfate, LacCer II3-sulfate; SM1a, gangliotetraosylceramide II3-sulfate; SB1a,gangliotetraosylceramide II3,IV3-bis-sulfate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 23, Issue of June 7, pp. 20386–20398, 2002Printed in U.S.A.

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dases, sulfatases, and sialidases. For several of these degrada-tion steps, the presence of one of the five known lysosomalactivator proteins is required (9). Defects in the lysosomalenzymes or activator proteins that degrade GSLs are the causeof severe human inherited diseases such as metachromaticleukodystrophy and the different forms of GM2 gangliosidosis.

Deficiency in arylsulfatase A in metachromatic leukodystro-phy leads to lethal demyelination in the central and peripheralnervous systems. This disease is characterized by the lysoso-mal accumulation of the sulfatides SM4s, SM4g, and SM3(10, 11).

Defects in the �-hexosaminidase isozymes (Hex S, (�/�); HexA, (�/�); and Hex B, (�/�)) or in the GM2 activator protein leadto GM2 gangliosidosis, in which ganglioside GM2 and relatedglycolipids accumulate in lysosomes mainly of neuronal cells.The GM2 gangliosidoses include Tay-Sachs disease (B-vari-ant), due to mutations in the HEXA gene encoding the �-sub-unit of �-hexosaminidase, Sandhoff disease (0-variant), due tomutations in the HEXB gene encoding the �-subunit, and theAB-variant characterized by mutations in the GM2A gene. Forall three enzyme defects, the severe infantile forms result inrapidly progressing neurodegeneration, culminating in deathbefore age 4 years (12).

For the study of these human disorders, mouse models havebeen established that lack enzymes of GSL biodegradation(13–18). In addition, mutant mice deficient in enzymes of GSLbiosynthesis have been generated (19–23).

The aim of the present investigation was to determine howmouse kidney sulfatides were affected by genetically transmit-ted deficiencies in the metabolism affecting the brain typegangliosides and sulfatide SM4s (Fig. 1). Mouse models of thefour GM2-gangliosidoses (Hexa�/�, Hexb�/�, Hexa�/� andHexb�/�, and Gm2a�/�), metachromatic leukodystrophy(ASA�/�), as well as, UDP-GalNAc: �-1,4-GalNAc transferasedeficiency (GalNAcT�/�), were compared for their kidney sul-fatide component profiles by nano-electrospray ionization tan-dem mass spectrometry (nano-ESI-MS/MS).

EXPERIMENTAL PROCEDURES

Mutant mice used: Hexa�/� (13), Hexb�/� (15), Gm2a�/� (17),ASA�/� (14) and GD3S�/� as well as GD3S�/� and GalNAcT�/�(“GM3-only”) mice (23). Hexa�/� and Hexb�/� were produced byinterbreeding Hexa�/� and Hexb�/� mice as described (16). PCR wasemployed for genotyping (24).

The human Tay-Sachs liver material was from a girl that died at theage of 2 years and 10 months. The girl was affected with the classicalform of Tay-Sachs and symptoms were apparent from 8 months of ageincluding hyperaccusis. The control liver material was from a healthy42-year-old male donor who died in an accident.

Materials

All chemicals and solvents were of p.A. grade. Gold-sputtered boro-silicate glass capillaries, type D, were purchased from Teer CoatingsLtd. (Worcestershire, UK). Lyso-sulfatide (lyso-SM4s), sulfatide(SM4s), GlcCer, GalCer, LacCer, GM3, Gg3Cer, and bovine brain gan-gliosides were obtained from Matreya Inc. (Chalfont, PA), sphingosyl-phosphorylcholine, Forssman glycolipid, triethylamine, myristic acid,nonadecanoic acid, heptacosanoic acid, N-hydroxysuccinimide, N,N�-dicyclohexylcarbodiimide, anthrone, taurodesoxycholate, orcinol, abso-lute tetrahydrofuran, as well as, absolute N,N-dimethylformamide,both over molecular sieve from Sigma-Aldrich (Deisenhofen, Germany),SCDase (sphingolipid ceramide N-deacylase) from Takara Shuso (Otsu,Shiga, Japan), DEAE-Sephadex A-25 from Amersham Biosciences(Uppsala, Sweden), ammonia solution 25% p.A., LiChroprep RP-18 andLiChroprep Si 60 Silica Gel from Merck (Darmstadt, Germany), anddialysis tubes, Visking type 27/32 from Roth (Karlsruhe, Germany).GM2 was isolated from human GM2 gangliosidosis brain.

Methods

Purification of Sulfated GSL from Kidney Tissue—SM3 was ex-tracted from human kidney. SM2a and SB1a were isolated from ratkidney. In general, 100 g of tissue was homogenized on ice in 100 ml ofdistilled water with an Ultra Turrax T25 basic from IKA Labortechnik(Staufen, Germany) (6 � 2 min of homogenizing at 24,000 rpm withpauses of 2 min in between). The homogenate was freeze-dried andsubsequently extracted with acetone. The freeze-dried tissue then wasextracted for GSL, 2 times with chloroform/methanol/water (C/M/W)(10/10/1) and once with C/M/W (30/60/8). The combined C/M/W extractswere concentrated and dialyzed against 5 � 5 liters of distilled water.The dialyzed extract was lyophilized, dissolved in C/M/W (30/60/8) andloaded on DEAE A-25 column to separate neutral and acidic lipids.Elution was with a stepwise gradient of 20, 80, 200, 500, and 1000 mM

methanolic potassium acetate (KAc). SM3 was eluted with the 200 mM,SM2a with the 80 mM, and SB1a in the 500 mM KAc fraction. Thefractions were desalted by dialysis with 5 � 5 liters of distilled waterand lyophilized. From the corresponding fractions, the sulfated GSLswere further purified by repeated silica gel flash column chromatogra-phy with the appropriate mixtures of n-hexane/isopropyl alcohol/wateror C/M/W as running solvent systems.

Quantification of Purified GSL TLC Standards by Anthrone Reaction(25)—Commercially available, or from tissue isolated and purified,GSLs were dissolved in C/M/W (10/10/1). Aliquots in the range of 5–20nmol were dried in an 1.5-ml test tube with a gentle stream of nitrogen.100 �l of water and 500 �l of anthrone reagent were added. Then thecups were sealed and clamped in between two metal plates so that thelids could not open. One of the plates had appropriate holes for thelower part of the cups to fit through. The cups were incubated for 15 minat 100 °C and then cooled in a water bath at room temperature forfurther 20 min. Calibration curves were obtained with samples contain-ing a mixture of the free sugars in the appropriate equimolar ratioequaling the ratio of the individual GSL to be quantified (as an exam-ple: for SB1a quantification, Glc, Gal, and GalNAc were mixed in theratio 1:2:1). 300 �l of each sample was placed into a flat-bottomed

FIG. 1. Metabolic and structuralcomparison of gangliosides and sul-fatides of the ganglio-series. Enzymesthat are boxed in the figure were affectedby the investigated mouse models. For en-zyme abbreviations see the abbreviationfootnote.

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transparent 96-well microtiter plate. Absorption was measured at620 nm.

Synthesis of GSL Standards for Nano-ESI-MS/MS—Lyso-SM3, lyso-SM2a, and lyso-SB1a as well as lyso-GM3 and lyso-GM2 were obtainedby treatment of the purified compounds with SCDase according to Ref.26. The crude products were purified by silica gel column flash chro-matography using an appropriate mixture of C/M/W as running solventsystem. (Prior to activation 2-hydroxy fatty acids were esterified withacetic anhydride as follows: 2-hydroxy-myristinic acid was dissolved in200 �l of anhydrous and alcohol-free chloroform � 50 �l of aceticanhydride � 50 �l of 0.1% N,N-dimethylaminopyridine in chloroform.The reaction mixture was incubated for 60 min at 37 °C. Then 500 �l oftoluol were added and the sample dried under a gentle stream ofnitrogen at 37 °C. The sample was dissolved again in 200 �l of toluoland dried again as before.)

Fatty acids (65 �mol) were dissolved in 4 ml of dry tetrahydrofuranunder nitrogen gas and activated with 0.82 equivalents of dicyclohexyl-carbodiimide and 0.93 equivalents of N-hydroxysuccinimide. Reactiontook place overnight at room temperature.

For condensation, about 100 nmol of lyso-GSL was dissolved in 2 mlof dry N,N-dimethylformamide and 4 �l of triethylamine. Then 2 ml ofthe activated fatty acid were added. The reaction mixture was incu-bated at room temperature for 2–5 days and monitored by TLC. Uponthe long incubation some GSL were also acylated at hydroxyl groups.This resulted in a smear on TLC, running faster than the GSL stand-ard. The reaction mixtures were, therefore, treated with 0.1 M metha-nolic KOH for 2 h at room temperature. This mild base treatment wasalso necessary to remove the acetate ester from the 2-hydroxy group ofSM4s (18:1,h14:0). The crude products were purified by silica gel col-umn flash chromatography.

