Preparation of a Uveitogenic Peptide by Chymotryptic Digestion of Bovine S-Antigen

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1 Preparaton of a Uvetogenc Peptde by Chymotryptc Dgeston of Bovne S-Antgen Yosho Kamada,* Mrsunor Yamada,* Noveen D. Das,t Don 5amuelson4 Vctor R. Leverenz,* and Htosh Shch* Lmted proteolyss of bovne S-antgen wth a-chymotrypsn resulted n the accumulaton of three peptdes of MW 24,000, 6,000, and 2,000 daltons, respectvely. By ELSA (enzyme-lnked mmunosorbent assay), MW 24,000 peptde was found to react wth ant-s antbodes, but the other two peptdes dd not react wth the antbodes under the assay condtons. The reactve peptde was separated from the smaller peptdes by gel fltraton on Sephadex G-75 and Sephadex G-50. When the MW 24,000 peptde was njected nto Lews rats, severe to mld uvets was produced n all njected anmals. The results ndcate that the pathogenc determnant s on the MW 24,000 peptde. nvest Ophthalmol Vs Sc 26: , 985 Expermental allergc uvets (EAU) s an autommune dsease nduced n varous laboratory anmals by njecton of a soluble retnal antgen (S-antgen). The dsease s characterzed by nflammaton of the uveal tract and loss of retnal photoreceptors. " 3 A blateral uvets nduced n prmates by the antgen shows characterstcs resemblng those of human uvetc dseases. 4 Studes on serum levels of ant-s antbodes n uvets patents suggest a possble nvolvement of S-antgen n certan types of human uvets. 56 Uvets patents showed cell medated responses, but not humoral responses, to S-antgen. 5 Wth the excepton of the pneal gland, 7 the retna s the only tssue n whch S-antgen s located. n the retna, the antgen has been localzed to the vsual photoreceptors by mmunohstochemcal technques Usng monoclonal antbodes to bovne retnal S-antgen we have demonstrated that S-antgen s assocated wth rod photoreceptor outer segments. 0 S-antgen has been purfed to a homogeneous proten of MW about 50,000 daltons from the retna of several speces ncludng human retna. 3 " 2 A recent paper reports chemcal cleavage of bovne S-antgen nto several peptde fragments by treatment wth cyanogen bromde. 3 The fragments possess an- From Eye Research nsttute,* Oakland Unversty, Rochester, Mchgan; and the Department of Ophthalmology,! College of Medcne, and the Department of Comparatve Ophthalmology,:): College of Veternary Medcne, Unversty of Florda, Ganesvlle, Florda. Supported n part by Natonal Eye nsttute grant EY (Dr. Shch). Submtted for publcaton: December Reprnt requests: Dr. Htosh Shch, Eye Research nsttute, Oakland Unversty, Rochester, M tgenc reactvty toward ant S-antgen serum but t s not known whether they are uvetogenc. n the present work, we have dgested bovne S-antgen wth a-chymotrypsn and separated a peptde (MW 24,000) whch retans both antgenc and mmunopathogenc actvtes. Preparaton of S-antgen Materals and Methods Bovne retnal S-antgen was prepared as descrbed 0 by a modfcaton of Wacker's method.'' Sodum dodecyl sulfate-polyacrylamde gel electrophoress (PAGE) of S-antgen and ts proteolytc dgests was carred out n 0 percent polyacrylamde gels (5.5 X 0 mm) usng a contnuous buffer system of M Trs-acetate, ph 6.6, and 0. percent SDS, essentally followng Wacker et al." For electrophoress ng sample was used per gel. Proten was determned by the method of Lowry et al. 4 Dgeston of S-antgen wth a-chymotrypsn Ffty parts (wt) of purfed S-antgen were mxed wth one part (wt) of TLCK-treated