the metabolism of “histidine-rich” protein in normal and … · 2017. 1. 29. · the metabolism...

Tnn JOURNAL or INVESTIGATIVE DErnnroLooyCopyright 1968 by The Williams & Wilkins Co.

Vol. 91, No. 5Printed in U.S.A.

THE METABOLISM OF "HISTIDINE-RICH" PROTEIN IN NORMALAND PSORIATIC KERATINIZATION*

JOHN J. VOORHEES, M.D.,t SIBA G. CHAKRABARTI, PHD.AND I. A. BERNSTEIN, Pn.D.

Historically, the concept of a unique proteinsynthesized exclusively in the stratum granu-losurn, originated in our laboratory in 1964 (1)to explain the autoradiographs of Fukuyama,et al. (2) which showed the initial incorporationof intraperitoneally injected histidine and gly-cine in the granular and upper spinous layersof the newborn rat epidermis, whereas methio-nine, leucine, and phenylalanine first appearedin the lower cells of the epidermis. Supportfor this hypothesis was provided by Reavenand Cox (3) who showed the presence of co-pious amounts of histidine in keratohyalin asindicated by the Pauly stain, a relatively specifichistochemical reaction for protein-bound histi-dine.

Subsequently, Hoober and Bernstein (4) iso-lated a sulfur-poor, "histidine-rich" protein(HRP) from rat epidermis which Gumucioet cii. (5) then obtained from a tissue prepara-tion composed of stratum corneum and stratumgranulosum. This protein has been furtherpurified by Chakrabarti and Bernstein (6) andshown to contain predominantly serine, threo-nine, arginine, glycine, alanine, aspartic acid(and/or its amide), glutamic acid (and/or itsamide), tyrosine and histidine. No sulfur-con-taining amino acids were found nor did theintraperitoneal injection of cystine-S result inthe labelling of this protein (6). Fukuyamaand Epstein (7) studied the localization of sixadditional tritiated amino acids in the epidermisof the newborn rat and found that arginine andserine were also incorporated initially in theupper viable cellular layers while lysine and

* From the Departments of Dermatology, In-dustrial Health and Biological Chemistry and theInstitute of Industrial Health, The University ofMichigan Medical Center, Ann Arbor, Michigan48104.

t Carl Herzog Fellow of the American Dermat-ological Association.

Presented at the 29th Annual Meeting of theSociety for Investigative Dermatology Inc., June16—18, 1968, San Francisco, California.

Supported by Research Grant 1 ROl AM 10225and Training Grant 5 TOl AM 05268, NationalInstitute of Arthritis and Metabolic Diseases,USPIHS.

valine appeared in the lower layers. Prolineand tyrosine were evenly distributed in thebasal, spinous and granular layers.

Our laboratory has recently reported pre-liminary data on the in vitro synthesis of HRPin minced epidermis of the newborn rat (8)and on the isolation of keratohyalin proteinfrom the granular layer of the same tissue(9). Using ultrastructural autoradiography,Cox and Reaven (10) and later Fukuyama andEpstein (11) detected an increasing quantity oflabelled histidine within keratohyalin. If HRPis a precursor of keratohyalin (11), then thepresence or absence of HRP might coincidewith the presence or absence of keratohyalin.The data presented in this paper support thisprediction. No significant synthesis of HRPwas detected in keratohyalin-free psoriaticplaques as compared with that found in theuninvolved epidermis of the psoriatic. A smallamount of synthesis occurred in a healing psori-atic plaque.

MATERIAL AND METHODS

Isolation of HEP from necropsy material. Skinspecimens obtained at necropsy from various sitesincluding the palm, sole, abdomen and arm, weremechanically defatted and stirred in 1 N NH4OHfor 30 minutes at room temperature. The epidermiswas then gently teased from its dermal mooringsand pooled. This pooled epidermis was submittedto the published procedure for the isolation ofHRP (4) as diagrammed in Fig. 1. Figs. 2 and 3are histologic pictures of the epidermal materialfrom which the HRP was obtained. The speci-mens were fixed in 10 percent formalin and stainedin the usual way with hematoxylin and eosin (Hand E) or by a modification of the Pauly reac-tion (3). The intense concentration of melanin inthe basal zone indicates a separation plane nearthe dermal-epidermal junction. Amino acid analy-sis was done automatically on material hydrolyzedin 6 M HC1 at 108—110° in vacuo for 24 hours bythe ion exchange method of Spackman, et al. (12).