Quantification of the Synthesized GSL MS Standards—Aliquots ofthe synthesized GSL MS standards in the range of 0.3–1.0 nmol werespotted on TLC using a Linomat IV from CAMAG (Muttenz, Switzer-land). On adjacent lanes corresponding GSL TLC standards were spot-ted in different concentrations to obtain calibration curves. After devel-opment in chloroform, methanol, 0.2% aqueous CaCl2 (60/35/8) GSLbands were developed with orcinol/sulfuric acid spray reagent at 110 °Cfor 20 min or with 10% CuSO4 in 8% H3PO4 at 150 °C for 20 min. Theamount of the GSL compounds was determined by densitometric scan-ning of each lane at a wave length of 440 nm (Shimadzu CS-9301 TLCscanner). For each GSL the C14, C19, and C27 fatty acid containingcompounds were mixed in an equimolar ratio resulting in the ready-to-use MS standard of known concentration.

Extraction of GSLS from Murine Kidney for Mass SpectrometricAnalysis—Kidneys wet weight was determined, and the kidney homog-enized on ice in 5 ml of distilled water with a Ultra Turrax T25 basic(6 � 30 s of homogenizing at 24,000 rpm with pauses of 30 s in between).

GSL MS standards were transferred to glass tubes and the solventwas evaporated with a gentle nitrogen stream. An aliquot of the aque-ous kidney homogenate, equal to 20 mg of organ wet weight, was added,and the sample was sonicated for 5 min. Thereafter, the sample waslyophilized and extracted 2 times with 2 ml of acetone. The residualpellet then was extracted twice with 1.5 ml C/M/W (10/10/1) and with 2ml of C/M/W (30/60/8) for GSL. Neutral and acidic GSL of the combinedC/M/W extracts were separated on DEAE A-25 columns (bed volume:300 �l). The flow-through and wash yielded fraction 1, containing theneutral GSL. Acidic GSL, collected as fraction 2, were eluted with 4 mlof 500 mM methanolic potassium acetate. Solvent was evaporated andacid GSL (fraction 2) desalted with RP-18 (200 mg RP-18 material percolumn) column chromatography. Fraction 1 was dissolved in 100 �l of

5 mM methanolic ammonium acetate, and fraction 2 in 100 �l of meth-anol. If necessary, samples were further diluted for nano-ESI-MS/MS.

Extraction of GSLS from Human Liver for Mass Spectrometric Anal-ysis—Human liver GSLs were extracted in analogy to the mouse kidneyprotocol. Since the Tay-Sachs liver was stored frozen for more than 25years it lost barely any weight by freeze drying. Therefore GSL concen-trations were calculated per mg dry weight. Extraction without MSstandards was carried out with 150 mg dry weight, introducing MSstandards with 20 mg.

Determination and Characterization of Sulfatides and GSLS byNano-ESI-MS/MS—All analyses were performed with a triple quadru-pole instrument (VG micromass (Cheshire, UK) model Quattro II)equipped with a nano-electrospray source operating at an estimatedflow rate of 20–50 nl/min. Usually, 10 �l of a samples, dissolved inmethanol or methanolic ammonium acetate (5 mM), was filled into agold-sputtered capillary. The capillary was positioned at a distance of1–3 mm in front of the cone. The source temperature was set to 30 °Cand the spray was started by applying 800–1200 V to the capillary. Foreach spectrum 20–50 scans of 15–30 s duration were averaged. Alltandem MS experiments were performed with argon as collision gas ata nominal pressure of 2.2–2.7 � 10�3 mbar. The parameters for the conevoltage and the collision energy of the different scan-modes are listed inTable I.

Evaluation of the Nano-ESI-MS/MS Data and Quantification of Lip-ids—Quantitative spectra were measured with an average mass reso-lution of 1200 (ion mass/full width half-maximum). Peak height valuesof the first mono-isotopic peak of each compound were taken for eval-uation. From the peak intensities of the corresponding internal stand-ard lipids a linear trend was calculated. The obtained calibration curverepresented the intensity of the internal standard molar amount at agiven m/z value. In addition, a linear trend for n�2 molecular isotopicsignal intensities (molecules containing either two 13C-atoms or one34S-atom, and, thereby, shifted by m/z 2 upwards) was calculated fromthe internal standards. If necessary, signal intensities were correctedfirst from influence of n�2 signal overlap. This overlap appears if lipids,that contain one additional double bond, are present. Then their n�2signal overlaps with the main signal of the lipid without this doublebond. From the corrected intensity ratio (sample lipid/internal standardtrend) and the amount of internal standard added the quantity of theindividual molecular species (e.g. SM4s (18:1, 16:0) or SM4s (18:1, 24:1)etc.) was calculated. From the sum of individual molecular species thenthe amount of a lipid (SM4s) resulted. Endogenous SB1a, GM3, andGM2 were correlated to the sole corresponding standard.

Extraction of GSLS from Murine Kidney for TLC Analysis—For TLCanalysis, 50 mg of kidney wet weight were extracted as above using theappropriate volumes. The neutral and acidic GSL fractions were eachtaken up in 100 �l of C/M/W (10/10/1). Aliquots according to the Figurelegends were spotted on TLC plates with a Linomat IV from CAMAG(Muttenz, CH). A pre-run was performed with chloroform/alcohol (C/A)(1/1). Then the plates were dried and GSL were separated with therunning solvent chloroform, methanol, 0.2% aqueous CaCl2 (60/35/8), ifnot otherwise noted. Bands were detected with orcinol/sulfuric acidspray reagent at 110 °C for 10–20 min.

Carbohydrate Constituent Analysis—Carbohydrate constituentswere released by acid hydrolysis after hydrofuran treatment, convertedinto their corresponding alditol acetates and analyzed by capillaryGC/MS as detailed elsewhere (27).

Carbohydrate Permethylation Analysis—For determination of link-age positions of monosaccharide constituents, glycolipids were per-methylated and hydrolyzed (28). Partially methylated alditol acetates

TABLE IParameters used with the different scan-modi of nano-ESI-MS/MS

[Polarity] and scan modus Charge ofdetected ions Measured substances Cone Collision energy

V eV

[�] Precursor ion m/z 97 (�) Sulfatides by [HSO4]� fragment 70–105 60–115[�] Precursor ion m/z 220 (�) Sphingolipids containing HexNAc 125 58[�] Precursor ion m/z 405 (�) Sphingolipids containing (HexNAc)2 125 50[�] Precursor ion m/z 322 (�) IV6-GlcNAc�-Gb4Cer compared to Forssman glycolipid 125 58[�] Product ion (�) Fragments of sulfatides 70–105 40–60[�] Product ion (�) Fragments of Forssman glycolipid and IV6GlcNAc�-Gb4Cer 125 58[�] Precursor ion m/z 264 (�) Neutral sphingolipids containing C18-sph or C18-phyto by

fragment from sphingoid base35–70 44–90

[�] Precursor ion m/z 204 (�) Sphingolipids containing HexNAc 55–90 65–75[�] Precursor ion m/z 407 (�) Sphingolipids containing (HexNAc)2 75 40[�] Product ion (�) Fragments of Forssman glycolipid or IV6GlcNAc�-Gb4Cer 70 40

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obtained after sodium borohydride reduction and peracetylation wereanalyzed by GC/MS using the instrumentation and microtechniquesdescribed previously (29, 30).

RESULTS

Synthesis of Internal Standards for Nano-ESI-MS/MS—Forquantification of GSLs by nano-ESI-MS/MS an appropriateinternal standard must be applied (24). Therefore, sulfatidesand gangliosides with unusual fatty acid composition (myristicacid (14:0), 2-hydroxymyristic acid (h14:0), nonadecanoic acid(19:0), and heptacosanoic acid (27:0)) were synthesized fromthe corresponding lyso compounds. The latter were preparedenzymatically from the corresponding sulfatides, SM4s, SM3,SM2a, and SB1a, and gangliosides, GM3 and GM2 usingSCDase (for SM2a, see Fig. 2A). Coupling the lyso-GSL to afatty acid as described under “Experimental Procedures,” weproduced SM4s (18:1,14:0), SM4s (18:1,19:0), SM4s (18:1,27:0),SM3 (18:1,14:0), SM3 (18:1,19:0), SM3 (18:1,27:0), SM2a (h18:0,14:0), SM2a (h18:0,19:0), SM2a (h18:0,27:0), SB1a (h18:0,19:0), and recently GM3 (18:1,19:0), GM2 (18:1,14:0), and SM4s(18:1,h14:0). The sphingoid of human kidney SM3 consistedsolely of C18-sphingosine (data not shown), whereas rat kidneySM2a and SB1a contained a mixture of C18-sphingosineand C18-phytosphingosine as verified by nano-ESI-MS/MS(Fig. 2B).

Densitometric quantification of the product bands of lyso-SM2a from the orcinol/sulfuric acid-stained TLC (Fig. 2A) re-vealed a ratio of 57:100 for sphingosyl-lyso-SM2a (18:1) tophytosphingosyl-lyso-SM2a (h18:0). This was in good agree-ment with the ratio of the corresponding peaks in the nano-ESI-MS/MS spectrum (Fig. 2B) showing a sphingoid ratio of 59(18:1):100 (h18:0).