bovne pancreas a-chymotrypsn (Sgma; St. Lous, MO) n 00 mm potassum phosphate buffer, ph 7.6, contanng 0. percent polyethylene glycol 400 (PEG-400) and 0.02 percent sodum azde and the mxture was ncubated at 37 C for a gven length of tme. The reacton was stopped by addton of phenylmethylsulfonyl fluorde (PMSF) n 2 percent ethanol (to a fnal concentraton of mm). For tme-course experments, the dgest was dssolved n SDS buffer and loaded on polyacrylamde gel for electrophoress. After electrophoress, 274 Downloaded From: on 07/3/208

2 No. 9 UVETOGENC PEPTDE FROM BOVNE S-ANTGEN / Komodo er ol. 275 the dsk gel was slced n segments of 4 mm n thckness, placed n a seres of small tubes, extracted overnght at 4 C each wth 0.5 ml of 50 mm sodum bcarbonate, ph 9.6, contanng 0.02 percent NaN 3. The extracts were then subjected to ELSA. For purfcaton of antgenc peptdes, proteolyss was stopped after 6 hr of dgeston and the dgest was dalyzed aganst 50 mm ammonum bcarbonate for 24 hr at 3 C. Purfcaton of Chymotryptc Peptde on Sephadex Dalyzed peptde sample (-2 mg) was centrfuged at 80,000 X g for 30 mn and the supernatant was appled to a Sephadex G-75 (Pharmaca; Pscataway, NJ, partcle sze = urn) column (.6 X 80 cm). The column was eluted wth 50 mm ammonum bcarbonate to collecte 2.5 ml fractons at a flow rate of 0 ml/hr. For determnaton of molecular weght, the column was calbrated wth standard protens [ovalbumn (45,000) carbonc anhydrase (29,000) and cytochrome C (2,400)] purchased from Sgma. ELSA-postve fractons from ths column were pooled and frozen slowly at -20 C. The frozen sample was allowed to thaw at room temperature. The early crop of thaw contaned enrched peptdes. The peptde mxture (-2 mg) thus concentrated was centrfuged at 80,000 X g for 30 mn to remove aggregates and the clear supernatant was placed on a Sephadex G-50 (Pharmaca, partcle sze = /m) column (.6 X 94 cm). The column was eluted wth 50 mm ammonum bcarbonate to collect.2- ml fractons at a flow rate of 3.6 ml/hr. Enzyme-lnked mmunosorbent Assay (ELSA) A 20-/l alquot of gel extract was mxed wth 80 x\ of sodum bcarbonate and ELSA was carred out by a modfcaton of the publshed procedures. 56 n bref, the well of polystyrene mcrottraton plate (Dynatech; Alexandra, VA) was coated wth sample, washed wth 0 mm potassum phosphate, ph 7.0, contanng 0.05 percent Tween 20 and 50 mm NaCl, and shaken dry. The proten adsorbed to the well surface was then reacted wth rabbt ant S- antgen serum at 37 C for 2 hr, washed wth phosphate buffer, and shaken dry. Fnally, the well was reacted wth peroxdase-labeled goat antrabbt gg at 37 C for 90 mn, washed, and then ncubated wth a mxture of 0.04 percent o-phenylenedamne and 0.0 percent H 2 O 2 n 0. M ctrate buffer, ph 5.0, at room temperature for 5 mn. The reacton was stopped wth 50 Al of 8 N H 2 SO 4 and the optcal densty at 490 nm was determned wth a Bo-Tek mcroplate reader Model EL 307 (Burlngton, VT). Purfed ant- S antbodes were used n some ELSA assays. Rabbt serum was saturated wth ammonum sulfate to 40 percent at ph 6.5 and centrfuged. The precptate was dssolved n 0 mm potassum phosphate, ph 7.5, dalyzed aganst the buffer, centrfuged and further purfed by on-exchange chromatography on DEAEagarose. Ant-S antbodes were eluted at M NaCl. nducton of Expermental Autommune Uvets (EAU) Female albno rats (Lews, 8-2 wk) were purchased from Harlen-Sprague-Dawley (Walkersvlle, MD). Two rats were njected