Intradermal injections and biopsy procedures.Seven microcuries of tritiated L-histidine (4.7c/mmole) were injected intradermally into eachsite. At varying intervals, the control injectionsites were excised and the epidermis obtained bytreatment with 1 N NH4OH as described above.

344

PROTEIN METABOLISM IN PSORIATIC KERATINIZATION 345

EPIDERMAL PROTEIN FRACTIONATION AND ISOLATION SCHEME

RADIOACTIVE HUMAN EPIDERMIS PLUSNONRADIOACTIVE RAT EPIDERMIS

UREA TREATED EP I DERMAL HOMOGENATE

UREA EXTRACT UREA INSOLUBLE PPT.

UREA SOLUBLE NONDIALYZABLE UREA SOLUBLE DIALYZABLE

EPIDERMAL PROTEIN

iN PER CHLORIC ACID SOLUBLE .JN PER CHLORIC ACID

EPIDERMAL PROTEIN INSOLUBLE PPT.

pH 4.5 EPIDERMAL PROTEIN PPT. pH 4.5 SUPERNATE

SOLUBILIZED IN .02M Na2CO3

1

Psoriatic and healing psoriatic tissues were ob-tained by a scalpel incision parallel to the surfaceof the skin and at a level which allowed punctate,free capillary bleeding in the wake of the surgicalslice. The remaining psoriatic dermis was excisedand this, plus punch biopsies (4 mm) taken adja-cent to the biopsy sites, were processed for II andE staining, the Pauly reaction and autoradiog-raphy. The separated epidermal samples and thepsoriatic slices were submitted to the isolationprocedure for HRP as outlined in Figure 1. Non-radioactive rat epidermis was added to the smallbiopsy specimens to facilitate isolation of HRP.Autoradiography was done using Eastman KodakNTB-3 liquid emulsion and an exposure of 4weeks. Separate sections were stained with II andE and the Pauly reagent. Radioactivity in the pro-tein fractions was detected by liquid scintillationspectrornetry as previously reported (4). Proteinwas measured by the method of Lowry, et al. (13).

RESULTS

Isolation of "histidine-rich" protein (HRP)from necropsy skin. The HEY obtained byprecipitation at pH 4.5, followed by solubiliza-tion in sodium carbonate, was submitted to

chromatography on Sephauex G-5O where nei-ther the protein nor the Blue Dextran markermolecules entered the Sephadex matrix, thusbeing excluded from the column simultaneously.The results of this procedure are given in Figure4. The protein fraction excluded on SephadexG-5O was retarded and resolved into two peaks(Fig. 5) when chromatographed on SephadexG-100. This chromatographic behavior, in con-junction with previous analytical Wtracentrifu-gation of newborn rat HEY, places the mo-lecular weight of human HEY at about 30,000.In tracer studies done in the rat, the firstSephadex G-100 peak contained the HEY.Figure 6 compares the amino acid compositionof this peak and the HRP obtained from theepidermis of the newborn rat. The first 9 aminoacids are comparable qualitatively, but notquantitatively, in man and the rat. The next5, with small amounts in man and none in therat, possibly reflect inclusion of several earlyfractions of the second Sephadex G-100 peak inthe amino acid analysis. Notable by their ab-

SM UREA HOMOGENIZATION

CENTRIFUGATION

NH4OH DIALYSIS x 3

LYOPHILIZATION

iN PCA SOLUBILIZATION

ISOELECTRIC PPT.

GEL FILTRATION CHROMATOGRAPHY ________ AMINO ACID ANALYStS BY

USING SEPHADEX G-50 AND G-IOO ION-EXCHANGE CHROMATOGRAPHY

Fie. 1. Flow diagram of fractionation and isolation procedure for epidermal protein asmodified from the technique of iloober and Bernstein (4). PCA = perchloric acid.

346 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 2. II and E stained section of epidermis separated in 1 N NILOR from necropsyskin. Magnification: 180 x.

sence are the sulfur-containing amino acids,cystine and methionine.

Tracer experiments. Seven tracer experiments,in three psoriatic volunteers, were carried outto establish concomitance of keratohyalin andHRIP. Tritiated histidine was injected intrader-mally into these sites: psoriatic plaques, a heal-ing psoriatic plaque, and uninvolved controlsites, including the palm. At appropriate inter-vals shown in Table I these sites were excisedfor biochemical fractionation and autoradiog-raphy.