Each standard solution was quantified by densitometricscanning of the orcinol/sulfuric acid- or CuSO4/phosphoric acid-stained TLC band. On TLC, standards with C14, C19, or C27fatty acid migrated sequentially faster as compared with oneanother (data not shown). For mass spectrometric quantifica-tion of each sulfatide, the three respective fatty acid derivativestandards were mixed in an equimolar ratio. Since the concen-tration of the different sulfated GSLs, i.e. SM4s, SM3, SM2a,and SB1a, was not identical in murine kidney (Fig. 3, top, lane3), different amounts of corresponding MS standards wereadded to the kidney samples. For most samples, 432 pmol ofSM4s, 156.6 pmol of SM3, 102.6 pmol of SM2a, and 152.2 pmol

of SB1a-MS standards were added (Fig. 4). Correlating theendogenous sulfatide signals to those of the correspondingstandards levels of kidney sulfatides were quantified as de-scribed under “Experimental Procedures.”

At Higher Collision Energies 2-Hydroxy Fatty Acid-contain-ing Sulfatides Are Measured in the Precursor Ion Mode(m/z �97) with the Same Abundance as Sulfatides ContainingNon-hydroxy Fatty Acids—Since sulfatides SM4s with a 2-hy-droxy fatty acid give rise to additional product ions (due to abreak between the carboxyl-carbon- and the �-carbon-atom ofthe fatty acid), this might affect the relative abundances of thecommon fragments (e.g. [HSO4]� used for quantification) (31,32). These additional fragments could be detected for SM4s(2hFA) at collision energies of 50–60 eV with not more than 7%of the intensity of fragment m/z �97 ([HSO4]�). At collisionenergies of 90–115 eV that were used to quantify SM4s in theprecursor ion mode, these fragments were not detectable or hadan abundance smaller than 0.2%. For SM3 and SM2a addi-tional fragments due to a 2-hydroxy fatty acid could also bedetected at collision energies of 65–70 eV with up to 7% abun-dance relative to m/z �97. But none of these fragment ap-peared at collision energies of 90–115 eV, which were relevantfor quantification.

For SM4s a 2-hydroxy fatty acid containing standard SM4s(18:1,h14:0) was synthesized and mixed in an equimolar ratiowith SM4s (18:1,14:0), SM4s (18:1,19:0), and SM4s (18:1,27:0).From the peak intensities of the three non-hydroxy fatty acidcontaining SM4s a linear trend was calculated. At low collisionenergy (60 eV) the measured intensity of the 2-hydroxy stand-ard SM4s (18:1,h14:0) reached 90% of the linear trend whereasat 90 eV it differed no more than 2% from the linear trend.

Linearity of the Mass Spectrometric Method in Comparison toTLC Densitometry—To test the linearity of the mass spectro-metric method, a constant amount of SM4s standard (272pmol) was mixed in several samples with different amounts ofbovine brain sulfatide (8.5 to 17.7 nmol). The values obtainedby mass spectrometry as plotted against the amounts usedshowed that linearity was achieved from 35 to 8830 pmol (Fig.5). The average concentration evaluated from the 9 data pointsin this range differed by 1.7% from the theoretical value with astandard deviation of 8%. Since bovine brain sulfatide is amixture of sulfatides with different ceramide compositions,values obtained for some representative individual sulfatides

FIG. 2. Generation of lyso-SM2a by enzymatic digestion of rat kidney SM2a. SM2a was purified from rat kidney and hydrolyzed for 24 hwith the enzyme sphingolipid ceramide N-deacylase (SCDase) from Pseudomonas sp. as described under “Experimental Procedures.” A, reactionproducts of the aqueous phase were taken up in C/M/W (10/10/1, v/v), separated on TLC with running solvent C/M/0.2% aqueous CaCl2 (45/45/10)and stained for sugars with orcinol/sulfuric acid. Lane 1, purified SM2a from rat kidney; lane 2, SCDase digest of SM2a. Whereas GSL stainedpurple, taurodesoxycholate (TDC), used in the assay, turned light blue after several hours at room temperature, and no remaining SM2a stainingcould be observed in the digest. Densitometric quantification of the product bands revealed a ratio of 57: 100 for sphingosyl-lyso-SM2a tophytosphingosyl-lysoSM2a. B, nano-ESI-MS/MS precursor ion m/z 97-spectra of rat kidney SM2a (i) and its products of SCDase digestion(spectrum ii) corresponding to lanes 1 and 2 of A), respectively. m/z �97 represents the fragment [HSO4]� produced in the collision chamber. Bythis scan only compounds bearing a sulfate group (m/z �97) are detected and plotted in the spectrum. Therefore no TDC m/z �1019.5, carryinga sulfono- (giving rise to [�SO3]� with m/z 80) but not a sulfate group, is detected. A ratio of 59:100 for lyso-SM2a (18:1) to lyso-SM2a (h18:0) wasdetermined.

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were also plotted in this diagram to demonstrate linearity forthe individual species. The results indicate that individualsignal intensities down to 2.5% and up to 1000% of the stand-ard signal intensities were in the range of linearity.

Kidney of Wild Type Mice Contain the Sulfated GSLS SM4S,SM3, and SB1A—Acidic GSLs were isolated and separated onTLC as described. Staining with orcinol/sulfuric acid revealedGSLs with migration rates comparable to SM4s, SM3 and SB1aand GM3 (Fig. 3A, lane 3). Except for the compound migratingwith GM3, the TLC bands also stained with azur A indicatingthat they are sulfated glycolipids (data not shown). Quantifi-cation by nano-ESI-MS/MS revealed SM4s, SM3, and SB1a tomake up 83, 10, and 7% of the sulfated GSLs of mouse wholekidney in close agreement with an earlier report by Tadano-Aritomi and co-workers (8) (Fig. 6A; Table II). As comparedwith their data, however, the present analyses showed an �1.5times higher concentration of sulfated GSLs. SM2a and SB2,that are present in rat kidney, could not be detected in thekidney of wild type mice. Considering the limiting backgroundnoise of the mass spectra obtained, the concentration of SM2awas calculated to be less than 1.4 pmol/mg wet weight corre-sponding to less than 0.3% of total sulfated GSL.

With regard to mouse kidney sulfatide ceramide composi-tion, C18-sphingosine was the most prominent sphingoid withless than 6% of additional C18-phytosphingosine and 60–70%fatty acids of C22- and C24-aliphatic chain length. In addition,fatty acids of C-16, C-18, C-20, C21, C-23, C26, and C28 chainlength were also detected. More than 75% of the fatty acidswere saturated and the amount of 2-hydroxylated fatty acids,�60% of the total, was twice as high for SM4s than for SM3 andSB1a with �30%. 2-Hydroxylation was identified by both, mo-lecular mass in mass spectrometry, as well as, the additionalfragments m/z 522, 540, and 568, that appeared in the corre-sponding product ion spectra of SM4s (data not shown). Thesefragments have been reported to be characteristic for sulfatidewith 2-hydroxy fatty acids (31, 32).

Gg3Cer, Gb4Cer, and IV6GlcNAC�-Gb4Cer Accumulate inHexa�/� and HexB�/� Kidney—Neutral and acidic GSLs ofdouble mutant Hexa�/� and Hexb�/� mice were isolated. Ascompared with the wild type mouse, TLC of the neutral GSLsrevealed two double and one single bands that stained in-tensely with orcinol/sulfuric acid indicating the accumulationof three glycolipid components (Fig. 7, lane 1). The upper dou-ble band had a TLC migration rate corresponding to Gg3Cer,and the lower with Gb4Cer. Both GSLs are known to accumu-late in these mice. The lower single TLC band, designatedcompound X, showed a migration between Forssman glycolipidand Gg4Cer.

Investigating the neutral GSL fraction in nano-ESI-MS/MSwith a precursor ion scan of m/z 264 significantly increasedsignals for neutral GSL with the sequence Cer-Hex-Hex-Hex-NAc (as in Gg3Cer) and Cer-Hex-Hex-Hex-HexNAc (as forGb4Cer) were detected, as compared with wild type kidney. m/z�264 represents the protonated and dehydrated C18-sphingo-sine base, which is obtained as a characteristic fragment ofneutral GSLs under these conditions. The ascribed sequencewas confirmed from the collision induced fragments obtainedfrom these molecules (data not shown). Scanning for higherneutral GSLs in nano-ESI-MS/MS, we also used a precursor ionscan of m/z �204. m/z �204 represents a protonated and de-hydrated HexNAc residue which should be present at the ter-minus in all storage compounds of this mutant mouse. By bothof these scans, signals for a GSL containing 3 Hex and 2HexNAc residues could be identified that were not present inwild type kidney. Thus, the third accumulating GSL, compoundX, contained five sugar residues.

Comparing the collision induced fragments of the protonatedstorage compound X with that of protonated Forssman glyco-lipid by nano-ESI-MS/MS indicated that the characteristicfragments were identical (Fig. 8A). From these data the structurefor compound X could be assigned as HexNAc-HexNAc-Hex-Hex-Hex-Cer.

