The Fibrous Proteins In Various Types Of Ichthyosis · THE FIBROUS PROTEINS IN VARIOUS TYPES OF...

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THE JOURNAL OF I NVESTI GATIVE DERM ATOLOGY, 65:228- 230, 1975 Copyright © 1975 by The Williams & Wilkin s Co. Vol. 65, No .2 Printed in U.S.A. THE FIBROUS PROTEINS IN VARIOUS TYPES OF ICHTHYOSIS H. P. BADEN , M.D., L. A. GOLDS MITH , M.D., AND L. D. LEE , P H.D. Departm ent of Dermato logy, Harvard M edical Schoo l and Massachu set ts General Ho sp ital, Boston, Massachus etts Th e str at um corne um of indi v idu a ls with ichth yos is v ul gar is, sex -linked ichth yos is, lame ll ar icht hy osis, a nd epide rm ol yt ic hype rk er atos is has been s tudied. An a x-ray diffr act ion pattern ha s been observed in a ll s pecimen s and the so lubili ty of the a fibrous prot eins a ppear s to be the same as in normal stratum co rn e um. Sodium dodecyl s ulfate (SDS) - polyacrylamide el ectrop hor es is of the fibrous proteins sh owed variable pa tterns within the different types of ichthyosis, while amino ac id a nal yses of the protein s were quite s imil ar to those from n orma l st ratum co rn eum. These d ata su ggest th at the fibrou s protein s in the ic hth yosis are n ot ab n orma l, but further st udies on the indi v idual polypeptide chains are neces sary to rule out more su btle differences. Ichth yos is refers to a gr oup of h ete rogen eo us di seases which h ave in common a markedly th ick- e ned st r at um corne um. A gen et ic bas is h as been demon st r ated in m ost pat ients [1], but acq uir ed ichth yos is h as b ee n reported in assoc iat ion with drug th erapy [2] a nd malignanc y [3]. Since str u c- tural prot eins are th e principa l constit ue nts of the st r atum co rn e um , a n ab n or ma li ty in th eir stru c- tur e cou ld be an imp or ta nt facto r in the pa thog e n- es is of th ese di seases . As a r es u lt of recent a dvances in our kn ow ledge of the ma cro mole c ular chemistry of keratiniza tion it is n ow po ss ibl e to ca rry out de finitive st udi es on hum an st ratum corneum. The purpo se of thi s paper is to d esc ribe o ur observa- ti o ns on the phy sicoch em ica l prop e rties of th e princip al st ru ct ur al pr ote in of st r atum co rneum, the a protein , in seve ral types of ichthyosis. MATERIALS AND METHODS Mat erials. All chemi ca ls use d were of reage nt grade except iodoacetic ac id whi ch was crystalli ze d fr om anh y- drous ether and petrole um et her. Stratum co rn e um wa s sc raped fr om the skin with a scalpel blade and stored at - 20°C in a desiccator. Samples fr om 4 patient s with ichthyosis vu lga ri s, 3 with sex-linked ichthyosis, 2 with epidermolytic hype rk eratosis, 7 with lame ll ar ichthyosi s, and membranes fr om 2 co ll odion ba bi es were st udi ed. Extrac tion proce dur es. A 20 % homogenate of the va ri ous tissues was prepared in 6 Murea in 0.1 M Tr is, pH 9.0, (Tris-ur ea) and st irred at roo m temperature for 24 hr. Foll owi ng ce ntrifu ga tion the extr act ion wa s repeated a second time. T he undisso lv ed pell et was then ex tr acted in Tri s- urea with 0.1 M merce ptoethanol under nitr oge n at ro om temperature ove rni ght . The suspension was ce ntrifuged and an aliqu ot of the supern ata nt treated with iodoac et ic ac id to give the S-carboxymethyl (SCM) deriv at ive [4\; the a lk ylated and remaining untreated Manuscript re ce ived Janu ary 20, 197 5; in revi se d form March 3, 1975; acce pted for publication Mar ch 3, 1975. This work wa s supported by Grant No. AM 06838 from t he Nat ional In st it utes of Health. Reprint requ ests to: Dr. H. P. Baden, Departme nt of Dermatology, Massachusets Ge neral Hos pital, Bosto n, Massachusetts 02114. extracts were di alyzed aga in st distilled wate r a nd lyophi- li ze d. X -ray diffraction . X-ray diffraction analys is was done us in g ni ckel-filtered co pper Ka radiation (X = l. 54 A) at 40 kv at a spec im en to film distan ce of l.50 cm. Regenerated filaments for x-ray diffraction analysis were prepared by disso lvin g t he prote in in 80% formic acid picking up the solution on the tip of forceps, and st retc hing while drying at roo m temperature. Amino acid analys is. Samp les for amino ac id analysis were hydrolyzed in 6 N HCI for 24 hr under vacuum at 110°C and run on a Beckman 116 amino ac id ana lyzer. The ana lyses were done in duplicate and the d ata expressed as residues per 100 re sidues not including cyst in e. Electrophoresis. Disc electrophores is in urea was done by the method of Davis [5] with the addition of urea at a co ncentration of 6 M to the ge ls and buffer. Sod iu m dodecyl sulfate (SDS) electrophoresis em pl oyed the same running ge l and buffer but with the addition of 0.1% SDS instead of urea. The samples for SDS elect rophoresis wer e heated at 50°C in 1% SDS and 1% mercaptoethan ol for 30 min ju st prior to being use d, which resulted in co mplete equilibr at ion . Sul fur co nte nt. Tot al sulfur co nt ent wa s determ in ed gravim et ri ca ll y foll owing its ox id at ion to sulfate and the addit ion of barium (Be lm ont An alyt ical La b) as previ- ously described [6]. RESULTS X-ra y diffraction. X-ray diffraction analyses of sca les from patients with various types of ich- thyosis show a patterns s imilar to th ose from nor- mal patients, with 5.14 A m er idion al a nd 9. 8 A eq u ator ial re f1 ect ions. There is a n add itional meri- dional reflection at 4.15 A which can be e limina ted by prior extract ion of the tissue with a chl oroform- methanol mixture (3/1) [7] . E xtraction of tissu e. The so lubili ty of the stra- tum co rn eum proteins was st udi ed by first ex- tracti ng the tissue with Tris - ur ea a nd then with the same buffer with the a dditi on of 0.1 M mer capto- et han ol. * The yield with th e Tris- ur ea buffer for • The extracted prote in s were dialyzed, lyophi li zed, and weighed . 228

