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CONTRACT NO: DAMDl7-91-Z-1027

TITLE: GLYCOSPHINGOLIPIDS AS PUTATIVE RECEPTORS OFSTAPHYLOCOCCAL ENTEROTOXIN 1 IN HUMAN KIDNEYPROXIMAL TUBULAR CELLS

PRINCIPAL INVESTIGATOR: SUBROTO CHATTERJEE

ORGANIZATION ADDRESS: The Johns Hopkins UniversitySchool of Medicine600 North Wolfe StreetBaltimore, Maryland 21205

REPORT DATE: DECEMBER 26, 1992 STYPE OF REPORT: MIDTER.M REPORT

PREPARED FOR: U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFORT DETRICKFREDERICK, MARYLAND 21702-5012

Distribution authorized to U.S. Government agencies only,proprietary information, February 17, 1993. Other requests forthis document shall be referred to Commander, U.S. Army MedicalResearch and Development Command, ATTN: SGRD-RMI-S, FortDetrick, Frederick, Maryland 21702-5012.

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REPORT DOCUMENTATION PAGE..

I AGENCY USE ONLY Leae Wajnt) 2. REPORT OATE J REPORT TYPE ANO OATES COvERED

]Dec. 26, 1992 iMidterm 1 Jun 91 - 30 Nov 92 44. TITLE ANG SUIBTITLE S. FUNDING NUMBERSGlycosphingolipids as Putative Receptors of DAMD17-91-Z-1027Staphylococcal Enterotoxin B in Human KidneyProximal Tubular Cells 61102A

6. AUTHOR(S) 3M161102BS12 AASubroto Chatterjee DA335896Wan Yang Shi

1. PAFORMNG ORGANIZATION NAME($) ANO AOORESS(ES) I. PERFORMING ORGANIZATIONThe Johns Hopkins University REPORT NUMBERSchool of Medicine600 North Wolfe StreetBaltimore, Maryland 21205

9. SPONSORING, MONITORING AGENCY NAME(SI ANO ALDOUESS4Es) .o.. PONSORING MONITORING

4U.S. Army Medical Research & Development Command ACIECY REPORT NUMBER

Fort DetrickFredericK, Maryland 21702-5012

I. SUPKIEMENTARY NOTES

i24. OISTRIBUTION AVARI.AS.ITY STATEMENT IZb OISTRIBUTION CODE

Distribution authorized to U.S. Governmentagencies only, proprietary information,

-February 17, 1993,

13. ABSTRACT Ma.4 M200rVa,~'O r1*e)

We have investigated the binding of 1251 staphylococcal enterotoxin-B (SEE)to glycosphingolipids (GSL) from cultured human kidney proximal tubular cellsemploying GSL overlay technique and microplate quantitative assay.

Structural studies of the putative GSL receptor forSEB was performedemploying high performance liquid chromatography (HPLC), gas liquid chromato-graphy, mass spectrometry (GC-MS) of Trimethylsilyl derivatives of methyl-glycosides. fatty acids and sphingosines.

Permethylation analysis of sugars was carried out employing GC-MS. Ourresults are; I. Out of twelve GSL present in human kidney, only a diglycosylceramide specifically bound to SEB. 2. The chemical composition of the putativereceptor: cirbohydrates, glucose, galatose, sphingosines, d18:2, d23:U, fattyacidd, C16:O, C18:1

14. SUBJECT TERMS 15. NUMBER OF PAGESRA 1; Glycosphingolipids; Receptors; Kidney proximal _

tubular cells; High performance thin layer 16. F..CE CODE_'tchromatography

11. SECURITY CLASSIFICATION 1B. SECURITY CLASSIFICATION 19.7 SECURITY CLASSiFICATION 20. IIMITATIGN OF ABSTRACT'Of REPORT OF THIS PIAGE OF ABSTRACT

Unclassified Unclassified Unclassified Unlimited%SN75 8-0 .S550 .38 19)

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FOREWORD

Opinions, interpretations, conclusions and recommendationsare those of the author and are not necessarily endorsed bythe U.S. Army.

V Where copyrighted material is quoted, permission hasbeen obtained to use such material.

./ Where material from documents designated for lim!teddistribution is quoted, permission has been obtained to urethe material. /S

Citations of commercial organizations and trade namesin this report do not constitute an official Department ofthe Army endorsement or approval of the products or servicesof these organizations.

J In conducting research using animals, theinvestigator(s) adhered to the "Guide for the Care and Useof Laboratory Animals," prepared by the Committee on Careand Use of Laboratory Animals of the Institute of LaboratoryAnimal Resources, National Research Council (NIH PublicationNo. 86-23, Revised 1985).

J1 For the protection of human subjects, theinvestigator(s) have adhered to policies of applicableFedeal Law 45 CFR 46.

In ccnductinq research utilizing recombinant DNAtechnology, the investigator(s) adhered to currentguidelines promulgated by the National Institutes of Health.

PI Signature Date

&oessiou Fow.S

CONUS x~sG-JUL 90 DTTC QUALITYMP!: DTIS AE

Unarnnot-medU-it I fc,,* 23n

j3i1By t•Coenisntrbution/

I AvalladoDtA $pecal

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Grant No. DAMD17-91-Z-1027

MID TERM PROGRESS REPORT

From June 1, 1991 - November 30, 1992, researchactivities have been aimed at the following areas.

