Skin Calcium-Binding Protein Is a Parvalbumin of the Panniculus Carnosus* Pam ela Hawley-Nelson, M.S. , M artin W. Berchtold, Ph.D., H em·ik Huitfeldt, M .D., Jack Spi egel , Ph.D., and Stuart H . Yusp a, M.D. Laboraco ry of Cellular Ca rcinogenes is and T umor Promotion, Nati onal Cancer Institute (PI-1 -N , HI-I , SHY), lkthcsda, Ma rybnd; Depart ment of Cell Biology. Baylor College of Med icine (MWB) , Houston, Texas; and Department of Biology. Catholi c University of Ameri ca (PI-1 -N , JS) , Was hington, D.C. , U.S.A. Skin calcium-binding protein (SCaBP) is a ca lcium binding that the l\1,. 13,000 PV/SCaBP cross-reacting antigen was protein purified from w hole rat skin. It has a molecul ar res tricted to the hypodermal tiss ue removed by scrapin g. weig ht o f approx11nately 12,000 daltons but migrates at l\1,. Immunoflu orescent stain ing of Bouin-fixed skin sections 13,000 on sodium dodecyl sul fa te (S D S)-polyacrylamJde w ith these antisera confirmed the locali za ti on ofPV /SCaBP gels. On nitrocellulose blots of SDS-polyacrylamide gels, to the panniculus ca rnosus, a h ypodermal m uscle layer. 6 different antisera to SC aBP reacted equally wel l with N ewborn mouse skin does no t conta in this antigen. Ad­ SCaBP and parvalbumin (PV), an 11 ,500-dalton calcium­ ditional polypeptides of M ,. 10, 500 and 12,000 on SDS gel s bin ding pro tein purifi ed from rat skeletal muscle, which of extracts from the epidermis of newborn and adult rats also migrates at M ,. 13, 000 on SDS-polyacrylamide gels. and n'li ce were found to be immunoreactive with anti­ Rabbit antiserum to muscl e PV also recognized both PV SCaBP serum. T hese polypeptides were not recognized by and SCaBP, and either protein absorbed specific antibodies the PV antise rum, and the reacti vity o f anti-SCaBP fo r against either antigen fro m bo th types of antise ra. Soluble these antigens was not absorbed by purified PV o r SCaBP. protein extracts fr01n whole adult rat and m ouse skm con­ O ur results indica te that SCaBP is antigeni ca ll y indistin­ tain ed a M ,. 13,000 protein w hi ch w as recogni zed on ni­ gui shable from PV and is locali zed in the ad ult rodent trocellulose bl o ts of SDS gels by both antisera . Blots of panniculus ca rnosus, and th at antisera to SCaBP are poly­ extracts from epidermis, dermis, whole skin, and skin scraped specifi c, recognizin g epidermal proteins in addition to on the dermal side to remove hypodermal tissue revealed SCaBP/PV. ) ln lJcst Dcnnatol 86:157-162, 1986 alcium io n concentrati on has been shown to contro l T he present study was undertaken to com pa re SCaBP and PV, the differentiati o n of m o use epiderm al keratinocytes and to determ ine their tiss ue distributio n by immunohistochem­ 111 culture [1] and this regul a n o n can be altered m istry and immuno bl o t analysis, the latter technique having the ca rcinogen-treated o r m ali gnantly transformed ker­ adva ntage o f providing in fo rmation o n m o lecul ar weig ht and atinocytes [2,3]. It w as of great interest, therefo re, antigenicity simultaneously. We presen t da ta dem o nstra tin g that Cwhen a skin ca lcium-binding p rotein (SCal3 P) was identified anci PV and SCaBP are antigen ica ll y indistinguishable and that the purified fro m w ho le adult rat skin [4,5], and an tisera to this pro­ PV / SCaB P antigen is locali zed in the panniculus ca rnosus of adult tein were raised in rabbits. The locali za ti on of SC:~ BP anngen, roden t skin. Additio nal antigens of unknown nature, with M,. determined b y indirect immuno flu o rescence of frozen skin sec­ 10, 500-12,000 an sodium dodecyl sul fate (SDS)-polyacrylam ide tions, was rep orted to be in basal cells of the epidermis [6]. Its gels, are recognized b y an ti-SCaBP serum and are localized in molecular weight (1 2,000 daltons), isoe.le ctric po in t (5.0), and the epidermis of newbo rn as well as adul t rodent skin. T hese amino acid composition were similar to ano ther calcium binding antigens m ay account fo r previo us reports [6] of SCJBP in epi­ protein, p arvalbumin (PV) IS]. PV was known to be a maj o r dermis, and of regulated syn thesis of low-m olecular weight an­ compo n ent o f fast-tw itch skeletal muscle (7] and has been shown tigens in epidermal ce ll cultures r<.