wth S-antgen and fve rats were mmunzed wth peptde. S-antgen (about 80 Mg per rat) or MW 24,000 peptde (about 300 ng per rat) was emulsfed n complete Freund's adjuvant and njected nto the footpads of a rat. Two control anmals receved salne soluton mxed wth the adjuvant. Two control anmals were njected wth the peptde treated for 3 mn at 00 C and two anmals receved S-antgen treated for 3 mn at 00 C. The eye was examned wth an ophthalmoscope. The anmals were klled 5 days after treatment. All procedures nvolvng anmals conform to the ARVO Resoluton on the Use of Anmals n Research. Hstology of EAU nduced by Chymotryptc Peptde Ffteen days post mmunzaton the anmals were klled by cervcal dslocaton and the eyes enucleated. A small ncson was made near the optc nerve and the globes were mmedately fxed n 0 percent buffered formaln. For hstology, the globes were then dehydrated through a graded seres of alcohol, cleared n cederwood ol, and embedded n paraffn (Peel-A- Way; Scentfc Products; Ocala, FL). Fve-mcron sectons were cut n an AO 820 mcrotome (Amercan Optcal Corp; Buffalo, NY) and mounted on clean mcroscope sldes. The sldes were staned wth hematoxyln and eosn, examned and photographed n a Nkon Labophot mcroscope (Nkon nc.; Garden Cty, NY) equpped wth a UFX camera. Results Chymotryptc Dgeston of S-antgen Tme-dependent changes n the profle of chymotryptc dgeston of bovne S-antgen are shown n Fgure. Coomasse-blue staned proten bands ndcate that part of S-antgen was cleaved to two major peptdes of MW 42,000 and 26,000 wthn an hour of dgeston. n 2 to 4 hr, both peptdes were further degraded and three peptdes of MW 24,000, 6,000, and 2,000 became preemnent. The three peptde bands faded gradually durng prolonged dgeston, ndcatng further breakdown nto smaller fragments whch probably electrophoresed out of the gel. Downloaded From: on 07/3/208

3 276 NVESTGATVE OPHTHALMOLOGY b VSUAL SCENCE / Seprember 985 Vol hrs t 2 4 \ t Fg.. Chymotryptc dgeston of S-antgen. Changes n peptde composton durng dgeston of bovne S-antgen wth achymotrypsn. Dgeston tme vared and s ndcated on the rght of fgure. The bottom gel shows Coomasse-Blue stan of protens of known molecular weghts. 0» t Tl To determne whch peptde contans antgenc determnants, the polyacrylamde gels were slced n segments after electrophoress. Peptdes extracted from the segments were then tested by the enzyme-lnked mmunosorbent assay (ELSA) usng ant-s rabbt serum. As demonstrated n Fgure 2, the antgenc actvty assocated wth S-antgen was markedly reduced after hr of chymotryptc dgeston, although the newly formed peptdes of MW 42,000 and 26,000 showed consderable actvty. The actvty then shfted from MW 26,000 peptde to MW 24,000 peptde n 4 to 6 hr of dgeston and slowly decreased thereafter. The rabbt ant S-antgen used was not absorbed before utlzaton. Therefore, part of the actvty n the ELSA could be attrbuted to contamnants (eg, autolyzed chymotrypsn fragments) whch showed nonspecfc reactvty toward the ant S-antgen serum. However, the amount of such contamnants was probably small. We have also tred other proteases for dgeston of S-antgen. Thermolysn (S:enzyme = 50:, w/w) converted S-antgen to MW 47,000 peptde (major) and MW 42,000 peptde n 24 hr. Subtlsn (S:enzyme = 50:, w/w) produced peptdes of MW 36,000 and 26,000 n an hour and quckly degraded these peptdes nto smaller fragments durng the subsequent ncubaton perod. Dgeston of S-antgen wth papan (S: enzyme = 20:, w/w) gave rse to a peptde of MW 47,000 n an hour and no accumulaton of smaller std 4 K peptdes was observed. Thus, a-chymotrypsn seemed to be more useful than these proteases for preparaton of smaller peptdes wth antgenc actvty n suffcent quanttes for further studes. Separaton of ELSA-postve Peptde by Gel Fltraton on Sephadex Columns From the results of Fgure and Fgure 2, t s evdent that a peptde of MW 24,000 wth antgenc actvty accumulates transently durng chymotryptc dgeston of bovne S-antgen. To solate ths peptde, we dgested S-antgen wth a-chymotrypsn for 6 hr and purfed the dgest (e, a mxture of peptdes) on a Sephadex G-75 column (Fg. 3). The frst peak collected n the vod volume contaned a trace of Santgen and MW 42,000 peptde as determned by SDS-poyacrylamde gel electrophoress (data not shown). n a separate experment, S-antgen (2 mg) was loaded on the G-75 column. S-antgen was recovered n fractons 50 to 76. No trace of Santgen was detected by ELSA n the fractons collected after fracton 77, Therefore, the second peak was consdered to be completely free of undgested S-antgen. The second peak (ndcated by two connected arrows n Fgure 3) showed postve reactvty toward ant-s antbodes as determned by ELSA (Fg. 3, top) and contaned three major peptdes of MW 24,000, 6,000 and 2,000, respectvely (Fg. 3, Downloaded From: on 07/3/208

4 No. 9 UVETOGENC-PEP-TDE FROM BOVNE S-ANTGEN / Komoda er ol. 277 OD hr Fg. 2. Antgenc actvty of chymotryptc peptdes as determned by ELSA after electrophoress. Tme of dgeston s ndcated on rght. 6 hr 8 hr o J.0 2 hr to J o 0 hr o oocpc^oooo<3 OQOOOOO 2 4 K f 2 4 K bottom). The fractons of peak 2 were pooled and further purfed on a Sephadex G-50 column. Of the three peaks separated on the column, only the largest peptde (e, MW 24,000) was found to possess antgenc reactvty under the assay condtons (Fg. 4). The largest peptde showed postve reactvty toward purfed ant-s antbodes. Suffcent quanttes of the MW 24,000 peptde were pooled from several runs of G-50 gel fltraton and used for mmunzaton of anmals. The result ndcates that the peptde of MW 24,000, though less than a half n sze of S-antgen, stll retans the antgenc determnants. Producton of Expermental Autommune Uvets n Lews Rats wth Chymotryptc Peptdes of S-antgen The pooled fractons of antgenc peptde were concentrated by lyophlzaton, emulsfed wth complete Freund's adjuvant and njected nto Lews rats. The eyes were examned from 0 days after treatment. None of the sx control rats showed any ndcaton of ocular nflammaton. However, all of the fve rats whch had receved an njecton of peptde exhbted n both eyes sgns of ocular nflammaton ndstngushable from those observed n the S-antgennduced uvetc eye. The general descrpton of pathology was as follows. The dseased eyes showed marked panuvets, vascults, and focal and dffuse retnts. All affected specmens showed accumulaton of subretnal flud. Many lymphocytc cells, hstocytes and plasma cells were found throughout the uvea and were especally numerous n the exudatve flud beneath the retna. Fgures 5C and 5D show the affected areas n the anteror uvea and the retna, respectvely. Cellular nfltraton n the aqueous s evdent and photoreceptor outer segments are entrely lost. Fgures 5A and 5B are photographs of sectons from control eye. Both MW 6,000 peptde and MW 2,000 peptde, when njected wth complete Freund's adjuvant (200- Downloaded From: on 07/3/208