Establishina hum&n kidnev 2roxilal cell (PT Ce1lculture in the laboratory from human cadaver kidneys.

Essentially the same procedure :1) as described in theresearch project was followed to culture human PT cells.Confluent cultures of PT cells were subculturod employingdigestion with trypsin and passaged up to 2-3 times.Confluent cultures of PT cells were harvested and storedfrozen for the isolation of GSL.

Isolation of GlvcosDhinaoliDids IGSL) frol _M&akidney/PT cells and other tissues.

GSL were isolated from human kidney or PT cells by theprocedure described previously. (2) GSLs were also preparedfrom rat kidneys, human brain, erythrocytes and humanintestine as above. The GSLs were separated by th.in layerchromatography on silica gel-G HPTLC plates (Merck) usingchloroform-pethanol-water (55;25:4 v/v) as the solventsystem, and were identified using aniline diphenylamine(DPA) reagent. Figure 1 shows the separation of humankidney GSL on a high performance thin layer chromatography(HPTLC) plate. The major GSL species observed in humankidney were glucosylcaramide (GlcCar), lactosylceramide(LacCer), trihexosylcaramide (-bo%3Cer) andtetrahexosylceramide (Gbose4Cer) (Figure ).

SEB Receptor Identification

itgphylococcal enterotoxin-B (SEB) was radiolabelledwith ' I using lodogen and was used for overlayingprocedurea (3). GSL from human kidney or PT cells wereseparated as described above and identified using DPAflgqent (Fig. 2A). A duplicate plate was incubated with

I-SEE for binding to the individual glycosphingolipids.Autoradiograms of the plate were then analyzed for bgdingactivity. Figure 2B, an autoradiogra, shows that 14I-SEBbound to human kidney q, corresponding in chromatographicmiqration to LacCer. I-SEE binding to GSLs derived fromrat kidney, human brain or human intestine was not observed.

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Grant No. DAMDl7-91-Z-1027

Binding of SEB to purified kidney GSL (SEB Receptor. U

The GSL fraction showing binding with 1251-SEB on HPTLCplate was purified as described and referred to as SEBreceptor (SR), The binding of SEB to t1 receptor wasassayed by measuring the attachment of I-SEB to thisputative receptor (SR) immobijied on microtiter plates(Immunolon wells) (4). The I-SEB binding to GSL derivedfrom human kidney (HKGSL), the SR, LacCer and the GSLderived from rat kidney (RKGSL) is shown in Figu1 5 3. HK GSLand the SR, both, showed significant binding to I-SEB0Maximal binding of SEB to HKGSL and SR occurred at aconcentration of 100 ng/well and 20 ng/well, respectively.At this concentration, the SR binding to1jB was 5-fold morethan HK GSL. A significant decrease in I-SEB binding to •HKGSL was observed at a concentration of 100 ng/well andabove. A similar inhibition was observed with SR at theconcentration of more than 20 ng/well. This observeddecrease in binding could be due to the formation ofmultilamellar layers in the well at these GSL concentrationswhich have been reported to result in the inhliAtion of the,[geptor binding (4). Synthetic LacCer also bound some

I-SEB; at a GSL concentration of 20 ng/well it bound12 5 -SEB in the order of 26 fold less than purified SEBreceptor GSL, (Figure 3). Increasing the concentration ofintnetic LacCer in the assay mixture did not increase its

I-SEB binding further, showing saturable bindin 2f thisGSL at low concentrations. RKGSL did not bind to ?2?f-SEBat these concentrations (Figure 3). SEB did not bind toLacCer prepared f-om rat kidneys (data not shown).

Specificity of 12;1ISEB Binding

The specificity of SEB binding to SR was assessed Sfurther employing various structurally definedglycosphingolipids in Table 1. These were GalCer, GlcCer,Gbose4Cer, GM1, GTjb, and sulfatide (S04-GalCer). At GSLconcentrati 9on the order of 10-1000 ng/well, a marginalbinding of L I-SEB to 1gse GSLs were seen. At lowconcentrations of GSL, I-SEB did not bind to any of theseGSLs. (Data not shown).

Characterization of SEB ReceDtor

The putative GSL receptor for SEB (SR) is at presentbeing characterized further.

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Grant No. DAMD17-91-Z-1027

Hinh Performance Liauid ChromatoaraDhv (HPLC) of SEBReceptor

Purified SEB receptor GSL was perbenzoylalated by themethod of Ullman and McCluer (5) and a suitable aliquot wassubjected to HPLC on a Spherisorb Si-S column with detectionat 230 nm as described (6). In figure 4 (B) a HPLCchromatogran of the purified SR is presented. It resolvedinto two main peaks, with retention times on the order of8.49 min. and 8.74 min, respectively. The standard LacCer(stearoyl LacCer) under similar conditions, also resolvedinto two peaks, having lower retention times on the order of8.14 and 8.39 Figure 4 (A). The SR GSL was quantified usinga standard curve prepared with authentic LacCer. p

Gas ChromatoaraDhv-Mass Spectrometry (GC-MSa of SEB Receptor

The SR was subjected to acid catalysed methanolysiA.The methylglycosides, methyl fatty acids and methylshingosines were derivatized employing trimethylchlorosilane Sand analyzed on an Ion Trap Detector-800 (ITD-800) GC-MSusing DB-5 capillary column (0.25 X 30 m). Table 2 showsthe percent fatty acid, sphingosine composition and sugarratio of SR. The GC-NS chromatogram of SR is shown inFigure 5. The preliminary data shows it to contain mainlythree fatty acids, namely methyl palmitate (C16; molecularweight 270.46); methyl elaidate (C 18:1; molecular weight296.48) and methyl stearate (C18; molecular weight 298.51);two sugars, namely glucose and qalactose and threesphingosine bases (d18:2, d22:2, d23:0). turther GC-M3analysis oi this GSL is in progress.