Transcript of The Fibrous Proteins In Various Types Of Ichthyosis · THE FIBROUS PROTEINS IN VARIOUS TYPES OF...

Page 1: The Fibrous Proteins In Various Types Of Ichthyosis · THE FIBROUS PROTEINS IN VARIOUS TYPES OF ICHTHYOSIS H. P. BADEN, M.D., L. A. GOLDSMITH, M.D., AND L ... Sulfur % 0.95 0.03 0.96

THE JOURNAL OF I NVESTIGATIVE DERM ATOLOGY, 65:228- 230, 1975 Copyright © 1975 by The Wi lli ams & Wilkins Co.

Vol. 65, No. 2 Printed in U.S.A.

THE FIBROUS PROTEINS IN VARIOUS TYPES OF ICHTHYOSIS

H. P. BADEN , M.D., L. A. GOLDSMITH, M.D., AND L. D. LEE , P H.D.

Department of Dermatology , Harvard M edi cal Schoo l and Massachusetts General Hospita l, Boston, Massachusetts

The stratum corneum of individua ls with ich t hyosis vulgaris, sex -linked ich t hyosis, lame lla r icht hyosis, and epiderm olytic hyperke ratosis has been s tudied. An a x-ray diffraction pattern has been observed in a ll specimens and t he so lubili ty of the a fibrous prote ins a ppears to be t he same as in normal stratum corn eum. Sodium dodecyl sulfate (SDS)- polyacrylamide electrophoresis of the fibrou s proteins showed variable pa tterns within the different types of ichthyosis, while amino ac id a nalyses of the proteins were quite similar to t hose from normal stratum corneum. These d ata suggest t hat t he fibrou s proteins in the ichthyos is are not ab norma l, but further studies on t he indi vidual polypeptide cha ins are necessary to rule out more subtle differences.

Ichthyosis refe rs to a group of heterogeneous diseases which have in common a markedly th ick­ened stratum corneum. A genetic basis h as been demonstrated in m ost patients [1], but acquired ichthyosis h as been reported in assoc iat ion with drug therapy [2] a nd malignancy [3]. S ince stru c­tural proteins are the principa l constituents of t he stratum corneum , a n abnorm a li ty in their stru c­ture could be an importan t factor in t he pa thogen­esis of t hese di seases . As a resu lt of recent advances in our knowledge of the macromolecular chemistry of keratinizat ion it is now possible to carry ou t d efinitive studies on huma n stratum corneum. The purpose of this p a per is to descri be our observa­tions on the phys icochem ical properties of the principa l structura l prote in of stratum corneum, t he a protein, in several types of ich t hyosis.