Since in Forssman glycolipid the terminal HexNAc residue is�-glycosidically linked and not a substrate for �-hexosamini-dase, it is assumed not to accumulate in the GM2 gangliosido-sis mice. In addition, the TLC band of compound X did notco-migrate with the Forssman lipid standard. Compound X wasfurther analyzed by nano-ESI-MS/MS. Comparing the collisioninduced fragments of the deprotonated compound X andForssman glycolipid in the negative product ion mode of nano-ESI-MS/MS, distinct differences were observed. First, the stor-age compound did not yield a fragment of m/z 154 that ap-peared in Forssman lipid standard from sheep erythrocytes(Fig. 8B, ii) or from chicken heart (data not shown). Second, afragment with m/z 322, not present in Forssman lipid, ap-peared with compound X (Fig. 8B, i). This is a terminal frag-ment produced by ring cleavage between C2-C3 and C5-oxygenring of the subterminal HexNAc residue. To ensure that this

FIG. 3. TLC of acidic GSL from kidneys of mutant mice. AcidicGSL were extracted from kidney homogenate, lipids corresponding to 4mg of kidney wet weight separated on TLC and stained with orcinol/sulfuric acid as described: top, lanes 1 and 9, and B, lanes 1 and 10:ganglioside standards, from top to bottom: GM3, GM2, GM1, GD1a,GD1b, and GT1b; A, lanes 2 and 8; and B, lanes 2, 5, and 9: sulfated GSLstandards, from top to bottom: SM4s, SM3, SM2a, SB2, and SB1a. Top,lane 3, wild type; 4, Gm2a�/�; 5, Hexa�/�; 6, Hexb�/�; 7, Hexa�/�and Hexb�/�. Bottom, lane 3, ASA�/�; 4, ASA�/�; 6, GD3S�/� andGalNAcT�/�; 7, GM3 only GD3S�/� and GalNAcT�/�; 8, GD3S�/�and GalNAcT�/�. TLC, top, shows the strong storage of SM2a inHexa�/� (lane 5) and Hexa�/� and Hexb�/� kidney (lane 7) andminor storage of SM2a in Hexb�/� (lane 6) and Gm2a�/� kidney (lane4, faint band), whereas no SM2a could be detected in wild type kidney(lane 3). The acidic GSL pattern of Hexa�/� and Hexb�/� kidney isidentical to that of Hexa�/� kidney (data not shown). TLC, bottom,shows (i) strong storage of SM4s, SM3, and SB1a in ASA�/� kidney(lane 2), with SM4s and SM3 not separated and (ii) complete lackof SB1a with corresponding accumulation of SM3 in kidney ofGalNAcT�/� mice (lane 7).

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fragment was not due to impurities, the neutral GSL fractionwas scanned for compound X using this fragment in a nano-ESI-MS/MS precursor ion mode. The storage compound withthe same ceramide pattern (C18-sphingosine combined with16:0, 22:0, 24:1, and 24:0 fatty acids) as described before with aprecursor ion scan of m/z 220, representing the deprotonatedterminal HexNAc-residue was again detected (Fig. 8A, iii andiv). Since Forssman glycolipid from chicken heart had a dis-tinctly different ceramide composition, including ceramide

(C18-sph,18:0) and (C18-sph,20:0) (Fig. 9A, ii), it was admixedwith the GSL fraction containing compound X. Both com-pounds could be detected when scanning the mixed sampleeither in nano-ESI-MS/MS total negative ion mode (Fig. 9B, i),or with collision induced fragments (m/z 405, Fig. 9B, ii, or m/z220, Fig. 9B, iii) that appear in the product ion scans of bothcompounds. In contrast, by scanning with the compound Xcollision-induced fragment the m/z 322 only compound X couldbe detected; no signals for Forssman glycolipid appeared (Fig.

FIG. 4. Nano-ESI-MS/MS spectrum of a MS standard mixture for sulfatide determination. Synthetic sulfated GSL standards weremixed as follows: C14-, C19-, and C27-SM4s: 4.8 pmol/�l each; C14-, C19-, and C27-SM3: 1.74 pmol/�l each; C14-, C19-, and C27-SM2a, 1.14pmol/�l each; and C19-SB1a, 1.69 pmol/�l. The mixture was scanned by nano-ESI-MS/MS in negative mode using a precursor ion scan with m/z�97 (corresponding to [HSO4]�) specific for sulfated compounds (31, 32, 40, 53, 54). Aliquots of this mixture later were added to the kidney GSLsamples for quantification.

FIG. 5. Quantification of SM4s bynano-ESI-MS/MS. Bovine brain sul-fatide (SM4s) from 8.5 pmol to 17.7 nmolwas mixed in 220 �l of methanol withSM4s-MS standard, containing C14-,C19-, and C27-SM4s, each in an amountof 272 pmol. Samples were measured witha precursor ion scan specific for sulfatedlipids as described under “ExperimentalProcedures.” Besides the total sulfatide,the amount of different ceramide compo-sitional SM4s species are plotted. Thenumber indicates the m/z value of the sin-gle sulfatide with: m/z 806, SM4s(18:1,h18:0); m/z 862, SM4s(18:1,22:0); m/z888: SM4s(18:1,24:1); m/z 890, SM4s(18:1,24:0); m/z 904: SM4s(18:1,h24:1); m/z916, SM4s(18:1,26:1). Data points for 806,862, 904, and 916 are more or lessoverlapping.

FIG. 6. Comparison of kidney sul-fatides in wild type, Hexa�/� and“GM3-only” mice by nano-ESI-MS/MS. Kidney homogenate was mixed withMS standards for sulfatides and extractedfor acidic GSL as described. Sulfatideswere detected by nano-ESI-MS/MS innegative mode using the precursor ionscan m/z 97. Spectrum A, wild type; B,Hexa�/�; and C, GM3-only (GD3S�/�and GalNAcT�/�) mice. Signals corre-spond to 20 mg wet weight kidney, and432 pmol of SM4s, 156.6 pmol of SM3,102.6 pmol of SM2a, and 152.1 pmol ofSB1a internal standards, each. Signalsfor internal standards are pointed out inspectrum A). Mass per charge (m/z) rangefor mouse kidney endogenous sulfatidesignals: SB1a, 692 (18:1,16:0) to 756 (18:1,h24:0); SM4s, 776 (18:1,16:1) to 944 (18:1,28:1); SM3 938 (18:1,16:1) to 1080 (18:1,26:0); and SM2a, 1143 (18:1,16:0) to1283 (18:1,26:0). Signal m/z 713(18:1,19:0) is due to SB1a internal stand-ard (see Fig. 4). Clearly, accumulatingSM2a is detected in high amounts in B,whereas in C, no kidney SB1a could bedetected (*).

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9B, iv). Therefore, compound X with the structure HexNAc-HexNAc-Hex-Hex-Hex-Cer, must be different from Forssmanglycolipid. For a further investigation of the nature of com-pound X, the glycolipid was isolated by preparative TLC. Sub-sequent carbohydrate constituent analysis by GC/MS revealedthe monosaccharides Gal, Glc, GlcNAc, and GalNAc in the ratio(2.0:1.25:1.0:0.9). And additional permethylation analysis iden-tified 4-substituted Glc, 4-substituted Gal, 3-substituted Gal,6-substituted GalNAc, and terminal GlcNAc (data not shown).

It is known that in mouse kidney a characteristic globo-/neolacto-series glycolipid occurs, Gal�1–4(Fuc�1–3)GlcNAc�1–6(Gal�1–3)Gb4Cer, which could be detected by nano-ESI-MS/MSin wild type and in mutant kidney samples (data not shown).Therefore, it appears highly likely that compound X is an accu-mulated degradation product of this glycolipid with its remnantN-acetylhexosamine-terminal core structure IV6-GlcNAc�-Gb4Cer.

Besides C18-sphingosine and non-hydroxylated fatty acids ofC16 up to C24 aliphatic chain length, hydroxy fatty acids, aswell as, phytosphingosine were determined in Gb4Cer andGg3Cer. This explains the appearance of TLC double bands forboth of these glycolipids (data not shown).

SM2A Accumulates in HexA�/� and HexB�/� MouseKidney—Separation of the acid GSL fraction on TLC and stain-

ing with orcinol/sulfuric acid revealed a new prominent bandrunning at the level of SM2a that does not appear in wild typekidney. No significant increase of a band at the level of GM2was observed.

Whereas quantification of SM4s, SM3, or SB1a by nano-ESI-MS/MS showed no significant changes in concentrations, alarge amount of SM2a (239 pmol/mg wet weight) was identifiedin kidney from a 9-week-old mutant mouse. This corresponds toan SM2a increase of at least 172-fold as compared with kidneyfrom a wild type mouse (Table II and Fig. 10). No significantchanges in the ceramide compositions of SM4s, SM3, and SB1a,or accumulated SM2a compared with wild type were detected.

SM2A but Not Neutral GSLS Accumulate in Hexa�/�Kidney—TLC analysis of the neutral GSLs of Hexa�/� micekidney showed no significant differences as compared with wildtype (data not shown). In contrast, the acidic GSL componentprofile was characterized by a prominent band running at thelevel of SM2a that was not present in lipids of wild type kidney(Fig. 3, top, lane 5). However, no significant increase of a TLCband at the level of GM2 could be observed. Similar to TLCanalysis, quantification of the sulfated GSL by nano-ESI-MS/MS revealed no significant changes in SM4s, SM3, or SB1aconcentrations (Fig. 6B). However, in the case of kidney from19- and 20-week-old Hexa�/� mice, large amounts (248 � 18

TABLE IISM2 accumulation in kidney of �-hexosaminidase or GM2 activator protein-deficient mice as determined by nano-ESI-MS/MS

The standard deviation of the method is smaller than 8%.