The following papers and abstracts were publishedduring June 1, 1991 - November 30, 1992.

Publications:1. Chatterjee, S.; Jett, M. "Glycosphingolipids:The •

putative receptor for staphylococcus-aureus enterotoxin-B inhuman kidney proximal tubular cells." Mol.Cell. BioChem. lfl:pp.25-31 (1992)

2. A manuscript relevant to the work presented here isunder preparation.

3. A review article entitled "Glycosphinqolipids asputative receptors.* is under preparation.

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Grant No. DAMD17-91-Z-1027

Abstracts:1. ChatterJes, S.; Jett, M. "Glycosphinge~llpids as

putative receptors for Staphylococcus-aureus toxin-B incultured human proximal tubular cells." FASEB. J. a :pp.2,629. (1991).

References:

1. ChatterJee, S.; Trifillis, AL.; Regec, AL.; (1987) Can.J. BioChem. Cell. Biol. 15: pp.1,049

2. Esselman, WA.; Laine, RA. and Sweeley, CC. (1972)Methods Enzymol. 2: pp.140

3. Karlsson, KA. and Stromberg, N. (1987) Methods Enzymol.=3: pp.220

4. Karlsson, KA. and Stromberg, N. (1987) Methods Enzymol.U8: pp.227

5. Ullman, MD. and McCluer, RH. (1987) Methods Enzymol. •138: pp.117

6. Chatterjeo, S. and Yanni, S. (1987) LCGC: pp.571

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Table 3.. Structure of Glygsphi nglligids Used to DetermineSpeCi JCitV Of AALSZinpana

1. Lactosyl Ceramide Gal(B1-4)Glc-Cor

2. Glucosyl Carazide Gic-Cer

3. Galactosyl Ceaaide Gal-Car

4. Globoside

GalNAc (Bl-4:Gal( Bl-4)Gal(BI-4) Glc~l-lCer

S. Trisialoqanglioside:

NeuAc2-3Gal(Bl-3)GalNAc(Bl-4)Gal(Bl-4)Glc(1-1)Car

-N*UAc2-6 - NeuAC2S

S. Monosialoqanqlioside:

XeuAc2-3Gal (Bl-4) Glc(B1-1)Cer

7. Galactosyl Sulfatide S0 4 -Gal-Cer

Grant No. DAMD17-91-Z-1027

Table 2. GC-MS Data of Purified Receptor GSL

1. Carbohydrates: Galactose 10.28 nmo.

Glucose 14.12 nmol

Gal!Glc 1:1:37

2. Sphingosine:

d18:2 d22:2 d23:0% 45.34 8.04 46.62

3. Fatty Acid •

C16 C18:1(8-ene) D18% 23.96 30.78 39.24

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Grant No. DAMD17-91-Z-1027

Legends to iaures

Figure #1 - HPTLC of human kidney glycosphingolipids (GSLs)on an iPTLC (silica gel G) plate using chloroform-methanol-water (65:24:4 v/v). The plate was dried in air anddeveloped with aniline diphanylamine reagent. Lane I -humankidney GSL; Lane 2 - cultured proximal tubular cell GSL.

Figure #2 - Binding of 1 2 51-labelled Staphylococcalenterotoxin-B (SEB) to glycosphingolipids (GSLs) separatedby high performance thin layer chromatography (HPTLC). A -GSLs detected wich aniline diphenylanine reagent; B - GSLfitected by radi autoqraphy (exposure time:l8hrs.) employing

I-SEB (1 X 10 cp/ml; incubation for 4 h at roomtemperature). Lanes I and 6 - Human kidney GSL; Lanes 2 and5 - PT cell GSL; Lanes 3 an4 4 - rat kidney GSL,

Figure #3 - Bindinq of 1 2 5 I-abelled staphylocozcalentertoxin-B (SZB) to glycomphingolipids (GSL) coated onmicrotiter wells. Data are expressed as Aean values of Striplicate determinations. The X-axis indicates the amountof GSL coated to wicrotiter wells.

Figure #4 - L".C tnalysis of perbenzoylated (GSL) A -LacCer; B - Staphylococcal enterotoxin-B receptor (SR).GSIs were benzoylated, dried, suspended in hexane andinjected.

Fiqure #5 - Mass chrozatoqrams (SA - SC) of staphylococcal4nterotoxin-B receptor (SR). 5 (A) mass chromatoqram of SRcarbohydrates and sphingosene; 5 (3) mass spectrum of SRsphingosines; 5 (C) mass chromatoqram of fatty acids.

Figure #6 - Mass spectrum (6A - 6C) of SR fatty acids. 6 (A)C16, methyl palitate; 6 (B) C18:l, methyl slaidate; 6 (C)C18:0, methyl stearate

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Glycosphingolipids: The putative receptor forstaphylococcus aure-as enterotoxin-B in humankidney proximal tubular cells

Subroto Chatterjee' and Marti Jett' DParrn&'nf of Pchaincr. John.% HopkI)ins. ViivflhI.%' Shool of Miedicine. and 'Depa~rtment of Pailuilogv.Walter Reed 1rmY Rg'erch In.%anse. WaNhiligtOll. D.C.