MATERIALS AND METHODS

Materials. All chemi ca ls used were of reagent grade except iodoacetic acid whi ch was crystallized from anhy­drous ether and petroleum ether. Stratum corneum was scraped from the skin with a scalpel blade and stored at - 20°C in a desiccator . Samples from 4 patients with ichthyosis vu lgaris, 3 with sex-linked ichthyosis, 2 with epidermolytic hyperkeratosis, 7 with lamellar ichthyosis, and membranes from 2 collodion babies were studied.

Extraction procedures. A 20% homogenate of the various tissues was prepared in 6 M urea in 0.1 M Tr is, pH 9.0, (Tris-urea) and st irred at roo m temperature for 24 hr. Followi ng centrifugation the extraction was repeated a second time. T he undissolved pellet was then extracted in Tris-urea with 0.1 M merce ptoethanol under nitrogen at room tem pera ture overnight. The suspension was centrifuged and an aliquot of the supernatant t reated with iodoacet ic ac id to give the S-carboxymethyl (SCM) derivative [4\; the alkylated and remaining untreated

Manuscrip t received January 20, 1975; in revised form March 3, 1975; accepted for publication March 3, 1975.

This work was supported by Grant No. AM 06838 from the National Institutes of Health.

Reprint requests to: Dr. H. P. Baden, Department of Derm atology, Massachusets General Hospital, Boston, Massachusetts 02114.

extracts were di alyzed against distilled water and lyophi­lized.

X -ray diffraction . X-ray diffraction analysis was done using ni ckel-filtered copper Ka radiation (X = l.54 A) at 40 kv at a specimen to film distance of l.50 cm. Regenerated filam ents for x-ray diffraction analysis were prepared by dissolving the protein in 80% form ic acid picking up the solution on the tip of forceps, and stretching while drying at room temperature.

Amino acid analys is. Samples for amino acid ana lysis were hydrolyzed in 6 N HCI for 24 hr under vacuum at 110°C and run on a Beckman 116 amin o acid analyzer. The ana lyses were done in duplicate and the data expressed as residues per 100 residues not including cystin e.

Electrophoresis. Disc electrophoresis in urea was done by the method of Davis [5] with the addition of urea at a concentration of 6 M to the gels and buffer. Sod ium dodecyl sulfate (SDS) electrophoresis em ployed the same running ge l and buffer but with the addi t ion of 0.1% SDS instead of urea. T he samples for SDS electrophoresis were heated at 50°C in 1% SDS and 1% mercaptoethanol for 30 min just prior to being used, which resulted in complete equilibration .

Sulfur content. Tota l sulfur content was determ ined gravimetrically following its ox idat ion to sulfate and the addition of barium (Belmont Analytica l Lab) as previ­ously described [6].

RESULTS

X-ray diffraction. X-ray diffraction a na lyses of scales from patients with various types of ich­thyosis show a patterns s imilar to those from nor­ma l patients, with 5.14 A m er idiona l and 9.8 A equator ial ref1 ect ions. There is an addit ion al meri­dional reflection at 4.15 A which can be eliminated by prior extraction of th e t issue with a chloroform­methanol mixture (3/1) [7] .

E xtraction of tissue. The solubili ty of the stra­tum corn eum proteins was studi ed by first ex­tracti n g the tissue with Tris- urea a nd th en with the same buffer with the addition of 0.1 M mercapto­ethanol. * The yield with the T ris- urea buffer for

• The extracted proteins were dialyzed, lyophi lized, and weighed .

228

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Aug. 1975

mal stratum corneum is 20 ± 6 gm/lOO gm a nd n~~h the Tris-urea- mercaptoethanol buffer is 45 ± ':(1 The values obtained .for. the ichthyotic st~at~m

. eum samples are wlthm 1 standard devIatIOn corn I I X d'f'f' . f the mea n norma va ue . -ray I ractlOn pat-o s of filamen ts regenerated from the Tris- urea-tern . soluble proteins sh~w nbo eVldden~e 0hf anTa. pattern b t an a pattern IS 0 serve m t e f1s - urea-

U captoethanol-soluble proteins. mer D' I h . f' Electrophoretic patterns. ISC e ectrop ores IS 0

1

1 2

FIG. Sodium dodecyl ~ulfate ~SDS) polyacr~ l a mide electrophoretic patterns of the c< flbrous prote l.n Isolated from stra tum corneum . Pat.ter.n 3 was seen 111 norm al

t m corneum whIle, within the varIOUS types of f~~~h~osis, examples of each of the three patterns have been observed.