>. l OJ. to be present in relative abundance in adult rat skin [8], locahzed to the h y po dermal muscl e, the panniculus ca rnosus. MATERIALS AN D M ETH ODS Proteins Purified adult rat skin SCal3 J> was a gift of D r. J ana Pavlovitch, Paris, France, and was p repared as descri bed previ­ Manuscript received April 4, 1985; acce pted for publica ti on Au gust 26, o usly [4,5]. Purified adult rat muscle PV an d bovin e b rain c:t l­ 1985. This work was perfo rm ed in pa rtial ful fi ll me nt of the req uirements fo r m odulin were from Calbioch em. Adul t Sprague-Dawlcy rat and the Doctor of Philosophy deg ree of the Biology Departmcm, Ca tholic B ALB/ c m o use skin was fractionated by the procedure of Ka­ University of Ameri ca (PH-N). MWB is supported by a Swiss Nati onal wamura ct al [11] . Shaved skin was treated w ith N air and then Foundati on Fellowship for Youn g Adva nced Scicmis ts (83.0 11 .082). rem oved fro m the anim al. T he dermal side was scr:t ped vigor­ *A preliminary report of this work has been published (] Cell Bioi ously w ith a scalpel to remove the hypodermal materia l; the scraped 99: A430, 1984). skin was fl oated on 1% trypsin in sa line for 1 h at 37°C. T he Reprint reques ts to: Pa mela Hawley-Nelson, M.S., Natiomtl Ca nce r epiderm is was rem oved fro m the dcrm is by sera ping on the ep­ Institute, Building 37, Room 3A23, Bethes da, Marylan d 20892. Abbreviati ons: idermal side w ith a scalpel. N ewbo rn m o use skin was no r scraped PV : parva lbumin but was fl oated on 0. 25% try psin overnig ht at 4°C, and the ep­ SCaB P: sk in ca lcium-bin di ng protein idermis and dermis were separated w ith fo rceps. Cytosol extracts SDS: sodiu m dodecyl sul fa te were prepared fro m full-thickness skill, skin fro m w hi ch the hy- 0022-202X/86/$03.50 Copyri ght © 1986 by T he Society for In vestiga ti ve De rmatology, Inc. 157 158 HAWLEY-N ELSON ET AL THE JOUHNAL OF INVESTIGATIVE DERMATOLOGY podermal material was removed, the hypodermal material,_epi ­ A B C 0 E F G H s p PSPSPS p s p s s p c p dermis, dermis, and leg muscle by ho m ogem zmg the ti ssue 111 20 c mM T ri s, pH 7.4, w ith 1 mM EDTA and 1 mM ph.enylmethyl­ sulfonyl fluoride, freezi ng and thawing 3 times and centnfugmg at 100,000 g for 30 min, retaining the supernatant. Cell cultures - 25kd of primary newborn mouse epiderm al cells were prepared as de­ -25kd- scribed J1 2]. After 3 days of cu lture in 0.07 mM calcium medium, cell s were scraped from the dish and homogenized in the sa me - 13kd- - 13kd buffer as described for tiss ue fractions. All cytosol preparations were as sa yed for protein concentrati on by the BioRad protein assay kit. Antisera Six different unfracti o nated rabbit anti-SCal3P sera were a gift of Dr. Jana Pavlovitch, Paris, France, and were pre­ pared as previously described J4 ]. Except w here otherw ise noted, Figure 1. Parvalbumin and SCa BP cross-rea ct antigenicall y. One mi­ anti-SCaBP was from rabbit no. 3. Unfractionatcd antiserum to crogram each of purif1ed calcium-binding proteins parvalbumm (P), SC~P (5), and ca lm odulin (C) we re el ectrophoresed on reducmg SDS 15 Yo PV as well as anti-PV lgG purified from the antiserum on a PV polyac rylamide gels and transferred (blotted) to nitrocellulose. Pa11el ,4 affi nity column were prepared as described [8]. Affinity-purified was stai ned with amido bla ck. SCal31' and PV C01111 gratc at M, 13,000. anti-PV lgG was reconstituted and used at 2.2 p.g/m l_ (final di­ Pa nel s B- 1-1 we re rea cted with eq ua l dilu tions of antise ra and visualized lution). In the undiluted anti-PV serum the concentratiOn of this wi th a peroxidase rea ction as described in Mnrc ria ls and Meth ods. B =an~­ specific lgG was 1.6 m g/ml. Absorbed antisera were prepared by SCaBP, C=anti -SCal3P wh ich had been absorb ed w1t h PV D=ann­ in cubatin g 50 p.g antigen w ith 1 ml antiserum followed by cen­ SCaBP whi ch had been absorbed with SCaBP.