R~ctd .11 Aupi.1 1141. wicpted 13 Fdcrunar 11A)2

Abstract

We have investigated the inding of "'I-staphylococcal enterotoxin-B (SEB) in cultured human proximal tubularcel:.i. We found that the binding of '!'I-SEB to PT cells was time and concentration depentdent and competitively9inhibited by antibody agat.-st SEB. Preincubation of cells with trypsin and ncuraminidasc or with tetuin did not%igtnificantly impair the bindi rigof 'I-SEB to such cells. In contrast, treatment with endoglycoceramidase completelyii~hibitedi the binding of '2l-SEB to cells. Neutral glycosphingolipids exerted a concentration-dependlent inhibition oi'.ISEB binding to such cells. maximum inhihition (96% compared to control) occurred upon incubation of PT cellswith neutral glycesphingolipids. Taken together. our studies indicate thtat SEB spcifically hinds to a neutralgiv.cosphingolupid in PT ctells. In cositrast. staphviococcal -riterotoxin-A and toxic shock toxin (TST- I) are hound ito apw otein in % uch cells. (Mol Cell Biochem 113: 25-31. 19i92)

Ahhreiations. SEB - Staphylococcal Enterotoxin-B. SEA - Staphylococcal Enterotoxin-A. TS- I - Toxic Shocksyndrome Toxin: GSL - Glycosphingolipiu; PT - Proximal Tubular-. LPDS - Lipoprotein Deficient Serum; PBS -Phosphate Buffered Saline

KeY words. glycosphingolipids. kidney proximal tubular cells. staphykococcal enterotoxin-A. B. toxic shock syn-Jromne toxin

Introduction

Staphviococcal enterotoxin-B (SEB)' is an important (MC) molecules of murine fibroblasts transfecledpathogen which causes severe diarrhea and death in with lILA class Ill has been noted 12. 31. liowever.experimental animals and man I 1. Recently. HLA-DR localizition and biochemical tracer studies reveil thatin human TCB cell lines were found to have high affin- the kidney plays a major role in the uptake of the toxinitv hndn for SEB. SEA and TST-I 121. The hindingo(f prior to its potent effect on other organs 14. 51. In1)EA to ttass IV major histowompatability complex particular. 75'%. of radiolibeled toxin given to nokeN

Add,..it faolfIpnns S Chsiterice. C'MA NW4. lipd Rewirch. Aih.,ri'wkrumn Unit, tDeparimcni of Keduink-. Johin% Ilopkin% tirtis.ss~ow.t of !.tcdwn. NNO. N Woltt Strco. Blwwmre. .Mirflind 2P2"4. t:SA

200o from Dupont. News LnoIltnd Nuclear. All other Nio-SE, chemical% were purchased from Sigma Cheical Co..

S St. Low%.. Vihrio cholera neuraminidase and Rhodo-

~i4cocxus endogl% coe ram midase were pu rchased froiCal-CL ~hiochemn and G, niMC Corporation. Boston. respi~c.

ti clv. "Phorcast- polyacr~ limide gels and Rainbowprotein markers (Nit 2.350-'Nlr -Ut(M) were purchase~d

c:o00 from Amersham Corixoration. Hluman brain ganglio-0 ~sides and human kidney neutral gly~osphingolipiI.s

E_ were pr.pared in our laboratory 171 awli characterize!lSEA emplommg HPTLC and 1IPLC techniques 181. Human

12 50lw density lipoproteins (LDL .119I.W gmidl) andT3T~ilipoprotein deficient plasma was obtained from the

plasma of normal human volunteers by KBr density* gradient ultracentrifugation 191. Lipoporotein-deficient,

0 1 2 2 wrum, (LPDS) was prepared from lipoprotein-delicien t-Toxin (jg/ml) plasma by precipitation with thrombin as described

Fit I 1101~q'.ua~~~aervnor.(Eii d'~ l'. Such preparations were free from glycosphingoli.,,,'n r*/ huoran pewtitmad iftunar OPT) (elk Huna~n FT MNit I X II pids and cholesterol.wercrwvseed nNiI, iS mm p4Asic pctn dishes ti fruvn n mediumcisnioning III",, feial cA~t wrum withoist inteheoties. Oni the sixlh da~kof cell groih. cdl% %ere kdJ medium tnn g I n mg rolin mi odqii proten & licient serum (L POSI and toctijtcd (or 2 hi i Surss- Preparation of SEB toxin and radio labeling wi "~Isefitv. fresh medium containing L"t) and ti-3 uli of >t-SEB

ts~dreacs~ty i~cm oi -SE scitc~a~i~tv 16 ~gl Commercially available SEB. SEA and TST-l wereand TST-I (siweiic .aettity. I(Mcplting) wd ahk Alclc iii onewt of1 labeled with 1 51 using iodo~gen I I 1 solubilized in %am-dishis. ro anoither et of diihies. pior to thie additio of t-toua tsit.-hiiii eesu- of awrcspiwdenS unaee toin wa &d and imtw ple buffer and subjected to polyacrylamide gel electro-in was eeitint" wd f-w hr at 371 C. Next. rnedeum %a% remod MaW phoreis on Phorcast gels at 12.5 milli amp/gel for 24 h at

the :,:I weir. waskd ten timnes with KacQl phth~itc hultercd room temperature. Appropriate standard proteins ofswline (FHS) for a perixod Ala .ao41 ts. known Mr were also elect rophoreseti simultaneouslyv.