Figure 7 demonstrates that tritiated histidinegiven by intradermal injection accumulates innormal human stratum granulosum as it doesin the stratum granulosurn of the rat. Figure 8shows the coincidence of maximum radioactivityand Pauly positivity in this same layer. Figure9 shows a control biopsy site. Involution of thearea beneath the biopsy site was documentedduring two months of continuous inpatient

observation. Figure 10 is a section of this healingpsoriatic tissue where there is the focal presenceand absence of keratohyalin. Figure 11 is asection of the psoriatic material used for bio-chemical fractionation showing the completeabsence of keratohyalin.

Table I gives the distribution of radioactivityin the various epidermal protein fractions. Thelast line of the table represents the control forthe psoriatic lesion while the other control re-lates to the healing psoriatic tissue. Two addi-tional control experiments and one additionalexperiment with psoriatic tissue were done. Thedata obtained in these experiments paralleledthose shown in the table.

The "pH 4.5" precipitate contains the HRP.As can be seen in Table I, about 20 per cent ofthe total 3H in the control epidermis was foundin this fraction whereas less than 0.1 per centappeared here in the case of the psoriañc le-sion. Interestingly, 2.5 per cent of the radio-

TISSUE:Normal Human EpidermisAutopsy Material

FRACTIONpH—4.pptHistidine—Rich" Protein

COLUMN30 x 2.cms

ELUANT.01 M NH4OH


Fio. 3. Pauly stained section of epidermis separated in 1 N NH4OH from necropsy skin.Magnification: 180 X.

SEPHADEX G—50 GEL FILTRATION CHROMATOGRAPHY

1200

900

ECa,

600

300

30 70ml Eluate

FIG. 4. Elution of the "pH 4." precipitated epidermal protein (normal human) fromSephadex G-50.

'I

I

TISSUE:Normal Human Epidemi,Autopsy Material

FRACTION:

pH 4.5 PrecipitateCOLUMN;

36 x .9 cnnELLIANT:

.1 N Acetic Acid

AMINO ACID MAN NEWBORN RAT

Residues I 100 Residues

Threon)rie 10.1 6.8

Aspartic NH2) 15.4 5.6

Glutamic (NH2) 12.0 14.1

Alanine tO. I 11.8Tyresine 43 9.4Glycine 16.8 15.3

Argiriine 10.0 9.0Histidine 8.3 6.9Serine 6.0 11.5

Valine 1.8Isolelicine I .2 —

Leucine I .6 —

Phenylalanine .6 —

Lysine j .3 —

Methionine — —

1/2 Cyst)ne — —

Fio. 6. Comparison of the amino acid composition of IIRP isolated from human andnewborn rat epidermis. See text for details of analytical method.


SEPHADEX G-IOO GEL FILTRATION CHROMATOGRAPHY

lao.

E

80

40

0 50 80ml Eluote

FIG. 5. Elution of "pH 4.5" precipitated epidermal protein (normal human) from Sepha-dex G-100. The sample chromatographed was material previously excluded from a columnof Sephadex G-50.

AMINO ACID COMPOSITION "HISTIDINE - RICH" PROTEIN

TABLE IDi1ribution of radioactivity in epidermal protein fractions derived from biopsies taken

after intradermal injection of hitidine—8H

HISTIDINE 3H IN EPIDERMAL PROTEIN FRACTIONS

BIOPSYSPECIMEN

TIMEAFTER

INJECTION

1&%DPM

UREA

EXTRACT

UREA

INSOLUBLE

PPT.

UREA

SOLIJ8LE

NONDIALYZA8LE

UREA

SOLUBLE

DIALYZABLE

PCAINSOLUBLE

PPT.

pH4.5PPT.

Hours DPM DPM % DPM % OPM % DPM % DPM % DPM %

Psoriasis I '/2 45,800 99.0 .8 .1 98.0 <.1 <.1

HeaIngP sot las is '/2 42,000 88.0 2.0 7.0 71.0 1.2 2.5

Control '/2 4,200 15.0 25.0 67.0 8.5 32.0 21.0

Control I /2 1,340 96.0 3.5 96.0 <.1 21.0 18.0

t = perchioric acid. For details of procedure see text and Fig. 1. DPM disintegrationsper. minute.

FIG. 7. H and E stained autoradiograph of human palm epidermis 1½ hours after intra-dermal injection of histidine-3H. Magnification: 540 X.

