Proc. Nati. Acad. Sci. USAVol. 80, pp. 1905-1909, April 1983Cell Biology

Differential immunological crossreactivity of HeLa keratinantibodies with human epidermal keratins

(epidermal proteins/two-dimensional gel electrophoresis/thin sections/indirect immunofluorescence/basalioma)

STEPHEN J. FEY, PETER MOSE LARSEN, RODRIGO BRAvo*, ARIANA CELIS, AND JULIO E. CELISDivision of Biostructural Chemistry, Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark

Communicated by Diter von Wettstein, December 27, 1982

ABSTRACT HeLa cells contain four keratin-like proteins hav-ing molecular weights of 50,000 (IEF 31), 48,500 (IEF 36), 44,000(IEF 44), and 43,500 (IEF 46), respectively. Mouse polyclonal an-tibodies prepared against two of these keratins (IEF 31 and 46)have been used in this study to identify human epidermal keratinswith common antigenic determinants. Using a sensitive immu-noprecipitation procedure we show that the IEF 31 antibodycrossreacts with three human acidic epidermal keratins, termedKI, K2, and K3, having molecular weights of 44,000, 47,500, and54,000, respectively. One of these keratins (Ki) comigrated withHeLa keratin IEF 44 and exhibited an identical one-dimensionalpeptide map. This protein is also abundant in basaliomas. In con-trast to these results, the EEF 46 antibody showed no crossreac-tivity with any of the human acidic or basic [35S]methionine-la-beled epidermal proteins. The lack of crossreactivity of thisantibody was further confirmed by indirect immunofluorescencestaining of cryostat sections from human split skin. These resultsemphasize both the similarity and diversity of antigenic deter-minants among HeLa and epidermal keratins.

Cytoskeletal preparations from Heba cells enriched in inter-mediate-sized filaments (7-11 nm) contain, in addition to vi-mentin, four keratin-like proteins [IEF 31, 36, 44, and 46; HeLanumbering system (1, 2)] with molecular weights ranging from43,500 to 50,000 (3-6). Mouse polyclonal antibodies have beenobtained against two of these keratins (IEF 31 and 46) (6) andhave been used to demonstrate the distribution of keratin-typefilaments in various cultured human cells of epithelial origin(6). It has been further shown that keratins IEF 31 and 46 haveantigenic determinants in common with each other and withkeratins IEF 36 and 44 (6).

Keratin-like proteins of epithelial nonepidermal cells havebeen shown to crossreact with various antibodies raised againstepidermal keratins (7-14), and this property and others such assolubility and phosphorylation have been used to identify ker-atins from nonepidermal cells (also called "cytokeratins") (7).Using a sensitive immunoprecipitation procedure, we have foundthat the IEF 31 antibody crossreacts with three acidic epider-mal keratins whereas the IEF 46 antibody shows no crossreac-tivity with epidermal proteins. These results emphasize boththe similarity and the diversity of antigenic determinants amongHeLa and epidermal keratins.

MATERIALS AND METHODSPreparation of Human Epidermis. Pieces of normal human

mammary skin (total or split skin; kindly provided by the PlasticSurgery Department, Aarhus Kommunehospital) were incu-bated overnight at 4°C with a 0.25% solution of trypsin in Hanks

balanced salt solution. After washing, the epidermis was liftedfrom the dermis with the aid of forceps.The procedures for labeling cells with [3S]methionine (15,

16), a mixture of 16 '4C-labeled amino acids (17), two-dimen-sional gel electrophoresis (5, 18), silver staining (19-21), indi-rect immunofluorescence (21-23), preparation of mouse poly-clonal antibodies (6, 24), and immunoprecipitation (6, 24) havebeen described in detail elsewhere.

RESULTSHeLa Keratins. Fig. 1 shows a region of a two-dimensional

gel fluorograph of proteins from asynchronous HeLa cells la-beled for 20 hr with a mixture of 16 14C-labeled amino acids.The positions of the four HeLa keratins (IEF 31, Mr = 50,000;IEF 36, Mr = 48,500; IEF 44, Mr = 44,000; and IEF 46, Mr= 43,500) (1, 2, 4-6, 25), actin, vimentin and a- and ,3-tubulinare indicated. The number given to each keratin correspondsto that assigned in the HeLa protein catalogues (1, 2) (e.g., seefigure 1 of ref. 2). We have shown that three of these keratinsare phosphorylated preferentially during mitosis (5, 25). Thepositions of some of the acidic satellite spots are indicated withshort arrows.

Crossreactivity of Human Epidermal Keratins with Anti-bodies Against Keratin IEF 31. Mouse polyclonal antibodiesraised against purified keratin IEF 31 recovered from two-di-mensional gels (6) were used for immunoprecipitation tests. Fig.2 shows a two-dimensional gel electrophoretic pattern of total[3S]methionine-labeled epidermal proteins used for immu-noprecipitation. The position of the putative keratins to be dis-cussed in this article (K1, K2, and K3) as well as of other majorcytoskeletal proteins of known identity are indicated as a ref-erence. The IEF 31 antibody immunoprecipitated three acidicepidermal keratins which have been termed KI (Mr = 44,000),K2 (Mr = 47,500), and K3 (Mr = 54,000) (Fig. 3). Large keratinsdo not label well with [3S]methionine and therefore we cannotcomment on their crossreactivity with the antibody.