issoecAii wrdie -sttvand roea crnitel wits IejPIrNAU inIM Following electrophoresis. a portion of the gel includingwe:rt riumucd in duptkate dishs 'P*An two hatcses o4 Pt t0 wi the standard molecular weeight proteins was sliced andanAi~zcd in du:p~icat ipecifi: tN*htkq i.e. tslndeng Ortr1W atisemc n; stained with coomassie blue at (AY*JC for 5-1(1mi. The

un fdoxon-tsindtng in the presence of unfahrd toxin wabaac. gel area corresponding ito Mr 290111) for SEB and SEAilatcd and plileed. and 24011) for TST-1 was sliced. eluted and dialyzed.

The material was freeze dried. ciolubilized and assessedlwas found in PT cells in the kidney Il11. Because of the for purity by SDS-PAGE analysis. Such prcparationsavailability of well characterized human kidney Pf cells were free from contaminating proteins.

*in Ilur laboralory [61. we have pursuied studics to deter-mine the biochemical nature of the receptor for SEB insuch cells. Competetive high affinity binding studies of (CellsSEB with SEA and rST-1 were pursued to revealwhether iech toxin% bound to similar or different bind- Cultured human PT cells wcre prepar~d from autopsying domains in FT cells, kidney as described previously [ft(. Cells were trypsi-

nized and sceded (I x 1(P) in 601 x 15 mm plastic Petridishes and grown for 6 days in medium containing 10"4~

Materials and methods fetal calf serum and no antibiotics, On the 6ith day.medium was remnoved. cells were washed %iul phos-

uwmpes an'd chemicals phate buffered saine. (PBS) and incubation continued~lfor 24 h iii mediem containing LPDS (I mg protein ml).

'-I(specific activity 644 MBq/,ug iodine) was purchased

-7I

,Q f~ill11Unless otherwise descrit _d in the text, the following.s.i %%as adopted to rn.:asurc ihe binding ol'"'I-SEB toU11'clis Medium %%.t% remos~ed fromn cells primed withLIDS Next. fresh medium (2 ml) and '-SUB (2,Lg E- 12nil) plus a lv.ent% fold excess of unlabeled SEll was;iddcd and incubation ctintinucd for 2 h a. 17 C, Next.the medium wa% discardcd and thi: cells Aashcd withim of PBS containing 0I.2 6, bovine serum albumin(maintained at 4*0~ and 5 time-, with PBS. The mono-

Saver %as soluh~ilized in I N NaOll. protein and radio-acti%,itv wasnmeasiured acco~rding to Lowry et al. I i2J andscintillation spectrometry. respectively. Specific bind-ing of "A toxin to PT cell% was calculated by subtracting!he data obtained in the absence of uniabcll'; toxin 4~~from :he data ohtained in the presence of 201 fold excessof unlabeiled toxin.

Incubtion of cells with en.zvmes 0 4 6

Cell% prcincubated w ith medium containing LPDS were Tim (hr)Ufurther incubated for 5min at 37*C with try!sin (0- Fit :. Effrv of __vii oftw he i hwming (f ''I-.SEB to PT549);Lg/ml). t reaction was terminated by removine ce/1. rhwbpoumcithis xpenitixwwasi.k-iiaio that dLenhcdthe enzyme solution from the dishes, washing tbe cells% in Fi. I "it thai M11%s were imeuhaled with 201 told csccsu oIt ihwith PBS anid incubation with so%-bean trvpsin inhibitor SEE anfd ± "ISEE 1it/ni) forw1.2. 4 and $hr at 37!c. Th s4wvtiifollowed by extensive washing wi'h PBS. Trypsin treat- Nndint: of '-SES io PT s.01% wascalkulaicd. as k-wrthtd in Fig. 1.ed ctlls were used in "' texin and in "l-LDIL binding Them .io craschctw ofep fch an aned inom uliat.isestudies j1131. Similarly, cell% verc iacubated with neura-minidaw (0.5 anits/ml to 2.01 unitsimlj .nd endoglycoc.erami-iase (0.13 milliunmts.-4)n mqlliunitsimi) for I h at370 C. washed an,! *he binding of 2Ql-SEB pursued as Resultsdesribed above.

8.nrling of "- Istaph vIococc ernerotoxin-11 (SEBi andother toxins h v cultured normal human prnsA;.Oal tubilar

licuhatou, of cells with glycosphingolipids (PT) cels

Cells preincubated with medium containing LPDS were The binding of '2'1-SEB to PT cells is shown in Fig. I.further incubated with fresh medium containing gly- Maximum high affinity birding occu~rred with 2Ag ofco-phingolipids and ''l-toxin mixture. Glycosphingoli- SEB and TST-l but not SEA per ml medium. Thepids were taken into -i sterile glasm tube and dried in N. binding of "l-SEB ito PT cell was saturated at higheratmosphere. Theii medium containing LPDS was add. eonenration: 5 jg/ml medium. Ihe ratio of binding ofed. sionicated and suitable aliquots added it) the assay 1ST-I1. SEA and iEB to PT cells was on the order ot. I :mixture. After incubatiun for 2 hr at 370 C the asay was 1.5: 3. respectively: The binding of '2l.SEB it) PT cellsterminated and the binding of toxin it) PT cell% inca- was time dependent (Fig. 2). For example. a linearsufcU. increase in the binuing of this toxin occurred up to 2hr

followed by a plateau after 4 hr. The inclusion of anit-body against SEB in the assay misture quantatiselvinhibited the binding of this toxin to PTells(Fig. 3). A