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the Tris-urea- merceptoethanol-ext racted proteins in urea gives patterns with rather poor resolution. Polyacry la mide electrophoresis in 0.1% SDS gives three quite clear patterns as shown in t he Figure. A number of components are present bu t one (A) predominates. Variable a moun ts of aggregated pro­tein can be noted by the staining at t he origin . Within the various types of ichthyos is, examples of each of these patterns have been observed. Pattern 3 was seen with norma l stratum corneum .

Amino acid analysis. Amino ac id a nalys is of t he Tris-urea-mercaptoethanol-soluble proteins from the various major ty pes of ichthyosis are shown in the T able. Although t here a re minor variations in some of the res idues, the resul ts tend to be rather cons istent. Also included are a nalyses of the solu­bili zed prote ins from the collodion membra nes of two children who later developed ich thyos is vul­garis. No resul ts on cystine content a re given as we have found such data on huma n scales to be rather inconsistent. Sulfur con ten t on some sam­pl es is shown as this a na lysis was very consistent.

DISCUSS ION

The chemica l basis of t he ich t hyotic di sorders is not known, excep t for t he harlequin fetus [8), where it has been shown t hat a cross-f3 fibrous prote in is present instead of t he usua l a one. In a previous study, minor differences were reported in peptide ma ps of sodium hydrox ide-solubili zed proteins from the stratum corneum of patien ts wi t h ichthyos is [9 J. The type of ich t hyosis was not iden t ifi ed a nd no conclusion could be reached as to t he s ignifi cance of t he changes. In t he major forms of ichthyosis described in thi s report, no consisten t diffe rences have been revea led in the x-ray diffrac­tion pattern a nd solub ili ty of t he stratum corneum

TABLE. Amino acid analysis e f the c< fibrous proteins isolated from th e strotum corneum of various . types of IchthyosIs"

