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Characterization of Monoclonal Antibodies Generated to the Cornified Envelope of Human Cultured Keratinocytes

Howard P. Baden, Joseph Kubilus, Scott B. Phillips
1987 Journal of Investigative Dermatology  
The co rnifi ed envelope of keratin ocy tes is an in so lubl e stru cture formed ben ea th th e pl as m a m em brane at th e base of th e stra tum co rn eum. It is made b y cross-linkin g precursor protein s by a mc mbrane-associatcd transglutaminasc . We have prepared mono cl o nal antib od ies to the co rnifi ed env elope of culturcd human kcratinocytes and used thesc to id cnti fy precurso r proteins usin g Westcrn bl o ttin g. T he corni fied en ve lope is a hi ghl y inso luble intracellu
more » ... luble intracellu lar structure th at is found in the epid ermis of m ammali an sk in , the extern al roo t shea th and cuticl e of hair, and nail (a lso hoo f) [1] [2] [3] . It is also prese nt in ce rtain stratifi ed ep ith eli a. Its inso lu bility in denaturin g solvents res ul ts from the prese nce of E-(y-g lutam yl)lysi ne cross-link s that are formed by a membrane-associated transglutaminase 14,5]. A so luble transglutamin asc su bstrate, in volu crin , has been isolated from cultured hum an keratinocytes and it has been postulated th at thi s is the major precursor of hum an co rnifi ed envelope both in vivo and in vitro [6] . Additi o nal precursors of the membrane have also bee n desc ribed [7-10], and a gro up o f inves tiga tors [1 O[ has suggested that one of these, term ed kcratolin in , is the major in vivo prec ursor of the hum an epiderm al corn ifi ed enve lope. The initial studies on the precursors of the envelope used labeled transglutaminase substrates [9 J or the disappea ran ce of protein s 171 and the formation of hi g h-molecul ar weig ht agg rega tes [ 11"[ . M e re rece ntl y in ves ti ga to rs have developed po lyclonal o r m o noclonal antibodies against w ho le cell s or cell ti'ag ments to identi fy putati ve precursors of th e envelope 18, 121. We report the use of cornified enve lopes de ri ved from human cultured keratin ocytes to ge nerate monoclonal antibodi es for dete ction of en ve lope precursors. A large num ber of antibo di es we re fo und by indirect immunoA uorescence, and m an y of these showed Manu script rece ived Feb ru ary 26, 1987; acce pted for publi cation Ma y I, 1987. This wo rk was suppo rted by Grant AR 06838 from th e National Institutes of H ca lth . Reprint req ues ts to: H oward P. Badc n, M. D. , Dcpartmc nt of Dcrmatology, Warrcn 5, Massachuse tts Gencra l Hos pital, Boston, MA 0211 4. Abbrev iat io ns: DTT: dithiot hrei to l EDT A: eth ylcnediam in e tc[ra cc tate FITC: phcn yliso thi ocya natc II F: illdirect inl I11 11l1 o Au o r csc C' ll cc PAGE: polyacrylamidc gel electrop ho res is PMSF: pheny lm cth ylsulfon yl flu o rid c SDS: sod ium dode cyl sul fatc We have un covered a numbe r of precursors includin g involllcrin and a 195 kD m em brane-associated protein , w hICh had previous ly been reported. Antibodies to these precurso rs, wi th th e exceptio n o f the one to in volucrin , reacted w ith the epidermis of other mammali an species, sugges ting stru ctural co nserva tion in at leas t so me envelope co mponents. J IlIll est D ennaro! 89: [454][455][456][457][458][459] 1987 the peripheral-t ype stain o bserved w ith in volu crin. The antigens res po nsi ble for so m e of these reacti o ns co uld be identified and these are described. MATERIALS AND METHODS Tissue Hum an foreskin keratinocytes were cu ltured using the meth od of Rheinwald and Green [1 3], w ith sli g ht m odification lI4]. Stratifi ed cultures 1-2 weeks beyond con flu ence were rinsed extensively w ith serum-free m edia and then harves ted w ith a plastic po li ce m an . Isolation of Cornified ElJvelope Human cultured 'keratinocytes we re ho m ogeni zed at a ratio of20: 1 (v/w) in 100 m M Tns-H e l, pH 9.0, containin g 2% sodium dodecyl sulfate (SDS) and 10 m M dithiothreitol (OTT), and the ho m ogenate was heated wi th stirring for 2 h at 55°C. The suspension was clarified b centrifu ga tion at 50,000 g for 30 min and the pellet reextracted as above 5 add itional times. Protein was undetectable in the fmal supern atant by the Low ry m eth o d (15) . The in solu ble residue was freed from SDS b y was hin g w ith 95% ethan ol, and then resuspended in 100 mM sodium bica rbo nate buffer. T o qu antitate the am o unt of corn ifi ed en velope protein , an aliquot was hydrolyzed for 24 h in 6 N H C I, and the free amin o acids were m eas ured by th e ninh yd rin meth od usi ng L-Ieucine as a standard (1 6 ). The mi crogra m of amino ac id was ass umed to equal th e nucrogram of cornifi ed envelo pe materi al. A suspensio n containing 50 f..Lg of purified cornified envelope in 0.2 ml of Freund's inco mplete adjuva nt was inj ected i. p. into 6 B ALB /c mi ce . The inj ec ti o ns were repea ted every o ther week until th e sera showed a reaction to hum an epidermis by indirect immunoflu o rescence (IIF) . The mice were res ted for 2 m onths during w hi ch time two animals di ed. The remaining 4 were immunized 0 11 three sll ccessive days , and o n the fourth day they were sacrificed, their spleens removed and pooled, and the cells fro m the spleens fused w ith N S-1 m Ollse m yelo m a cells in the presence of 37% polyethylene glycol (MW 1000). M o noclonal antibodies were developed as described prev io usly [17, 18] . Hbridoma cultures we re sc reened for antibodies that gave a reacti on with human ep id ermis by IIF. V8 Protease Digestion of Cell Envelopes Human cell envelopes were suspended at 0.5 mg / ml in 125 m M Tris, pH 6.8,
doi:10.1111/1523-1747.ep12460759 pmid:3668290 fatcat:xhmewcayg5ettjdscvn2dujq4q