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350 THE JOUENATJ OF INVESTIGATIVE DEEMATOTJOGY

FIG. 8. Pauly stained autoradiograph of normal human epidermis (taken from the siteindicated by the arrow in Fig. 9) ½ hour after intradermal injection of histidine-3H. Mag-nification: 180 X.

activity appeared in the "pH 4.5" precipitatefrom the healing lesion in which keratohyalinwas reappearing (Fig. 10). The data weresimilar for the "HC1O4-insoluble" fraction.While in the uninvolved epidermis most of theII in the urea extract was nondialyzable,nearly all the tritium in the psoriatic ureaextract—and to a slightly lesser extent in thehealing psoriatic tissue—was dialyzable andtherefore in small molecules.

DISCUSSION

The protein obtained in the "pH 4.5" pre-cipitate meets the following established criteriafor HRP as isolated from the rat: (a) solubilityin S M urea, (b) nondialyzability, (c) solubilityin 0.1 N HC1O4 at room temperature, (d)precipitability at pH 4.5, (e) exclusion onSephadex G-50, (f) slight retardation on Sepha-dex G-100, (g) and an unusual amino acidcomposition showing a high level of histidine.

Very little radioactivity appeared in the"pH 4.5" precipitate in the psoriatic as com-pared with the control tissue. A higher per-centage of the radioactivity appeared in thisfraction in the case of healing psoriasis butthe control level was not achieved. There areat least four possible explanations for thisfailure to detect HRP in psoriatic plaques.(a) It is possible that all of the injected radio-active histidine was quickly converted to uro-canic acid by histidase. (b) Possibly, as a resultof rapid turnover, radioactive HRP was syn-thesized and degraded before biopsies weretaken at 1½ hours. (c) High dermal bloodflow may have prevented injected histidinefrom reaching the potential keratohyalin com-partment. (d) Perhaps no HRP synthesis oc-curs in psoriatic plaques.

A two and one-half fold increase in histidasehas been reported (3) in the psoriatic epidermis.However, calculations using this figure would

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Fio. 9. Photograph made after control (at tip of arrow) and healing (within circle) biop-sies were taken. Note the absence of a post-surgical scar in the healing biopsy site.

predict a 60 per cent reduction in the controlvalue rather than the complete absence ofradioactivity in the "pH 4.5" precipitate. Fur-thermore, it has been shown (14) by auto-radiography of psoriatic explants exposed tohistidine-3H in vitro, that there is no accumula-tion of radioactive histidine in the area of thenormal granular zone over a 6-hour period.During this entire period of time, the radio-active precursor was present. The third pos-sible explanation for the failure to detectHRP appears to be excluded by the presence ofover 45,000 IDPM in the dialyzable fraction of

the psoriatic tissue and by the demonstration offixed radioactivity in the malpighian compart-ment as shown in the autoradiograph (Fig. 12).It would seem, therefore, that the presence of abiosynthetic block is a reasonable explanationfor our failure to detect HRP in psoriaticplaques.

That the "pH 4.5" precipitate is indeed theHRP was indicated by the results of co-chro-matography of radioactive human and non-radioactive rat HRP on Sephadex G-50 (Fig.13). The major peak of radioactivity and theprotein peak are superimposable and both are

4• ..%

-1Ic

FJG. 10. II and E stained tangential section of the surgical slice through the healing siteshown in Fig. 9. A indicates orthokeratosis with keratohyalin beneath. B indicates parakera-tosis with absence of keratohyalin. Magnification: 180 X.

FIG. 11. II and E stained section of psoriatic plaque used for biochemical fractionation.

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.11

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,:

itt S r

*fl

fS

•.

e.

t

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44

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044

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FIG. 12. II and E stained autoradiograph of punch biopsy specimen taken adjacent to theexcised psoriatic lesion. Magnification: 540 X.

COLUMN:

30x 2.5crnsELLJANT:

\t\l\

.01 M NH4OH

FIG. 13. Elution of "pH 4.5" precipitate from uninvolved human and non-radioactive ratepidermis co-chromatographed on Sephadex G-50.


SEPHADEX G—50 GEL FILTRATION CHROMATOGRAPHY

TISSUE:U')')

400

200

E

0a

Uninvolved Histidine—3H LabeledHuman Epidermis

Nonradicoctive Rat Epidermis CarrierFRACTION:

pH—4.5ppt.Hisfldine—Rich Protein

1200

900

Ca,

600 20a

300

0 3uml Eluote

70

*

enii• —

-

'a •ç_#.a cw• a —,'tr ;-e'

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I


excluded, with the Blue Dextran marker, exactlyas was the human necropsy HRP.