5 278 NVESTGATVE OPHTHALMOLOGY 6 VSUAL SCENCE / September 985 OD Vol ELSA - A / 03 -» / \ n K Fracton 250 Number Fg. 3. Gel fltraton on Sephadex G-75 of peptdes produced by lmted chymotrypsnolyss of bovne S-antgen. The lower fgure s a profle of gel fltraton. The fractons ndcated by two connected arrows were pooled for subsequent purfcaton on Sephadex G-50. The presence of three peptdes of MW 24,000, 6,000 and 2,000 n the pooled sample s shown by nset gel. The upper fgure s the antgenc actvty of fractons determned by ELSA. 300 Mg per rat) nto Lews rats, dd not nduce expermental uvets. Dscusson By lmted proteolyss of bovne S-antgen wth achymotrypsn a mxture of three major peptdes (MW 24,000, 6,000, and 2,000) accumulated, of whch MW 24,000 peptde was found to be ELSA-postve. t s not known whether the smaller peptdes were produced by further degradaton of MW 24,000 peptde or they derved from the other fragment remanng after the 24,000 peptde was generated from S-antgen. Whatever the orgn of the smaller peptdes s, these peptdes were not ELSA-postve under the assay condtons. However, the present result does not rule out the possblty that the smaller peptdes also contan antgenc determnants. After cyanogen bromde cleavage of bovne S-antgen a peptde of 25 amno acd resdues was solated.6 When tested aganst ant-s antbodes, the peptde demonstrated antgenc reactvty only after conjugaton to poly-l-glu-ala-tyr of MW 46,000. t s possble therefore that the peptdes of MW 6,000 and 2,000 may show mmunoreactvty toward ant-s antbodes f ther sze s ncreased by conjugaton to a carrer polypeptde. Snce evdence suggests that S-antgen has several antgenc determnants, 6 the antgenc determnant of the smaller peptdes, f present, may well be dfferent Downloaded From: on 07/3/208

6 No. 9 UVETOGENC PEPTDE FROM BOVNE S-ANTGEN / Komodo er ol Fg. 4. Gel fltraton of chymotryptc peptdes on Sephadex G-50. Open crcles: profles of gel fltraton; flled crcles: antgenc actvtes determned by ELSA. Note that the antgenc actvty s assocated only wth MW 24,000 peptde fracton Fracton from the antgenc determnant of MW 24,000 peptde. The present fndng on the producton of EAU n rats by an njecton of the MW 24,000 peptde Number ndcates that more than half of the polypeptde s not essental for the pathogencty of bovne S-antgen. Pathologc features of EAU produced by S-antgen and peptde are ndstngushable. Further studes are Fg. 5. Hstopathologc changes of anteror and posteror parts of eyes of control and peptde-nduced EAU Lews rats. A, Clary body and rs, control (orgnal magnfcaton, X93); B, retna and chorod, control (orgnal magnfcaton, X370); C, clary body and rs, peptde njected (orgnal magnfcaton, X93); D, retna and chorod, peptde njected (orgnal magnfcaton, X370). Downloaded From: on 07/3/208

7 280 NVESTGATVE OPHTHALMOLOGY & VSUAL SCENCE / September 985 Vol. 26 needed to compare quanttatvely the pathogenc potency of S-antgen and ts chymotryptc peptde. Pathogencty may declne as the antgen molecule becomes smaller. However, t should be possble to determne the mnmum sequence requrement for nducton of EAU by further cleavage of the MW 24,000 proten. For nducton of allergc encephalomyelts n Lews rats, the mnmum sequence requrement of myeln basc proten s eght amno acd resdues. 