- Vulgaris Sex· linked Epidermolytic Lamellar Collod ion Normal

Lysine 4.7 ± 0.7 4.0 ± 0. 3 5.0 ± 0.5 4.6 ± 0.2 4.7 ± 0.3 4.3 ± 0. 3

Histidine 1.1 ± 0.3 1.0 ± 0.1 1.4 ± 0.2 1.1 ± 0.2 1.1 ± 0.3 1.0 ± 0.2

Argin ine 4.5 ± 0.7 4.0 ± 0.1 4.6 ± 0.3 4.3 ± 0.4 4.4 ± 0.4 4.7 ± 0.2

Aspar tic acid 9. 2 ± 0.3 9.0 ± 0.4 9.5 ± 0.6 9.1 ± 0.3 9.3 ± 0. 3 9.4 ± 0.5

Threonine 3.4 ± 0.2 3.2 ± 0.3 3.4 ± 0. 3 3.7 ± 0.2 4.2 ± 0.1 3.9 ± 0.1

Serine 11.4 ± l.0 12.2 ± 0.8 11.3 ± 0. 3 11.7 ± 0.8 12.1 ± 0.1 11.5 ± 0.6

Glutamic acid 13.1 ± 0.7 13.2 ± 0.4 13.6 ± 0.2 13.8 ± 0.4 13.7 ± 0.1 13.7 ± 0.4

proline 1.8 ± 0.4 l.6 ± 0.3 1'.9 ± 0.6 2.2 ± 0.4 3. 1 ± 0.1 2.3 ± 0.2

Glycine . 21.6 ± l.4 23.3 ± 0.3 21.6 ± 2.2 21.0 ± 0.8 19.0 ± 0.6 18.4 0.5

Alan ine 5.9 ± 1.2 5.0 ± 0.6 4.9 ± 0.4 5.2 ± 0.3 5.8 ± 0.1 5.5 ± 0.2

Va line 3.3 ± 0. 3 3.6 0.4 3.6 ± 0.1 3.7 ± 0.4 3.8 ± 0. 2 3.7 ± 0.2

Methionine 1.1 ± 0. 2 1.0 ± 0.1 1.2 ± 0.1 1.2 ± 0. 2 1.3 ± 0.2 1.5 ± 0.1

Isoleucine 3.5 ± 0. 3 3.7 ± 0.3 3.4 ± 0.3 3.6 ± 0.3 3.0 ± 0. 1 3.2 ± 0.1

Leucine 8.9 ± 0.7 8.7 ± 0.3 8.3 ± 0.1 8.4 ± 0.3 8. 1 ± 0.1 8.4 ± 0.3

Tyrosine 3.4 ± 0.2 3.4 ± 0.4 3.2 ± 0.1 3.3 ± 0.3 3.0 ± 0.3 3.2 ± 0.2

Phenylalanine 3.2 ± 0.2 3.2 ± 0.1 3.1 ± 0.1 3. 1 0.4 3.4 ± 0.1 3.4 ± 0.1

Sulfur % 0.95 0.03 0.96 ± 0.02 0.91 0.03 0.93 ± 0.01 0.97 ± 0.02 0.94 ± 0.03

a Resid ues per 100 residues ± standard deviation.

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230 BADEN, GO LDS MITH, AND LEE

or the amino acid composition and electrophoretic pattern of the isolated fibrous prote ins. That the extracted fibrous prote in is ent irely norma l must be viewed wi t h some reservation in view of the recent observation that different polype ptide com­ponents can be detected in the a protein of epidermis [10].

The seve ral polypeptides which have been ob­served in the a fibrous prote in of cow snout epidermis have been shown to be different but t hey do have similar am ino acid analyses. Human fi­brous protein may also consist of severa l compo­nents. It would be poss ible to miss several such polypeptide chains and a lterations in them, how­ever, since SDS- polyacrylamide electrophoresis only detects differences in molecular weight.

A fur ther problem to be considered is t he varia ­bility observed in the SDS-acrylam ide elec tropho­retic patterns . In the case of cow snout epidermal ex polypeptides, it has been shown that there is a different ia l susceptibility to enzymatic hydrolysis. It is not unlikely that in the fina l stages of keratinization some modification occurs in the fibrous prote ins secondary to the release of hydro­lytic enzymes. In the case of the ex proteins of wool it has been found that such changes may not be detectable until the proteins a re t reated with denaturing solvents which then allows fr agments to be released [11]. Such changes in the polypep­t ide cha ins could be responsible for the vari abili ty in t he relat ive intensity of the electrophoretic components which have been described , consider-

Vol. 65, No . 2

ing the abnorm al keratinization whi ch OCC urs in t he various forms of ichthyosis.

REFEREN CES

1. Goldsmith LA, Baden HP: M a nagement a nd t reat­men t of ichthyos is. N Engl J Med 286:821- 823 1972 '

2. Winklemann RK , Perry HO, Achor RW, Kirby LJ: Cuta neous syndromes produced as side effec ts of t riapa ranol therapy. Arch Derm atol 87:372- 377, 1963

3. Bluefarb SM : Cutaneous manifestations of mU ltiple mye loma . Arch Derm atol 72 :506- 522, 1955

4. Gi llespie JM, Lennox FG: Keratin derivatives ex­tracted from wool with alkaline thioglycolate solu­t ions. Aust J BioI Sci 8:97-113, 1955

5. Davis BJ: Disc elect rophores is. II . Method and a pplication to huma n serum prote ins. Ann NY Acad Sc i 121:404- 427, 1964

6. Baden HP, Goldsmith LA, Fleming BC: A compara­t. ive study of the physicochemical properties of hum a n keratinized t issues . Biochim Biop hys Acta 322:269- 278, 1973

7. Goldsmith LA, Baden HP: A uniquely orien ted epiderma l lipid. Nature (Lond ) 225: 1052- 1053, 1970

8. Craig JM , Goldsmith LA, Baden HP: An abnorma l­ity of keratin in the harlequin fetus. Pediatrics 46:437- 440, 1970

9. Rothberg S: The eva luation of keratin fractions in normal and abnormal epidermis. J Invest Derma­tol 34: 197- 206, 1964

10. Baden HP, Goldsm it h LA, Fleming BC: Polypep t ide composit ion of epiderm a l prekeratin. Biochim Bio­phys Acta 317:303- 311, 1973

11. Baden HP: Enzymatic hydrolysis of t he a-protein of epidermis . J Invest Dermatol 55:184- 187, 1970