Mouse kidney Age SM4s SM3 SM2a SB1a SM

weeks pmol lipid/mg wet weight

Wild type 15 390 49 �1.4a 32 3000Wild type 52 380 47 �1.4a 31 4200Gm2a�/� 23 320 25 6.4 22 3300Gm2a�/� 23 370 25 7.9 21 2900Hexa�/� 19 340 33 230 38 3900Hexa�/� 20 280 41 270 33 5400Hexb�/� 13 240 38 25 23 3500Hexb�/� 18 310 52 25 28 3600Hexa�/� and Hexb�/� 9 500 24 240 36 2000Hexa�/� and Hexb�/� 15 360 41 130 39 4000

a No SM2a could be detected and background noise was calculated to 1.39 pmol/mg wet weight.

FIG. 7. Neutral GSL storage compounds in kidney of Hexb�/�, Hexa�/� and Hexb�/�, and Hexa�/� and Hexb�/� mice. NeutralGSL corresponding to 5 mg of kidney wet weight were separated on TLC with the running solvent chloroform, methanol, 0.2% CaCl2 (60/35/8) andstained with orcinol/sulfuric acid as described. Lanes 1, Hexa�/� and Hexb�/�; 2 and 8, neutral GSL standard, from top to bottom: GalCer (tripleband), LacCer (double band), Gg3Cer and Forssman lipid (double band, mixture from sheep erythrocytes and chicken heart); lane 3, wild type; 4,Hexa�/�; 5, neutral GSL standard, from top to bottom: GlcCer (double band), LacCer (strong double band), Gb3Cer (double band), Gb4Cer (strongband), Lc4Cer, nLc4Cer and Gg4Cer (strong band); 6, Hexb�/�; 7, Hexa�/� and Hexb�/�. Whereas there is no significant difference betweenHexa�/� (lane 4) and wild type mice (lane 3), Hexa�/� and Hexb�/� (lane 1), Hexb�/� (lane 6), as well as, Hexa�/� and Hexb�/� kidney (lane7) accumulate Gg3Cer and Gb4Cer. In addition, only Hexa�/� and Hexb�/� mice (lane 1) accumulate a third neutral GSL (compound X), runningbetween Gg4Cer and Forssman glycolipid. Fuc-LacNAc-Gb5Cer: Gal-�1,4 (Fuc-�1,3-)-GlcNAc�1,6(Gal-�1,3-)-GalNAc�1,3-Gb3Cer.

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pmol/mg wet weight) of SM2a were detected that correspondedto an average increase of at least 180-fold as compared with thewild type (Table II and Fig. 10). No significant changes in theceramide compositions of sulfated GSL compared with wildtype were detected The SM2a pattern was similar to that ofHexa�/� and Hexb�/� mice.

Accumulation of Gg3Cer and Gb4Cer but Not IV6GlcNAC�-Gb4Cer in HexB�/� Kidney—TLC analysis of the neutralGSLs of kidney from Hexb�/� mice showed storage of Gg3Cer,Gb4Cer, but no accumulation of GlcNAc�1–6Gb4Cer. There

was no significant difference in Gg3Cer and Gb4Cer TLC-bandintensities between Hexb�/� and Hexa�/� and Hexb�/�double mutant kidney (Fig. 7).

SM2A Accumulates in Hexb�/� Kidney—In the case of theHexb�/� mouse mutant, TLC of the kidney acidic GSLsshowed the appearance of a faint band migrating identically tothe SM2a standard (Fig. 3, top, lane 6). By nano-ESI-MS/MS,24.7 � 0.05 pmol of SM2a per mg wet weight was quantified ina 13- and 18-week-old mutant kidney corresponding to anaverage increase of at least 18-fold as compared with the wild

FIG. 8. Fragmentation patterns of compound X from Hexa�/� and Hexb�/� and of Forssman glycolipid from sheep erythrocytesby nano-ESI-MS/MS-product ion mode. A, fragments in positive mode: (i) compound X (18:1,24:0) with m/z 1542.9 and (ii) Forssman glycolipid(GalNAc-�1,3-Gb4Cer (18:1,24:0)) with m/z 1542.9. Comparing the fragments of both compounds demonstrates, that the sugar increments areordered in the same sequence: Cer-Hex-Hex-Hex-HexNAc-HexNAc. B, fragments in negative mode (i) compound X (18:1,24:0) with m/z 1540.9 and(ii) Forssman glycolipid (18:1,24:0) with m/z 1540.9. Fragment m/z 322 is generated only by compound X (i) and represents the terminal HexNAcwith a fragment of the sub-terminal HexNAc generated by a ring cleavage between C2-C3 and ring O-C1. On the other hand, fragment m/z 154can only be generated by Forssman glycolipid (ii), indicating that both compounds are not identical. C, structures and fragmentation schemes ofcompound X and Forssman glycolipid.

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type (Table II, Fig. 10). No significant changes in SM4s, SM3,or SB1a-concentrations were found. No significant differencesin the ceramide compositions of sulfated GSL compared withwild type were detected. The SM2a pattern was similar to thatof Hexa�/� and Hexb�/� mice.

SM2A Accumulates in Gm2a�/� Kidney—TLC analysis ofthe neutral GSL fraction from Gm2a�/� kidney showed nosignificant differences as compared with wild type kidney (datanot shown). A faint TLC band of the acidic GSLs, not seen inthe wild type, co-migrated with SM2a (Fig. 3, top, lane 4). Asdetermined by nano-ESI-MS/MS, kidney from 23-week-old mu-tant mice contained 7.1 � 1.8 pmol of SM2a per mg wet weightcorresponding to an average increase of at least 5-fold over wildtype (Table II and Fig. 10).

No significant changes in the ceramide compositions of sul-fated GSL compared with wild type were detected. The SM2a

pattern was similar to that of double mutant Hexa�/� andHexb�/� mice.

All Sulfatides Accumulate in Arylsulfatase A-deficientKidney—The neutral GSLs of ASA�/� mouse kidney werenot different from the wild type (data not shown). In contrast,TLC of the mutant mouse kidney acidic GSL fraction showedstrong increases in bands co-migrating with SM4s, SM3, andSB1a. All were stained by orcinol/sulfuric acid spray reagent(Fig. 3, bottom, lane 3) and with azur A (data not shown).

In a 11-week-old ASA�/� kidney, 11-, 4.4-, and 15-foldaccumulation of SM4s, SM3, and SB1a, respectively, was quan-tified by nano-ESI-MS/MS. Analysis of a kidney from a 1-year-old ASA�/� mouse demonstrated a further increase in theaccumulation of SM4s, SM3, and SB1a to about 80-, 40-, and60-fold, respectively. However, no further increase in the accu-mulation of these GSLs was seen in a 2-year-old ASA�/�

FIG. 9. Mass spectrometric differences between Forssman glycolipids and compound X from Hexa�/� and Hexb�/� kidney. A,compositions of Forssman glycolipid from chicken heart or from sheep erythrocytes, and from compound X, respectively, as measured by precursorion scanning. According to the fragmentation patterns of Forssman glycolipid and compound X in Fig. 7B, the dominant fragment m/z 405 wastaken to measure Forssman glycolipid from sheep erythrocytes (i) and from chicken heart (ii), and the dominant fragments m/z 220 (iii) and 322(iv) were taken to scan for compound X in the neutral GSL fraction of Hexa�/� and Hexb�/� kidney. m/z 220 represents [HexNAc � H�]�, m/z322 a terminal fragment derived by ring cleavage through the subterminal HexNAc (see text) and m/z 405 represents [HexNAc2 � H3O�]�.Compound X contains mainly the fatty acids: 16:0, 22:0, 24:1, and 24:0. Forssman glycolipid from chicken heart contains mainly the fatty acids:16:0, 18:0, 20:0, and 22:0, whereas that of sheep erythrocytes contains mainly 24:1 and 24:0 fatty acids. Forssman glycolipid was not detectableby the precursor ion scan m/z �322. B, nano-ESI-MS/MS-spectra of a mixture of chicken heart Forssman glycolipid (A, ii) with the neutral GSLfraction from Hexa�/� and Hexb�/� kidney (A, iii or iv). (i) total negative ion spectrum; (ii) precursor ion scan m/z �405; (iii) precursor ion scanm/z �220; and (iv) precursor ion scan m/z �322. Whereas both compounds, Forssman glycolipid and compound X, are detected with the precursorions of m/z �220 (iii) and �405 (ii) (although with different sensitivities), the precursor ion m/z �322 (iv) is specific for compound X; no Forssmanglycolipid (lack of m/z 1457 and 1485) is detected and the intensity pattern in (iv) returns to that of the pure neutral GSL fraction as shown (A, iv).

FIG. 10. Accumulation of SM2a inkidney of �-hexosaminidase or GM2-activator protein-deficient mice. Val-ues obtained by quantitative nano-ESI-MS/MS as described under “ExperimentalProcedures” are plotted in a logarithmicscale. For absolute values see Table II.For wild type, SM2a detection limit (1.39pmol/mg wet weight) was set to 100%since no SM2a could be detected in wildtype. Age of mice in weeks is indicated onthe y axis.