162h.8",..and .10.4"... recilsl compared tot control.(Taible I). tinder similir conditions. lr psin t5 i(Ipe1 fill)inhibited Ml4'%, of 'l-LDL binding to r I.Preincu-

G hation of PT cells with end gl~coccraimidaise did not

impair the binding ol '1-SEA or ''l-TST-I (data notCL shovkn). Preincubation with endoglyccweraniidaseC 12 (). 15-40~t milli unit, ml) completely inhibitled the binding

of '1-SEB to PT cells (Table 1),

Effects of g4co.vplaingohipids on tihe finding of"''l-Sl R

I~t F r cells

M Maximumt inhibition of binding. (54. 2%/ relative to con-U) troll occurred with UN()Aglml gangliosides (Table 2). InCP contrast, when 50Mg/mI neutral glycosphingolipids

were added in the incubation mixture. 96.7- inhibitlonof 'NI-SEB binding to PT cells occurred as compared tocontrol.

0 5 10 25

SEB-Antibody (psq/mI) DsusionFig. 3 Eflninafair a~o.muSEA im iAw b .g f'-SEB to PTarJz toc~Jiufed PTrtFvIsl. .1.ndlisgoalihadyAgAzm4SEB Our major findings in this report are: first, kidney prox-%aa~ddpmwiotli aid*kmfof 2psg/mI AoV'*-SE8. lncuh&1Mbatwf

cacd tot 4hr ir aod th ofihtiiaa 'ISEB to PT imal tubular cells have high affinity binding sites (recep-cetih was measured. The data mpepfts average valuem Plitm.Ad toes) for SEB: second. preincubation of cells with en-tra~m dulowma thum ftvr,m two haerhs o( Fr ceUk analyzed -n du- doglycoceramidase and/or human kidney neutral gly-piecate. cosphingolipids markedly inhibited the binding of SEB

to i'r cells. Third. in contrast. cndoglycoceramidase

fineor decrcai ir binding occurred upto Myl;VmI of ireatmet did not impeait the binding of 1231-SEA orSEB antibody. '1-TST-I to PT cells. Moreover, these toxins were

unable to competetively dispIace SEB from binding toPT cells.

Displaeable binding of toxu..s in PT cells Previous studies in experimental arimals and manhave suggested that the kidney. in general and proximal

Displaceable binding assays employing a fixed amount tubular cells in the kidney cortex in particular. may' playof '5 1-toxin and increasing concentrAtions of unlabeled a major role in the pathophysiology of SEB inducedtoxin revealed that, firs, unlabeled SEB was unable to toxemia j4. 51.compeic for the binding sites for 'I-SEA and 1I-TST We have found that PT cell can bind 12TISEB first.(Fig. 4A). Similarly, unlabeled SEA was unable to via a high affinity receptor mediated mechanism at lowcompete with the binding of'I-SEB to PT cel! recep- concentrations of toxin. as well as a nonsaturable recep-tors (Fig. 4B). tor-independscnt mechanism at high concentration of

t ~SEB (Fig. 1). Competition experiments with unlabeledSEB, and antibody against SEB which quantitativelyK Effects of trypsin, neuraminidase and inhibited '1-SEB bincling suggest that the receptors in

endoglycoceramidase on the binding of' I-SEB and such cells are specific for SEB. The inclusion oif 3-iodo-other toxins in P Tcelli; tyrosine (3 X III 'M). an inhibitor of deiodinase tin the

assay mixture, did not alter the binding of '>1-SEll inA Preincubation of ce!Is with trypsin and neuraminidase PT cells (data not shown). These findings suggest thatI. maximally decreased '1I.SEB binding in the order of the binding of '!1-SEB of PT cells is not due to the

100

00

a cis

so.s E (~MI ntble ar p/d

Fit 4. Cimaneitftve hindis Cof SEB. SEA ad 7T.IT- io PMelh. ThaL pnaaaI to( thr~c1ramc1 .a wam4Rarto the legnd t Fig. I cxpvt thait

(tminif the addit~ame at :t oxin SEA and TST-l (Fag. A) untaheIed SEB (II-51111t; ml) *j% Akkd. Stotttadlymtami~herw ofe u da'ulte'. "'tSEBnlusi ~go m unlitheled SEA was added anid the draplaceahic 4ading u,(1oxi wit. mcawurd (Fig. 8). The recaalibrepruicat data trout monec'pcnient analyred in duplicate.

remo,,ai of '21 by a deiodinase and the subsequent binding assays only moderately decreased I-"l-SEBlabeling of cells with '-"I. Rather. "I-SEB binding is binding. Under similar conditions, trypsin inhibiteddue ~o the presence of receptors in PYT cells. 110% of the binding of '11LDL to PT cells compared to

To investigate the nature of the SEB receptor on P"T control. Previously. digestion of rat liver membranescells. studies were pursued further. First. pretneubatton with trypsin was found not to impair the binding ofof cells with trypsain and neuraminidase followed by cholera toxin (141. Second. SDS-PAGE analysis of PT

cells incubi 'ed with '211-SEB for 2 hr revealed alabeled

Table 1. Effects oft y pu. .ewammine and eadooeramiate hand at the dye front. In no instance did toxin hind toan the binding of "'I-SED and 'I-LDL to morma l'man kidney any other ban~d visible in the coomassie brilliant bluePala l u ceuja. stained gel. Third. inclusion of fetuin (250 ,sglrl). a

serum glycoprotein in the assay mixture, did not impairEnzym the binding of - 4 -SEB to PT cells.

