Comparative Studies of Keratins Isolated from Psoriasis and Atopic Dermatitis

Millard Thaler, Kimie Fukuyama, William L. Epstein, Knute A. Fisher
1980 Journal of Investigative Dermatology  
K eratin proteins were extracted from scales of normal skin, clinically active psoriatic lesions, and atopic d ermatitis . Filaments prepared by in vitro assembly upon dialysis of the proteins agains t a low ionic strength buffer w er e comparatively characterize d by electron microscopy, SDS gel ele ctrophoresis, and amino acid analysis. Filame nts forme d u sing k eratin obtaine d from the skin of norma l individuals w e re thin and wavy, whereas those formed u sing k eratin isola ted from
more » ... scale s of psoria tic p a tients w er e straight and showe d a tendency t o a ssemble side by side . Fila m e nts of atopic d ermatitis w e r e indis tinguishable from those of normal individuals . F ila m ents of both normal and atopic de rmatitis cont a ined the protein band of 67 ,000 daltons , which w a s a b sent in filaments of p soriasis . In contrast, 2 prote in b ands of 54,000 and 57,000 daltons were only d e t e ctable in p s oriasis. Amino acid a n a lys is of these filaments furthe r d emons trate d that filaments of p s oria sis diffe r from t hose of normal individua ls in that they have a glycine conte nt that is 60% of normal. I n t he previo us s tudy [1] ker a tins from scales of norma l individuals a nd patie n ts wit h h y pe rke ratos is wer e extracted in tri s~u ream erca p toeth a n o l a nd a nalyzed by sodium d o d ecyl sulfate (SDS) gel electr o phor esis. W e fo und that a ba nd a pproxim ately 67,000 dal to ns is d etecta bl e in cornifie d cells of norma l individuals a nd in vario us h y p erkerato tic diseases , but not in s cales fro m un tr eated psoriatic lesio ns. S kerrow [2] a nd S kerrow a nd Hunter [3] also d etected t his a bnorma li ty a nd t h e re ap pearance of th e normal protein ba nd wi t h treatme nt. These s tudies ind icate t h at we m ay be a ble to bioch e micaUy ide ntify psoriasis and understand o ne aspect of t h e basic pathomech anism s associated wit h skin ch a nges in t his disease. T his study was d esign e d to furt he r ch a racterize proteins in scales fro m les io ns of psoriasis a nd atopic d erm atit is a s compared wi t h t h ose in norm al s kin. S ince it h a s been s h own t h a t ke ratin protein of cow [ 4] a nd r at [5] can be polym erized in vitro, we h ave s im ilarly p ur ifie d keratin protein of man fro m normal s kin, psoriasis and atop ic d erma t it is by prec ip itatio n of ker atin filam e n ts. After obse rva tion of ultrastruc tu ral featur es, the fil a m e nts wer e furth er compa r ed by ge l e lectrop hor etic patte rns a nd an1ino acid compos it ions. MATE RIALS AND METHODS Shin Sample~;; Scales were obtained from 12 patients with acti ve pso riatic lesions a nd 8 patients with clinicaUy di agnosed a topic derm a ti tis by scrapin g Manuscrip t Reprint requ ests to: Kimie Fukuyama, M.D. , P h.D. Department of Derm atology, HSE-1092, Uni ve rsity of California, San Francisco, California 94143. Abbrevia tions: SDS: Sodj um dodecyl sulfate 156 wi th the sid e of a microscope slide. Similar scraping was performed on the skin of 10 norm al individuals at the ankle, leg, knee, palm, elbow and forehead and these acc umulated co rnified cells were . used as con trols. All samples were stored at -20°C until used. The patients were not on any medica ti on for at. least 2 weeks prior to the stud y. Keratin Filam ent E xtr action and Polym eriz ation Approximately 200 mg of scales from each subj ect were serially homogenized a nd extracted as previously described [5] . The extracts, in 50 mM Tris-H Cl, 8 M urea, a nd 0.1 M 2mercaptoethanol , pH 9.0, were acidified wi th a fiv e-fold vo lume of 2.5 mM H Cl in aceto ne a nd the precipi tate was washed 3 limes in 0.1 M Tris-I-ICl, pH 8.0, 1 mM Clela nd's R eagent. It was then di ssolved in 6 M urea con taining 25 mM 2-mercaptoethanol, and centrifu ged at 250,000 g for 2 hr at 4°C to remove small cell particles. Th e supern atant. was adjusted to 1 mg prote in/ ml by th e Coomassie Brilliant Blu e tec hnique of Bradford [6] a nd then polymerized by the method of Steinert [ 4] . Filaments form ed were peUeted at 150,000 g for 1 hr at 4°C, redissolved in 6 M urea containing 25 mM 2-mercaptoethanol, and adjusted to 1 mg/ ml protein. The process was repeated to repolymerize keratin fil amen ts as described by Steinert [7]. E lectron Microscopy The repolymerized sampl es were diluted to a bout 50 /J.g protein per ml of 5 mM Tris-HCl buffe r co ntaining 25 mM 2mercap toethanol. A drop of the solu tion was placed on formva rcarbon-coated grids. Excess solu tion was removed with fil ler paper, and the grid stained wi th 0.7% aqueous uranyl acetate, pH 5.0, for 1 min . The specimens were examined usin g a S iemens E lmiskop JA at 80 kv; a ll photographs were ta ken at a magnificati on of 40,000 X. The magnification was calibrated using a crossrul ed gratin g replica (E. F. FuLlam Inc. ). S DS Acrylam.ide Gel E lectropho re~;;is The repolymerized sampl es nol used for electron mi croscopy were lyophilized, dissolved in 10 mM phosphate buffer, pH 7.0, with the final concentration of 1% SDS, 1% 2mercaptoeth anol a nd 15% sucrose, and heated for 4 min in a boiling water bath. Th e buffer system of' Weber-Osborn [8] was empl oyed using a 7.5% acryla mide ge l with 0.1% SDS and 0.1 M sodium phosphate, pH 7.2 in tubes 5 mm in di ameter and 12 em in lengt.h . T he samples were electophoresed at 8 mA per tube for 6 1 /2 hr and stained with Coomassie Brilliant B lu e. P rotein patterns and molecul a r weigh t determin a tions we re made by co mparing the migration of peptide bands to phosphorylase A (94 ,000), BS A (68,000), ca ta lase (60,000) , ova lbumin (4 3,000) , a ldo lase (40,000) , glyce raldehyde-3-dehyd.J·ogc nusc (36,000) , chymol rypsin (25,700), and cytochrome C (11,700) . A mino A cid Analysis T he repolymerized fi.l amctnls were dialyzed 24 hr with 3 changes of distill ed wa ler at 4 oc a nd lyophi li zed. To determin e the a min o acid co mposition, 1 mg was hyd.J·olyzed under vacuum with l cc constant boiling H CI for 24 hr in a 105°C oil bath, and a Beckman Model 119 Automa tic ana lyze r was used. All samples were run simul ta neo usly. RESULTS The polype ptid es of normal, atopic a nd psoria tic e pider mis assemble d into filam e nts which a veraged 4-5 nm in width . The normal fil a m e nts we re wa vy, and they we re ultrastruc turally indis tingu ish a ble from one r egio n of t h e body t o anoth er (Fig 1) . They sometimes form e d w id e r fibrils , esp ecially w h e n they wer e poly m erized at highe r con centrations, but t h e in ternal s tructure of th ese wid er f ibrils containe d no discernib le per io-
doi:10.1111/1523-1747.ep12522546 pmid:6157751 fatcat:fzowjtsu2ne4xok77f6fzxwvpm