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kidney. Sulfatide concentrations were very similar to those ofthe 1-year-old ASA�/� kidney (Table III and Fig. 11). Nosignificant changes in the ceramide constituent compositions ofSM4s, SM3, and SB1a compared with wild type were detected.

Mice Deficient in �-GalNac Transferase Lack SB1A in theKidney—No significant differences, as compared with wildtype, were observed by TLC for the neutral kidney GSLs ofGD3S�/� and GalNAcT�/�, GD3S�/� and GalNAcT�/�,and GD3S�/� and GalNAcT�/� mutants (data not shown).With regard to the acidic GSLs, the TLC profile of the GD3S�and GalNAcT�/� kidney was characterized by the disappear-ance of a band co-migrating with the SB1a standard (Fig. 3,bottom, lane 7). Quantification of the sulfated GSL in thesemutants revealed a 20% decrease of SB1a with a correspondingincrease in SM3 in GD3S�/� and GalNAcT�/� kidney ascompared with GD3S�/� and GalNAcT�/� mutant. Kidneysfrom GD3S�/� and GalNAcT�/� mutant mice showed anincrease in SM3 with SB1a being undetectable (Table IV).

SM2A Accumulates in Addition to GM2 in a Tay-Sachs Pa-tient’s Liver—Acidic GSL were extracted with and withoutinternal MS standards from a Tay-Sachs patient’s and a controlhuman liver. In both livers comparable amounts (�10%) ofGM3 were detected in good agreement with the data publishedby Nilsson and Svennerholm (33). In addition, significantamounts of SM2a and GM2 could only be detected in theTay-Sachs liver (Table V). The ceramide composition of allthree GSLs, GM3, GM2, and SM2a, was different from eachother, whereby that of GM3 and GM2 were comparable to thevalues reported earlier (33) (Figs. 12 and 13). With 57% GM2containing stearic acid (GM2(18:1,18:0)) was the main GM2species. In contrast to this, with 31% SM2a containing a C24:1-fatty acid (SM2a(18:1,24:1)) was the main SM2a species.

DISCUSSION

The acidic glycolipids, because of their negative charge andoften complex and seemingly systematic structures appear asparticularly enigmatic with regard to their physiological sig-nificance. Naturally occurring genetic mutants that affect theirmetabolism have so far been observed only for deficiencies incatabolizing enzymes causing glycolipid accumulations. For thestudy of such inherited sphingolipid storage diseases, and morerecently, with a more basic view on the possible elucidation ofGSL functions, mutant mice models have been created thatlack enzymes for the synthesis and degradation of GSL. It hasbeen shown that most of the introduced mutations severelyaffect the presence or concentration of gangliosides. Sulfatideshave not been systematically investigated. However, in view ofthe overall similarity between sulfatides and gangliosides,these mutant mouse models may also be expected to displayderangement of the cellular sulfatide component profile. Kid-ney, particularly in the mouse, is the organ in which sulfatidesare highly concentrated and have been associated with renaltransport and metabolism (1, 34). Therefore, an investigationwas initiated to characterize alterations of sulfatides in mutant

mouse models. The sulfatide components were characterizedand quantified by nano-electrospray tandem mass spectrome-try. This technique has already been successfully applied tocharacterize and quantify different sphingolipids (31, 32, 35–44). Internal standards were synthesized from lyso-sulfatidescontaining fatty acids of unusual chain length. Lyso-sulfatideswere obtained by enzymatic cleavage of fatty acids from theparent GSL compounds using sphingolipid ceramide N-deacyl-ase (SCDase) from Pseudomonas sp. (26). In the case of some-what alkali-sensitive GSLs such as SM2a and SB1a, the enzy-matic method of ceramide fatty acid cleavage offers advantageover the chemical cleavage.

Quantification of sulfatide by mass spectrometry was linearover more than 2 orders of magnitude with R2 greater than0.995 from 18 to 8830 pmol. In contrast, the conventionalquantification of GSLs by densitometric scanning on TLCplates was only linear in the range of 100 to 700 pmol (with R2

of 0.995). Accordingly, the linear range of quantification bynano-ESI-MS/MS was �70 times greater than that of densito-metric scanning. Furthermore, nano-ESI-MS/MS proved to bemore sensitive than chemical staining of GSLs on TLC. Inaddition, mass spectrometric quantification gives more de-tailed information about the quantitative ceramide composi-tions of each individual GSL species, which could vary betweendifferent samples. No differences beyond the accuracy of themethod could be detected when comparing the mass spectro-metric sensitivity of 2-hydroxy fatty acid-containing sulfatideSM4s (18:1,h14:0) with sulfatides containing non-hydroxylatedfatty acids (collision energy: 90–115 eV). Nevertheless, at lowercollision energies (50–60 eV) detection of sulfatides SM4s witha 2-hydroxy fatty acid was about 10% less sensitive than forsulfatides without 2-hydroxy fatty acid. At these energies, ad-ditional fragments due to a break between the carboxyl-carbon-and the �-carbon-atom of the fatty acid are produced (31, 32),which seem to influence the abundance of the sulfate fragment.At higher energies (90 to 115 eV) these fragments disappear forall complex sulfatides. Therefore extrapolation of the SM4sdata onto SM3, SM2a, and SB1a is justified and all sulfatidescan be correlated to standards with non-hydroxy fatty acids.

SB1a was absent in �-GalNAc transferase-deficient mice asexpected from an in vitro study suggesting that a single enzymesynthesizes both GM2 and SM2a (45). In the kidneys of theseknockout mice, SM3 accumulated to a level comparable withthe combined levels of SM3 and SB1a of wild type mousekidney. The total concentration of these two sulfatides takentogether, was also retained at the same level in GalNAcT�/�kidneys. Therefore, it appears plausible that SM3 is the pre-cursor for SB1a biosynthesis. First SM3 is converted to SM2aby the action of GalNAcT and, possibly, via formation of SM1ato SB1a. This would be in analogy to the synthesis of GD1a inbrain (9). The absence of SB1a in GalNAcT�/� mice is con-sistent with its structural ganglio-series derivation.

Sulfatides like all other GSL are degraded in the cellular

TABLE IIIAccumulation of sulfated GSL in kidney of arylsulfatase A-deficient mice as determined by nano-ESI-MS/MS

The standard deviation of the method is smaller than 8%.

Mouse kidney Age SM4s SM3 SM2a SB1a SM

weeks pmol lipid/mg wet weight

ASA�/� 10 230 48 NDa 17 3700ASA�/� 11 2,500 210 ND 260 3400ASA�/� 47 210 87 ND 19 3900ASA�/� 53 18,000 1900 ND 970 4600ASA�/� 98 310 37 ND 30 4100ASA�/� 95 19,000 1700 ND 890 2600

a ND, not detectable.

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lysosomes. For SM4s, the initial step in degradation is theremoval of sulfate by the combined action of arylsulfatase Aand the activator protein SAP B (10). Thus far this is the onlysulfatase known to act on sulfatides (1). Since SM4s, SM3, andSB1a all carry a sulfate group linked to a terminal sugarresidue, the cleavage of the sulfate group is expected to be thefirst step of their biodegradation. It was shown here that inASA�/� mice that SB1a together with SM4s and SM3 accu-mulates in the kidney. This finding demonstrates that for thein vivo catabolism of all three compounds the presence of aryl-sulfatase A is an absolute requirement. Compared with thecorresponding wild type levels, the enrichment factors of theindividual sulfatide components is roughly the same. It is con-cluded that there is no other sulfatase that acts on any one ofthese three sulfatides individually and ASA is probably theonly sphingolipid-sulfatase in murine kidney cleaving thegalactosyl-3-sulfate bond.

In analogy to the degradation of GM1a, SM1a, the product ofSB1a hydrolysis by arylsulfatase A, then is likely to be furtherdegraded to SM2a and SM3 with the lysosomal enzymes �-ga-lactosidase and �-hexosaminidase, respectively. An overview ofthe 4 different GM2 gangliosidosis mouse models investigated

including remaining enzyme activities and SM2 accumulationis given in Table VI. All mutant mice that were deficient in the�-, �-, or both subunits of this enzyme accumulated SM2aconfirming that SB1a is processed to SM2a, with the latterbeing degraded further by �-hexosaminidase. Highest accumu-lation occurred in mice lacking all hexosaminidase isomers(Hexa�/� and Hexb�/�) or lacking the �-subunit (Hexa�/�).For these two mutant mice, SM2a accumulation was comparableindicating that Hex B, the only relevant active enzyme isomerexpressed in Hexa�/� mice, did not act on SM2a. This is in goodagreement with previous in vitro studies (46, 47).

Hexb�/� mice, expressing only an intact Hex S isomer,accumulated 10 times less SM2a than Hexa�/� or Hexa�/�and Hexb�/� mutants. It is, therefore, concluded that Hex Splays an important and necessary role in the in vivo degrada-tion of SM2a. In contrast, Hex A is the pivotal enzyme for thedegradation of GM2 (47). Nevertheless, Hex A contributes toSM2a degradation in vivo: Hexb�/� mice left with only Hex S,but no Hex A and Hex B activity, accumulate SM2a to a lesserdegree. These findings are in agreement with in vitro studiesdemonstrating the ability of both, human Hex S and Hex A, todegrade SM2a in the presence of human GM2 activator protein(47).