(*g SEB ba"d taahwtui Recently the binding of SEB to humanTcells bearing

"I-SEB CowwM0 11.2 0 Tuahl 2. Effects of human btrain taqianhades aud human kidney

'Crypsm I soog'ml) 9. 6 14.3 neutral oycoqsng(Aiapdo the hmduasiiio -I-SEB innornmlhu-Try"u I tt~t~ml) 8.9 206 man ProximiaJ tubular cels.Trypsin (30I~sAWmi) 8.2 26.11Neuramsaidaw ' (0.5 isatsim!) 7.5 33.1 GyI~osphuAalipad Binading%Nearuadae (1.0 aaats/m) 05 24.2 (agSED botmid' InhiitioanNeuraaudase (2.0 unitsimt) 7.8 30.4 10, W etit(CM

(0. 15 millaunats; 0 It00 Coaarnl 12.0 41Endoglycocransidaic Ganglowdcs

(0.0 malhWINNOW 0 lii) ( XtlOW.M) 10.3 14.2Endoolvieenmidm (150,4/md) 9.2 23.4

(0. () imaUiurw) 0 111) tXII,4/Ml) 9.2 23.4LVI.DL couarol tOO0 0) tONt~M) 3.3 54.2

Tr% j~n (5411 jug ml1) 2.0 YAt INetral i .mmmphingmlopid-I 5ugwi) 33 72 5

*One mii t neuramtntdaic releass I psmolct ncuramanc acid. man. ( II ps ml) I. 91.~l7-One unit otendogiycoceranuaw iuenlases I manok of glucowe from (S'5A pinIt) (1.4 96~.7

bovane twain gangixioudesma.

p.irticulir VH .-qiencc% a% part of thcir receptors for giscosphingolipid receptor and to etabhlish structurer1,11 hi sgOomipatiilit s comnpl,.- protein -associied funct ii relat ionship%

inntri has been %ho~n (31. In another study a uniqucitc oni cla%-- 11 NIIIC proteins ito %hich SEA. SEl aindS

I SI- I hinds %%i a, iio%%n 121 The reasotis for the di~- Acknowledgementscr:paliwies, in the abose studies and ours is not clearprcscntl%. Since the amino acid sequence of TST-l This work wissupisrted hy.tgiant from NIIh # RO-l-beajr. t resemblance with SEA and SEll, the hind- DK-31722 and from the U S. Army ito Subroto C. hatter-

js~.o tl~ese three tosxins to an identical site afrwars c. Ph.D.no~n-specific. Moreover, SEll is cnly 28'% homnologous'o SEA and the binding of SEA to such sites ii l0-13timtes higher than SEll 121. We also found that un- Referenceslabeled SEllwas unable to di'.F lace SEA or TST- I fromhinding to PT cells. Similarly. SEA or TST-I were NrNnS.aceR.onssR iholotiofetfscrimenl

unable to displace SE B from binding to PT cells. More- entliicl:th n:o~tz: fs.p~~.d ~ero-oser. in our studies, pretreatment with trvpstn only tot~.~J ih.fin ii idnos~lhlkka Jneoii~ Lit 5n 1 ? W

partially decreased the binding of SEB to PT cellscorn- and H its 11LA-DR. %ature 3Y: 221-22.11. I'J5I)pared to preincubation of cells with giycco -phingofipids 3, Kaprg.cr J. Koiiin B. tberrvt tL. Ciciford EW. Biluter RD. tiost-

or endoglycoccramidase which dramatically dec,-easd ti,m A. Carrcl S. Posneti DN. Chi"i Y. Mlarracti P: B.Slistific

"l.SEB binding to PT cells (see below). Our studics 24iitiin K f 1 uman 1.1 19lW4 ipvitsa t~n. ~lead to the suggestion th it there is a clear dichotomy in 4 Czrocti FIG. Henikwnr EL. Ayali E. Bowm.,n tfli: Suhettu-regard to th,- nature of receptor for SEA. TS-T-! and la Itclzto Bisiicca rtrtsi in raimt kidney.SEll. !is case of SEA and'IST- I the receptor is clearly a Am J Ptvii 226i: t.333. 1474protein, in contrast. in cas~eof SEB. the receptor is most 5.%iri% EL. floduisai LF. Bcrwi WR: The unu-sual rote (if the

probably a glycosphingolipid. liadveyiruiini i B J Infet Dis It? 273.in*iveka? pSeseral gyeosphingolipids have been suggested to lil Ouierle S. Trifiiigi. AL. Repes: AL: Biochemicai aiid muirpl

serve as receptors for various toxin~s. For examti,Ie GM, iiical effct% si of geiarrucin in human Proximai iviulur 0:11%(ceramtde-glucose-galactose-NacetvI~ietaraminic acid) Effcisort tipid and liipiiprocin trciabtlisin Can J ttsichc.n Celland 'iortotriosyl-ceramide (cei-amidr-glucose-galac- 8*ut65I 1M4. l"t7S