Both K2 and K3 are major components of the epidermis asjudged by silver staining of two-dimensional gels loaded witheither a homogenate of pure human.wnamnmary epidermis (notshown) or with the protein content of a single cryostat sectionfrom human mammary split skin (10 Am thick, Fig. 4a). Therelative amount of K1 in the human epidermis is &xtremely lowas judged by silver staining but this protein is increased in hu-man basaliomas (Fig. 4b) (4, 13). The protein moving just aboveKI in basaliomas (indicated with a short arrow in Fig. 4b) mayalso correspond to a keratin (13).

Abbreviation: AMA,- transformed amnion cells.* Present address: European Molecular Biology Laboratory, Postfach10.2209, Meyerhofstrasse 1, 6900 Heidelberg, Federal Republic ofGermany.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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HeLa

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FIG. 1. Two-dimensional gel electrophoresis of '4C-labeled pro-teins from HeLa cells. IEF 31, 36,44, and 46 correspond to keratins (1,2, 4-6, 25). Some of their phosphorylated satellite spots are indicatedwith short arrows. v, Vimentin; at, ax-tubulin; ,Bt, ,B-tubulin; IEF, iso-electric focusing; NaDodSO4, NaDodSO4/polyacrylamide gel electro-phoresis.

K1 has a fast turnover as indicated by its rapid incorporationof [fS]methionine (Fig. 2) and is the- only epidermal keratinthat comigrates with a HeLa keratin (IEF 44) (not shown; com-

pare Figs. 1 and 2) (1, 2, 4, 6, 13). One-dimensional peptidemap analysis using Staphylococcus aureus V8 protease (26, 27)has shown that K1, K1I', and keratin IEF 44 are similar or iden-tical proteins (not shown). We have previously shown that ker-

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atin IEF 44. is phosphorylated preferentially -during mitosis (5,25) and therefore it is likely that K1' may correspond to a phos-phorylated form of K1. Similar one-dimensional peptide mapanalysis of keratins K2.and K3 and of the satellite spots mi-grating to their acidic side (K2' and K3') indicated that theselatter polypeptides may correspond to isoelectric variants, mostlikely phosphorylations (results not shown).

That K1, K2, and K3 correspond to keratins is further sug-gested by their solubility properties (23, 28) (not shown) and bythe fact that mouse polyclonal antibodies raised against purifiedpolypeptides K2, K3, and K3' recovered from two-dimensionalgels of human epidermis stain epidermis and keratin-like fil-aments in various human cultured cells of epithelial origin. Fig.5 a-c shows cryostat sections from human split skin treated withmouse polyclonal antibodies raised against purified keratins K3,K3', and K2. All three antibodies reacted with the epidermisalthough the K3 and K3' antisera showed a stronger staining ofthe suprabasal layers of the epidermis. The position of the basallayer was assessed by using an antiserum of unknown speci-ficity that preferentially stains this layer of the epidermis (Fig.5d). Both the K3 and K2 antisera stained a keratin-like fila-mentous system in clonally derived groups of transformed am-nion cells .(AMA) (Fig. 5 e andf). This result is similar to thatobtained with HeLa cells treated with monoclonal antibodiesraised against.PtK1 keratins or human epidermal keratins (13,29).

Comparison of the data presented above with those.availablein the literature suggested that keratins K2 and K3 may cor-

Human epidermis

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1906 Cell Biology: Fey et al.

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Proc. Natl. Acad. Sci. USA 80 (1983) 1907

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FIG. 3. Immunoprecipitation of [3S]methionine-labeled proteinsfrom human mammary epidermis with IEF 31 antibody. The positionof actin is indicated as reference.

respond to components K50 and K56a of human epidermis de-scribed by Moll et al. (13) and to some of the abundant inter-mediate-sized keratins described by various groups (4, 13, 30-32). Small keratins have also been described in epidermal cells(32, 33), human squamous cell carcinomas (34), basaliomas (4,13), and the outer sheath of human hair follicles (13).Lack of Crossreactivity of Epidermal Keratins with Keratin

IEF 46 Antiserum. In contrast to the results obtained with theantibody raised against keratin IEF 31, the antibody to IEF 46did not immunoprecipitate any basic (35) or acidic [3S]methi-onine-labeled epidermal protein (data not shown). This lack ofcrossreactivity was further confirmed by indirect immunoflu-orescence staining of unfixed or methanol/acetone-fixed cryo-stat sections from human split skin. Whereas the IEF 31 an-tibody stained the whole epidermis [preferentially the lowerlayers (Fig. 5g)], the IEF 46 antibody gave only backgroundfluorescence (Fig. 5h). Both antibodies, however, stained ker-atin-like filaments in HeLa cells (not shown) and AMA (Fig. 5i andj) which exhibit both proteins (6, 36). In favorable cases,it was possible to see that both antibodies stain what we believemay correspond to keratin organizing centers in AMA cells (Fig.