For the degradation of GM2 in mice two metabolic pathwayshave been described. One catabolic sequence, in humans the onlysignificant pathway, is the degradation of GM2 to GM3 by Hex Ain presence of GM2AP, and further hydrolysis of GM3 to LacCer.Another mode of degradation is the formation of Gg3Cer fromGM2, and further processing to LacCer by Hex A or Hex B, withGM2AP as a cofactor. Therefore, Hexa�/� mice accumulate 3–6times less GM2 than Hexb�/� or Hexa�/� and Hexb�/� mice(16, 17). Since, in contrast to GM2, Hexa�/� and Hexa�/� andHexb�/� mice accumulated SM2a to an equal extent, it is sug-

FIG. 11. Accumulation of sulfatidesin kidney of arylsulfatase A-deficientmice. Values obtained by quantitativenano-ESI-MS/MS as described under “Ex-perimental Procedures” are plotted in alogarithmic scale. For absolute values seeTable III. Age of mice in weeks is indi-cated on the y axis.

TABLE IVLack of SB1a in kidney of �-GalNAc transferase-deficient mice as determined by nano-ESI-MS/MS

The standard deviation of the method is smaller than 8%.

Mouse kidney Age SM4s SM3 SM2a SB1a SM

weeks pmol lipid/mg wet weight

GD3S�/� and GalNAcT�/� 17 260 31 NDa 24 4300GD3S�/� and GalNAcT�/� 17 240 42 ND 24 4700GD3S�/� and GalNAcT�/� 17 260 61 ND ND 4500GD3S�/� and GalNAcT�/� 20 370 48 ND 36 4500GD3S�/� and GalNAcT�/� 20 340 51 ND 26 4100GD3S�/� and GalNAcT�/� 20 590 73 ND ND 5200GD3S�/� and GalNAcT�/� 24 270 32 ND 28 3800GD3S�/� and GalNAcT�/� 24 230 37 ND 22 4600GD3S�/� and GalNAcT�/� 24 240 47 ND ND 4000

a ND, not detectable.

TABLE VAccumulation of SM2a in addition to GM2 in a human Tay-Sachs

liver as determined by nano-ESI-MS/MSThe standard deviation of the method is smaller than 8%.

GSL Tay-Sachs liver Control liver

pmol GSL/mg dry-weight

SM2a 46 �4.7 (ND)a

GM2 580 �9.5 (ND)GM3 630 520

a ND, not detectable.

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gested that there is no significant degradative pathway fromSM2a to Gg3Cer in mice.

GM2 activator protein stimulates in vivo not only the enzy-matic ganglioside hydrolysis but also the degradation of SM2a.This is concluded from the significant accumulation of SM2a inGm2a�/� mice, which again is in agreement with in vitroresults (46, 47). However, as compared with hexosaminidase-deficient mutant mice, the accumulation of SM2a was small(roughly 3.5 times lower than in Hexb�/�, and 35 times lowerthan in Hexa�/� and Hexa�/� and Hexb�/� mice). Thedegradation of SM2a to SM3 must still be operative in theabsence of GM2 activator protein in the Gm2a�/� mice. Inconclusion, SM2a is degraded by Hex S and Hex A with bothenzymatic hydrolases increased in effectiveness by GM2AP.

SM2a accumulation was also observed in a human Tay-Sachsliver. SB1a has only been described in a human hepatic carci-noma cell line (48). We conclude that this lipid is the degradative

precursor of the accumulating SM2a in the human Tay-Sachsliver. Thus, it appears that the human pathway for complexsulfatides is very similar to that of mice. Interestingly, the cer-amide patterns of the SM2a and GM2 of the human Tay-Sachsliver are very different. Therefore we conclude that SM2a andGM2 are metabolized in different pools, e.g. different cell types.

With regard to neutral GSLs as storage compounds in gan-gliosidoses, Gg3Cer and Gb4Cer are known to accumulate inSandhoff disease and Hexa�/� and Hexb�/� mice. Only inthe Hexa�/� and Hexb�/� double mutant mice was a thirdneutral glycolipid found to accumulate. Its structure,IV6GlcNAc�-Gb4Cer, was identified by nano-ESI-MS/MS, GC/MS, and permethylation analysis. It can be distinguished fromForssman glycolipid in the negative ESI-MS/MS product ionscan by an additional ring-cleavage fragment (m/z 322). It isassumed that the likely precursor of IV6GlcNAc�-Gb4Cer is thetypical mouse kidney octaosylceramide Gal�1–4(Fuc�1–

FIG. 12. Composition of accumu-lated SM2a in a human Tay-Sachs pa-tient’s liver as determined by ESI-MS/MS-precursor ion m/z 97 mode.

FIG. 13. Composition of GM3 and accumulated GM2 in a human Tay-Sachs patient’s liver as determined by ESI-MS/MS-precursorion m/z 87 mode. Peaks are labeled according to masses derived by gangliosides containing C18-sphingosine, since this is the main sphingoid inthese gangliosides according to Nilsson and Svennerholm (33). Nevertheless, gangliosides with C20-sphingosine (4% in GM3 and 21% in GM1 (33))and a corresponding smaller fatty acid (shorter by a C2H4-unit) give rise to the identical signals.

TABLE VIMouse models for GM2 gangliosidosis and their relative accumulation of SM2a

Ranking: ����, very strong; ��, medium; and �, low accumulation.

Mouse model Knocked out genes Remaining �-hexosaminidase(subunit combination)

Accumulation ofSM2a in mouse

kidney

Tay-Sachs disease (TSD) (15) Hexa�/� Hex B (�/�) ����Sandhoff disease (SD) (15) Hexb�/� Hex S (�/�) ��Combination of TSD and SD (16, 55) Hexa�/� and Hexb�/� None ����GM2 activator deficiency (AB variant) (17) Gm2a�/� Hex A (�/�) and Hex B (�/�) �

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3)GlcNAc�1–6(Gal�1–3)Gb4Cer (49–52). The fact thatIV6GlcNAc�-Gb4Cer does not accumulate in Hexb�/� mice,demonstrates that Hex S is able to degrade neutral GSLs witha terminal �1,6-linkage in vivo. In addition, one remaining wildtype allele for the hexosaminidase �-subunit in Hexa�/� andHexb�/� mice is sufficient to maintain a level of Hex S activitythat suffices for the complete degradation of IV6GlcNAc�-Gb4Cer (Fig. 11, lane 7). This extends the spectrum of glyco-compound substrates on which Hex S can act to include neutralGSLs with a terminal �1,6-linked N-acetylhexosamine.

The knowledge of all storage compounds and the resultingcellular changes due to GSL accumulation are a prerequisitefor a more complete understanding of the pathology of therespective human diseases. And, by analogy, knowledge of allGSL that are missing in mutant mice may be important for acorrect interpretation of the respective phenotypes. Further-more, the accumulation of GSL in pathologic tissues, such asSM2a in human Tay-Sachs liver, demonstrates the existence ofcertain GSL components in particular organs where they mayplay special functional roles.

Acknowledgments—We thank Astrid Potratz (Kekule-Institut furOrganische Chemie und Biochemie, University of Bonn, Germany) andFrank Schestag (Physiologisch Chemisches Institut, University ofBonn, Germany) for providing kidneys of Gm2a�/� and ASA�/� mice,respectively; as well as Jan-Eric Mansson (Institute of clinical neuro-science, Section of experimental neuroscience, Sahlgren’s UniversityHospital, Molndal, Sweden) for providing human Tay-Sachs liver tis-sue. We are especially grateful to Prof. F. T. Wieland (BZH, Universityof Heidelberg, Germany) for making the triple Quadrupole nano-ESI-MS/MS available to us. We thank Konrad Sandhoff for his interest inthis study and continuous encouragement during its proceedings.