Lose-g:alacltusc) serve its reepttors for cholera toxin and 7. Es.eimawn WJ. '.A.ne RA. Swceky CC: I-slation and Character-iU[we of Glyoin.C4tlIPed1. Motsods, Enzvrsot 281: Part BI.vcrozytotoxin. respectively 114. ifl. Similarly. a large 17.1

num~ber of corao y ocwriIng baVCtria and pathogen- ..- c S. Yanni 5; Anoilysts of neutral ti tco.hingtoipidsic bacteria have been- found to hind speu~.ticall) to lactco- A andsifeick,; by high performance: liuid chroonaiugraphysvlceramide (Ceram ide-glucose- gal actose) 1191. We in- LCGC 5: 571. 1997ivestigated whether glycosphingolipid% may also serve as 9. Chatetelc S. Kwitcniiieh ?0- Cityciisingiidu of human

a putative receptor for SEB in PT cells. First, we found pltatia tipiitwicinst. Lipuk% It: 462. t1976Il.Cilrec S. Sekerie CS and Kwiieroviefi PO: Alterationit in

that human kidney neutral glycosphingolipics are a pe- ell suufac giv .pingiitei and other lipid clasiscs of fihmr-tent inhibitor of SEB binding to FIT cells. Such findings biastin familial h~rercht*%icricnia Prix Naltl Acid Sc, ISAare in agreement to previous reports suggesting neutral 73: 4.349 19t76

glcspigliisas probable receptors, for toi t.1. Markwcti MK. Foxt CF: Surface scific Iailnalot (if membraneglycsphngolpid toxns. protinsisirti-and cukaryoiicuxllsi using 1. 3.4.6. icirachtuiro

Furthermore. preincubation of cells with endogly coc- 3~. Is. diphsnelwosiuurit Bioshemisirs 17 4.8117. 191Meramidase resulted in complete inhibition of "I SEB 12. Loury 0. Rivebrllugh MJ. Farr AL and Randall RJ: Proteinbinding to PT cells. This enzyme specifically cle. . es the meiaiurcmnent with the Foiti phenols reagent. J Buuii (heni 193:glycissyl moiety from glycosphingolipids 1221. Finally. 205. tlt~

our preliminary studies reveals direct binding of SEB to 1.1 Chitirtee S. Sckcrke CS. Kwiierovich PO: Effect% of tunicamv

ahuman kidney neutral glycosphingolipid. Taken to. ci11 tihe cell surface bindinst. iniernializaiion and degiradaion o flott ,ten'if% liqsVrotin, in hurtin Ilrh a'r r I ltt, cni

Lcthcr c:u, fintfings suggest that most prohahl a neutral li 4.I'

glycosphingolipid in PT cells serves as a putative recep- 14 cerca~sPttr~cintinue~iireirlisn lli

tor for SEll. Further studies are under way in our rrmni'.ne% Biochemistry 12: 3.547. 19t73

laboratory to determine the structure of the putative

-- 77

31

%%I tcr.irnidc is %FtIlkwiIk r eognilied hy the E.chirwhiu (to uiriepiith.al ellxs lof p I. phrit.'ecne Lnhlcilihije coI 1.1..' HI~khCM He ph%, Rcs Comm 152. 74 14MQ coI N"4. 1'l41

It, %loba-eh %1. tNflohu,:Rolle A. J.:,Awe, MI. Grand I&J, 'tI I lin- in (W. Kj rstiueK A. aruin, 6. St romnhtrg %. I h u in J.Ku'.th G, P.&lhvlvvsni% %) hihgcll.e diirrhej Esuiknx lItr .e ')ridl C. Mifrh% E: A rnel~ .ipprahiich loi the studh ' it tgohithc lopmentiallk rcg~ulitacel g~ilia~psd recueor Ia lulm %oglatief rwapl~i% lair %iruL%% llindine tofwndi %irus tii thin.jir chri'.

inoe n lteu Iluid srutlar% rc4%,nw .$l rahui 'm41 inesine J maitairam% FILIIS Icit 1711: 1I. 1994~1bCitai tDis 17 1.i123 l4JSll 11 Kro%.a', IIC. ~talrs DO1. (,inshurr V %tai pulma'umnar% Phislc.

11 HRanishaI R. Pic M. Iurihk-r cahrxienturk AI the Irjuail ge.nics hooearw~ hind spsrwifials it) the a.rh,ihai.ilc sequecerectptw for pwudrrmiana% acruaniv.-. Eur J M ihiol 4. If. (.iNAu hctai 1-4 6A hund in soim ghasaibipid%. Proc %at Aksad

I M IDcjlCD.SiromfxibrgN Nw hr(NemaL.~.rsaK.i 2 ito . Y.,maigat.a r A nwa~cI gblawisphangobapid &ad~iing cn-%I Peiiadaadalhndn fNicjgnarh.ah meh.mc askjaaos oit lhw linkagex NvAccrii the eiligtisaiikharodc andIioud gf'I.vbepids Ant %an LA-uuncnheick 53 42-3. 1%n7 -cramot.1 t4i ouiilre ild .,cideu gI~caphingtilirids, J Romd Chem

%hontworig Js Situi oft crtieiidrakuin.b~ of rewrplo tm humaen