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5k). These correspond to brightly fluorescent rings or dots lo-cated in a perinuclear region (37). The IEF 46 antibody alsofailed to react with potoroo (rat kangaroo, Dipodomys) PtK2 cells(Fig. 51) which stained strongly with the IEF 31 antibody (Fig.Sm).

DISCUSSIONOf the three human epidermal keratins immunoprecipitated byantibody IEF 31, only K1 comigrated with a HeLa keratin (IEF44) (6). Both IEF 44 and K1 showed at least one acidic satellitespot (phosphorylation) and exhibited identical one-dimensionalpeptide maps. We have reported (6) that IEF 44 (or K1) is alsopresent in various human cultured epithelial cells such as nor-mal and transformed amnion, Detroit 98 (bone marrow), EJ 19(bladder carcinoma), and Chang liver. The synthesis of thisprotein increases substantially upon spontaneous transforma-tion of human amnion cells (unpublished data) and after giantcell formation in HeLa cells (38).

Small keratins have also been described in epidermal cells(32, 33), squamous cell carcinomas (34), basaliomas (4, 13), ada-mantinomas (13), and hair follicles (13). Of these, we can onlycomment on the Mr 46,000 protein (K46) recently described byMoll et aL (13). Briefly, this protein, a keratin, is present spe-cifically in the outer sheath of hair follicles but not in inter-follicular epidermis. It comigrated with a HeLa keratin (theircomponent 4) (4, 13) and was an abundant component of basalcell epitheliomas. Whether this protein corresponds to our ker-atin K1 (or IEF 44) remains to be established. At present wedo not know the epidermal distribution of K1.

Although the IEF 46 antibody showed no reaction with ep-idermal proteins it did react strongly with various cultured hu-man epithelial cells known to contain this protein. Recently, wehave obtained evidence indicating that this antibody reacts withsuprabasal layers of the epidermis in basaliomas (Fig. Sn). Thisresult contrasted with the homogeneous staining observed withthe IEF 31 antibody (Fig. So). Because we have not detectedIEF 46 in basaliomas it is likely that this crossreactivity may bedue to the increased synthesis of K1 (or IEF 44) in these tu-mors. This observation is consistent with other studies in whichwe have found that the IEF 46 antibody shows a small but sig-nificant crossreactivity with AMA IEF 44 (6). The reason for thepreferential staining of the suprabasal layers is unknown.

In summary, the above results taken together illustrate sim-ilarities and divergency in antigenic determinants among HeLaand human epidermal keratins. Furthermore, they point to theneed to use various criteria to identify nonepidermal keratins.

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FIG. 4. Two-dimensional gel electrophoresis of human epidermal proteins. Silver staining of proteins from a single cryostat section of mammarysplit skin (10 pum thick) (a) and of human basalioma proteins (b).

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Proc. Natl. Acad. Sci. USA 80 (1983)

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FIG. 5. Fluorescence microscopy of cryostat sections and cell cytoskeletons treated with various antibodies. (a-d, g, and h) Human split-skincryostat sections (10 ,um thick) treated with the following mouse polyclonal antibodies: (a) anti-human keratin K3; (b) anti-human keratin K3'; (c)anti-human keratin K2; (d) antibody of unknown specificity; (g) anti-keratin IEF 31; and (h) anti-keratin IEF 46. (e,f, i, j, and k) Methanol/acetone-fixed transformed amnion cells (AMA) treated with: (e) anti-human keratin K3; (f) anti-human keratin K2; (i) anti-keratin EEF 31; (j) anti-keratinEEF 46; and (k) anti-keratin IEF 31, putative staining of keratin organizing centers (KOC). (1) Methanol/acetone-fixed PtK2 cells treated withkeratin IEF 46 antibody. (m) As in I but stained with keratin IEF 31 antibody. (n) Cryostat sections of a basalioma stained with EEF 46 antibody.(o) Same as n but stained with the EEF 31 antibody. (x250.)

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We thank Jytte Jacobsen for preparing the cryostat sections, P. Bjer-ring for supplying the basalioma, and 0. Jensen for photography. S.J. F.is a recipient of a fellowship from the Danish Cancer Foundation; P.M. L.is a recipient of a fellowship from the Aarhus University. R. B. was a re-cipient of a fellowship from the Danish Medical and Natural ScienceResearch Councils. This work was supported by grants from Euratom,the Danish Cancer Foundation, the Danish Medical and Natural Sci-ence Research Councils, the Carlsberg Foundation, and Konsul Johan-nes Fogh-Nielsens og Fru Ellen Fogh-Nielsens Legat.

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