REFERENCES

1. Ishizuka, I. (1997) Prog. Lipid Res. 36, 245–3192. Kobayashi, T., Honke, K., Kamio, K., Sakakibara, N., Gasa, S., Miyao, N.,

Tsukamoto, T., Ishizuka, I., Miyazaki, T., and Makita, A. (1993) Br. J.Cancer 67, 76–80

3. Kobayashi, T., Honke, K., Kuramitsu, Y., Hosokawa, M., Miyazaki, T., Murata,J., Saiki, I., Ishizuka, I., and Makita, A. (1994) Int. J. Cancer 56, 281–285

4. Bezouska, K., Yuen, C. T., O’Brien, J., Childs, R. A., Chai, W., Lawson, A. M.,Drbal, K., Fiserova, A., Pospisil, M., and Feizi, T. (1994) Nature 372,150–157

5. Trinchieri, G. (1989) Adv. Immunol. 47, 187–3766. Jennemann, R., Schulze, M., Bauer, B. L., Kurtz, C., and Wiegandt, H. (1994)

J. Biochem. (Tokyo) 116, 450–4567. Kabayama, K., Ito, N., Honke, K., Igarashi, Y., and Inokuchi, J. (2001) J. Biol.

Chem. 276, 26777–267838. Tadano-Aritomi, K., Hikita, T., Fujimoto, H., Suzuki, K., Motegi, K., and

Ishizuka, I. (2000) J. Lipid Res. 41, 1237–12439. Kolter, T., and Sandhoff, K. (1999) Angew. Chem. Int. Edit. 38, 1532–1568

10. von Figura, K., Gieselmann, V., and Jaeken, J. (2001) in The Metabolic andMolecular Bases of Inherited Disease (Scriver, C. R., ed) Vol. 3, pp.3695–3724, McGraw-Hill, Inc., New York

11. Harzer, K., and Benz, H. U. (1974) Hoppe Seylers Z. Physiol. Chem. 355,744–748

12. Gravel, R. A., Kaback, M. M., Proia, R. L., Sandhoff, K., Suzuki, K., andSuzuki, K. (2001) in The Metabolic and Molecular Bases of Inherited Dis-ease (Scriver, C. R., ed) Vol. 3, pp. 3827–3876, McGraw-Hill, Inc., New York

13. Yamanaka, S., Johnson, M. D., Grinberg, A., Westphal, H., Crawley, J. N.,Taniike, M., Suzuki, K., and Proia, R. L. (1994) Proc. Natl. Acad. Sci. U. S.A. 91, 9975–9979

14. Hess, B., Saftig, P., Hartmann, D., Coenen, R., Lullmann-Rauch, R., Goebel,H. H., Evers, M., von Figura, K., D’Hooge, R., Nagels, G., De Deyn, P.,Peters, C., and Gieselmann, V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,14821–14826

15. Sango, K., Yamanaka, S., Hoffmann, A., Okuda, Y., Grinberg, A., Westphal,H., McDonald, M. P., Crawley, J. N., Sandhoff, K., Suzuki, K., and Proia, R.L. (1995) Nat. Genet. 11, 170–176

16. Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C. J., Skop, E.,Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L. (1996)Nat. Genet. 14, 348–352

17. Liu, Y., Hoffmann, A., Grinberg, A., Westphal, H., McDonald, M. P., Miller,K. M., Crawley, J. N., Sandhoff, K., Suzuki, K., and Proia, R. L. (1997) Proc.Natl. Acad. Sci. U. S. A. 94, 8138–8143

18. Oya, Y., Nakayasu, H., Fujita, N., and Suzuki, K. (1998) Acta Neuropathol.(Berl.) 96, 29–40

19. Takamiya, K., Yamamoto, A., Furukawa, K., Yamashiro, S., Shin, M., Okada,M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K., Sakae, M.,Kishikawa, M., Shiku, H., and Aizawa, S. (1996) Proc. Natl. Acad. Sci.U. S. A. 93, 10662–10667

20. Bosio, A., Binczek, E., and Stoffel, W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,13280–13285

21. Takamiya, K., Yamamoto, A., Furukawa, K., Zhao, J., Fukumoto, S.,Yamashiro, S., Okada, M., Haraguchi, M., Shin, M., Kishikawa, M.,Shiku, H., and Aizawa, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,12147–12152

22. Yamashita, T., Wada, R., Sasaki, T., Deng, C., Bierfreund, U., Sandhoff, K.,and Proia, R. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9142–9147

23. Kawai, H., Allende, M. L., Wada, R., Kono, M., Sango, K., Deng, C., Miyakawa,T., Crawley, J. N., Werth, N., Bierfreund, U., Sandhoff, K., and Proia, R. L.(2001) J. Biol. Chem. 276, 6885–6888

24. Brugger, B., Erben, G., Sandhoff, R., Wieland, F. T., and Lehmann, W. D.(1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2339–2344

25. Spiro, R. G. (1966) Methods Enzymol. VIII, 4–526. Kurita, T., Izu, H., Sano, M., Ito, M., and Kato, I. (2000) J. Lipid Res. 41,

846–85127. Geyer, R., Geyer, H., Kuhnhardt, S., Mink, W., and Stirm, S. (1982) Anal.

Biochem. 121, 263–27428. Parente, J. P., Cardon, P., Leroy, Y., Montreuil, J., Fournet, B., and Ricart, G.

(1985) Carbohydr. Res. 141, 41–4729. Geyer, R., Geyer, H., Kuhnhardt, S., Mink, W., and Stirm, S. (1983) Anal.

Biochem. 133, 197–20730. Geyer, R., and Geyer, H. (1994) Methods Enzymol. 230, 86–10831. Hsu, F. F., Bohrer, A., and Turk, J. (1998) Biochim. Biophys. Acta 1392,

202–21632. Marbois, B. N., Faull, K. F., Fluharty, A. L., Raval-Fernandes, S., and Rome,

L. H. (2000) Biochim. Biophys. Acta 1484, 59–7033. Nilsson, O., and Svennerholm, L. (1982) J. Lipid Res. 23, 327–33434. Nagai, K., Ishizuka, I., and Oda, S. (1984) J. Biochem. (Tokyo) 95, 1501–151135. Reinhold, V. N., Reinhold, B. B., and Chan, S. (1996) Methods Enzymol. 271,

377–40236. Stroud, M. R., Handa, K., Salyan, M. E., Ito, K., Levery, S. B., Hakomori, S.,

Reinhold, B. B., and Reinhold, V. N. (1996) Biochemistry 35, 770–77837. Stroud, M. R., Handa, K., Salyan, M. E., Ito, K., Levery, S. B., Hakomori, S.,

Reinhold, B. B., and Reinhold, W. N. (1996) Biochemistry 35, 758–76938. Metelmann, W., Muthing, J., and Peter-Katalinic, J. (2000) Rapid Commun.

Mass Spectrom. 14, 543–55039. Metelmann, W., Vukelic, Z., and Peter-Katalinic, J. (2001) J. Mass Spectrom.

36, 21–2940. Whitfield, P. D., Sharp, P. C., Johnson, D. W., Nelson, P., and Meikle, P. J.

(2001) Mol. Genet. Metab. 73, 30–3741. Gu, M., Kerwin, J. L., Watts, J. D., and Aebersold, R. (1997) Anal. Biochem.

244, 347–35642. Liebisch, G., Drobnik, W., Reil, M., Trumbach, B., Arnecke, R., Olgemoller, B.,

Roscher, A., and Schmitz, G. (1999) J. Lipid Res. 40, 1539–154643. Sullards, M. C. (2000) Methods Enzymol. 312, 32–4544. Brugger, B., Sandhoff, R., Wegehingel, S., Gorgas, K., Malsam, J., Helms, J. B.,

Lehmann, W. D., Nickel, W., and Wieland, F. T. (2000) J. Cell Biol. 151,507–518

45. Nagai, K., and Ishizuka, I. (1987) J. Biochem. (Tokyo) 101, 1115–112746. Ishizuka, I., Nagai, K., Kawaguchi, K., Tadano-Aritomi, K., Toida, T.,

Hirabayashi, Y., Li, Y. T., and Li, S. C. (1985) J. Biol. Chem. 260,11256–11261

47. Hepbildikler, S. T., Sandhoff, R., Kolzer, M., Proia, R. L., and Sandhoff, K.(2002) J. Biol. Chem. 277, 2562–2572

48. Hiraiwa, N., Fukuda, Y., Imura, H., Tadano-Aritomi, K., Nagai, K., Ishizuka,I., and Kannagi, R. (1990) Cancer Res. 50, 2917–2928

49. Sekine, M., Suzuki, M., Inagaki, F., Suzuki, A., and Yamakawa, T. (1987)J. Biochem. (Tokyo) 101, 553–562

50. Sekine, M., Yamakawa, T., and Suzuki, A. (1987) J. Biochem. (Tokyo) 101,563–568

51. Sekine, M., Nara, K., and Suzuki, A. (1997) J. Biol. Chem. 272, 27246–2725252. Sekine, M., Taya, C., Kikkawa, Y., Yonekawa, H., Takenaka, M., Matsuoka, Y.,

Imai, E., Izawa, M., Kannagi, R., and Suzuki, A. (2001) Eur. J. Biochem.268, 1129–1135

53. Metzger, K., Rehberger, P. A., Erben, G., and Lehmann, W. D. (1995) Anal.Chem. 67, 4178–4183

54. Sandhoff, R., Brugger, B., Jeckel, D., Lehmann, W. D., and Wieland, F. T.(1999) J. Lipid Res. 40, 126–132

55. Suzuki, K., Sango, K., Proia, R. L., and Langaman, C. (1997) J. Neuropathol.Exp. Neurol. 56, 693–703

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Gieselmann, Richard L. Proia, Herbert Wiegandt and Hermann-Josef GröneRoger Sandhoff, Stefan T. Hepbildikler, Richard Jennemann, Rudolf Geyer, Volkmar

SPECTROMETRYMASSDETERMINATION BY NANO-ELECTROSPRAY IONIZATION TANDEM

Kidney Sulfatides in Mouse Models of Inherited Glycosphingolipid Disorders:

doi: 10.1074/jbc.M110641200 originally published online March 27, 20022002, 277:20386-20398.J. Biol. Chem. 

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