Since the biogenesis of keratohyalin appearsto be a genetically programmed event occur-ring normally in the human about 14 days afterthe 1<eratinocyte leaves the basal layer, itsabsence in psoriasis might be a result of thefailure to derepress the HRP and keratohyalincistrons. In molecular biologic parlance, thismay represent an aberration in the transcrip-tion of DNA into the HRP protein messengerRNA, or a failure in translation involving theabsence of the required amino acyl transferRNA's or the proper ribosomal configurations.Internal or external epigenetic phenomena mayexplain the fact that involved and uninvolvedepidermis can be contiguous. These possibilitiesare amenable to study.

The relation of such a biosynthetic block tothe pathogenesis of psoriasis is unknown.

SUMMARY

The sulfur-free, "histidine-rich" protein hasbeen isolated and characterized in human ne-cropsy epidermis and shown to be similarqualitatively to that isolated from the epi-dermis of the newborn rat. Using tritiatedhistidine as a tracer, it was possible to demon-strate the synthesis of radioactive HRP incontrol, uninvolved epidermis where kerato-hyalin was present and to show the synthesisof a lesser amount in healing psoriatic lesionswhere keratohyalin was again appearing. Noevidence was obtained for synthesis of HRPin the involved tissue in which no keratohyalinwas seen.

REFERENCES1. Bernstein, I. A.: Rekation of the nucleic acids

to protein synthesis in the mammalian epi-dermis, p. 471, The Epidermis. Eds., Mon-

tagna, W. and Lobitz, W. C., Jr., AcademicPress, New York, 1964.

2. Fukuyama, K., Nakamura, T. and Bernstein,I. A.: Differentially localized incorporationof amino acids in relation to epidermalkeratinization in the newborn rat. Anat.Rec., 152: 525, 1965.

3. Reaven, E. P. and Cox, A. J., Jr.: Histidineand keratinization. J. Invest. Derm., 45: 422,1965.

4. Hoober, J. K. and Bernstein, I. A.: Proteinsynthesis related to epidermal differentia-tion. Proc. Natl. Acad. Sd. (US), 56: 594,1966.

5. Gumucio, J., Feldkamp, C. S. and Bernstein,I. A.: Studies on localization of "histidine-rich" peptide material present in epidermisof the newborn rat. J. Invest. Derm., 49:545, 1967.

6. Chakrabarti, S. G. and Bernstein, . 4Futher characterization of "histidine—rich,perchloric-soluble" epidermal protein. Ab-stracts, 152nd Natl Meeting, Am. Chem.Soc., New York, September, 1966.

7. Fukuyama, K. and Epstein, W. L.: Epidermalkeratinization. Localization of isotopicallylabelled amino acids. J. Invest. Derm., 47:551, 1966.

8. Sugawara, K. and Bernstein, I. A.: Biosynthe-sis of epidermal "histidine-rich" protein in.vitro. Abstracts, 153rd Natl Meeting, Am.Cheni. Soc., Chicago, September, 1967.

9. Feldkamp, C. S. and Bernstein, I. A.: "Kera-tohyalin" protein in epidermal differentia-tion. Fed. Proc., 27: 335, 1968.

10. Cox, A. J., Jr. and Reaven, E. P.: Histidineand keratohyalin granules. J. Invest. Derm.,49: 31, 1967.

11. Fukuyama, K. and Epstein, W. L.: Ultra-structural autoradiographic studies of kera—tohyalin granule forniation. J. Invest. Derm.,49: 595, 1967.

12. Spackman; D. H., Stein, W. H. and Moore, S.:Automatic recording apparatus for use inthe chromatography of amino acids. Anal.Chem., 30: 1190, 1958.

13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L.and Randall, R. J.: Phenol measurementwith the Folin phenol reagent. J. Biol.Chem., 193: 265, 1951.

14. Fegeler, F. and Rahniann-Esser, M.: Auto-radiographische lJntersuchungen zum Pro-teinstoffwechsel der Epidermis gesunder unddurch Psoriasis vulgaris veränderter Haut.Arch. Klin. Exp. Derm., 227: 847, 1966.

the metabolism of “histidine-rich” protein in normal and … · 2017. 1. 29. · the metabolism...

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