7 Sten 8 presented the results of prelmnary experments at the 984 ARVO meetng n whch he prepared a mxture of peptdes (MW 0,000-30,000) by Staphylococcus aureus V8 protease dgeston of bovne S-antgen and produced EAU n Lews rats wth the peptdes. However, t s not clear whether a small amount of undgested S-antgen was present n the peptdes. Gregerson and Putterman 3 prepared seven major peptdes by cyanogen bromde cleavage of bovne S-antgen. t awats further studes to determne whether these peptdes, when njected nto anmals, nduce EAU. Key words: S-antgen, chymotryptc peptde, purfcaton, expermental autommune uvets, bovne retna Acknowledgments The authors thank Dr. Denns M. Callerwaert for allowng us to use hs mcrottraton plate reader, Ms. Pat Lews for preparng hstology sectons, Dr. Harry Engel for examnng the hstology specmens, and Ms. Roxanne M. Bowman for typng the manuscrpt. References. Wacker WB, Barbee JY, and MacDonald R Jr: Expermental allergc uvets.. Manfestatons produced n the gunea pg by mmunzaton wth homologous retna. nvest Ophthalmol 8:38, Lawwll T, Wacker WB, and MacDonald R Jr: The role of electroretnography n evaluatng posteror uvets. Am J Ophthalmol 74:086, 972. ' V v ;..:.,' 3. Faure JP: Autommunty and the retna. n Current Topcs n Eye Research, Vol 2, Zadunasky JA and Davson H, edtors. New York, Academc Press, 980, pp Nussenblatt RB, Kuwabara T, de Monastero FM, and Wacker WB: S-antgen uvets n prmates. Arch Ophthalmol 99:090, Nussenblatt RB, Gery, Ballntne EJ, and Wacker WB: Cellular mmune responsveness of uvets patents to retnal S-antgen. Am J Ophthalmol 89:73, Gregerson DS, Abrahams W, and Thrkll CE: Serum antbody levels of uvets patents to bovne retnal antgens. nvest Ophthalmol Vs Sc 2:669, Kalsow CM and Wacker WB: Pneal reactvty of ant-retna sera. nvest Ophthalmol Vs Sc 6:8, Kalsow CM and Wacker WB: Localzaton of a uvetogenc soluble retnal antgen n the normal gunea pg eye by an ndrect fluorescent antbody technque. nt Arch Allergy 44:, Usu M, Matsushma T, Nakayama S, Takano S, Saka J, and Muramatsu R: Antgencty of human outer segment for expermental uveo-retnts. n mmunology and mmunopathology of the Eye, Slversten AM and O'Connor GR, edtors. New York, Masson Publshng, 979, pp Das ND, Ulshafer RJ, Zam ZS, Leverenz VR, and Shch H: Radommunocytochemcal localzaton of retnal S-antgen wth monoclonal antbodes. J Hstochem Cytochem 32:834, Wacker WB, Donoso LA, Kalsow CM, Yankeelov JA Jr, and Organscak DT: Expermental allergc uvets: solaton, characterzaton, and localzaton of a soluble uvetopathogenc antgen from bovne retna. J mmunol 9:949, Benesk DA, Donoso LA, Edelberg KE, Magargal LE, Folberg R, and Merryman C: Human retnal S-antgen. nvest Ophthalmol Vs Sc 25:686, Gregerson DS and Putterman GJ: Preparaton, solaton, and mmunochemcal studes of the cyanogen bromde peptdes from a retnal photoreceptor cell autoantgen, S-antgen. J mmunol 33:843, Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Proten measurement wth the foln phenol reagent. J Bol Chem 93: 265, Saka J, Takano S, Takamura K, and Usu M: Antbody contents aganst S-antgen tested by ELSA n expermental and human uvets. Fola Ophthalmol Japonca 34:350, Tuyen VV, Faure JP, Thllaye B, dekozak Y, and Forter B: Antbody determnaton by ELSA n rats wth retnal S- antgen-nduced uveoretnts. Curr Eye Res 2:7, Hashm GA: Myeln basc proten: structure, functon and antgenc determnants. mmunol Rev 39:60, Sten PC: Expermental autommune uvets nduced by protease dgests of bovne S-antgen n Lews rats. ARVO Abstracts. nvest Ophthalmol Vs Sc 25(Suppl):66, 984. Downloaded From: on 07/3/208