Immunochemical Detection of Sulfur Mustard Adducts with Keratins

These antibodies showed binding to the stratum corneum of human skin exposed to .... skin keratins: Mass spectrometry-based non-invasive proteomic ana...
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Chem. Res. Toxicol. 2002, 15, 21-25

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Immunochemical Detection of Sulfur Mustard Adducts with Keratins in the Stratum Corneum of Human Skin Govert P. van der Schans,* Daan Noort, Roos H. Mars-Groenendijk, Alex Fidder, Lai F. Chau, Leo P. A. de Jong, and Hendrik P. Benschop TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Received January 18, 2001

As part of a program to develop methods for diagnosis of exposure to chemical warfare agents, we developed immunochemical methods for detection of adducts of sulfur mustard to keratin in human skin. Three partial sequences of keratins containing glutamine or asparagine adducted with a 2-hydroxyethylthioethyl group at the ω-amide function were synthesized and used as antigens for raising antibodies. After immunization, monoclonal antibodies were obtained with affinity for keratin isolated from human callus exposed to 50 µM sulfur mustard. These antibodies showed binding to the stratum corneum of human skin exposed to low levels of sulfur mustard, as evidenced by immunofluorescence microscopy. This approach opens the way for development of a detection kit that can be applied directly to the skin. To the best of our knowledge, this is the first example of immunochemical detection of adduct formation of toxic chemicals with skin proteins. A similar approach can be followed for skin exposure to environmental pollutants.

Introduction The need for reliable methods to detect nature and extent of poisoning with chemical warfare agents is evident from the recent use and threat of use of these agents in warfare and in terrorist attacks (1-4). As part of a program to develop methods for diagnosis of exposure to sulfur mustard, we developed immunochemical methods for detection of adducts of sulfur mustard to DNA of white blood cells, the respiratory tract and skin and mass spectrometric methods for detection of adducts of sulfur mustard to hemoglobin and albumin (5-7). Besides the respiratory tract, the skin is a major target for vesicants such as sulfur mustard. Proteins in the skin, particularly those in the stratum corneum, are readily accessible to such agents. Since keratins are the most abundant proteins in the skin, we intended to develop immunochemical methods for retrospective detection of skin exposure to sulfur mustard based on identified adducts with these proteins. Information on immunochemical assays for the detection of protein adducts is only scarcely available in the literature. In general, the antibodies have been generated by using adducted keyhole limpet hemocyanin or albumin as an antigen (8-12). Such an approach was followed in one of our previous studies by using sulfur mustard treated hemoglobin as antigen. However, these experiments did not result in antibodies recognizing adducts in sulfur mustard-treated hemoglobin (13). In a more systematic approach, haptens having sequential similarity to parts of the adducted protein surface were used for raising antibodies (14, 15). In the current study, it appeared that aspartic and glutamic acid residues in keratins are major alkylation sites. Therefore, we synthesized partial sequences of keratins * To whom correspondence should be addressed.

containing glutamine and asparagine adducted with a 2-hydroxyethylthioethyl (HETE) group at the ω-amide function and used these as antigens for raising antibodies. Such amide analogues are supposedly more stable in vivo than the corresponding ester derivatives. The antibodies obtained were evaluated for detection of sulfur mustard adducts in the skin.

Experimental SectionProcedures Materials. Caution: Sulfur mustard (2,2′-dichlorodiethyl sulfide) is a primary carcinogenic, vesicant, and cytotoxic agent. This compound should be handled only in fume cupboards by experienced personnel. Technical grade sulfur mustard was distilled before use to a gas chromatographic purity exceeding 99.5%. Synthesis of [14C]sulfur mustard was carried out as described previously (16). The chemical purity, determined with GC, was 99%; specific activity 56.4 mCi/mmol. Goat-anti-mouse-Ig(total)-alkaline phosphatase was purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). FITC-labeled1 (fluorescein isothiocyanate) goat-anti-mouse (GAM-FITC) was from Southern Biotechnology Associates (Birmingham, AL). Fetal calf serum (FCS) was purchased from LCT Diagnostics (Alkmaar, The Netherlands). N-Boc-1-tert-butyl-L-glutamate (Boc-Glu-OtBu) and N-Boc-1tert-butyl-L-aspartate (Boc-Asp-OtBu) were purchased from NovaBiochem (La¨ufelfingen, Switzerland). All other chemicals used were of an analytical grade and were purchased from Acros (Tilburg, The Netherlands) or Merck (Darmstadt, Germany). Human callus was obtained from chiropodists. Human skin resulting from cosmetic surgery was obtained from a local 1 Abbreviations: Boc-Asp-OtBu, N-Boc-1-tert-butyl-L-aspartate; BocGlu-OtBu, N-Boc-1-tert-butyl-L-glutamate; Ct, concentration times time; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; Fmoc, 9-fluorenylmethoxycarbonyl; GAM, goat-anti-mouse; HETE, 2-[(hydroxyethyl)thio]ethyl; PBS, phosphate buffered saline (0.14 M NaCl, 2.6 mM KCl, 8.1 mM Na2HPO4, and 15 mM KH2PO4, pH 7.4); PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline (20 mM Tris‚HCl, 150 mM NaCl, pH 7.4).

10.1021/tx0100136 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/04/2001

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hospital with consent of the patient and approval of the TNO Medical Ethical Committee. Microtiter plates (polystyrene, high binding) were purchased from Costar Europe Ltd. (Badhoevedorp, The Netherlands). Exposure of Human Callus to [14C]Sulfur Mustard or Unlabeled Sulfur Mustard. To a suspension of human callus (70-100 mg) in 0.9% NaCl (100 µL) was added a solution of 0.1 µM to 10 mM of [14C]sulfur mustard or unlabeled sulfur mustard in 2-propanol (100 µL). The mixture was incubated for 6 h at 37 °C. Isolation of Keratin from Human Callus. Human callus (100 mg) was soaked in Tris‚HCl buffer (5 mL, 20 mM, pH 7.4) overnight. After centrifugation (30 min, 400 rpm) the pellet was stirred in a buffer (5 mL; pH 7.4) containing Tris‚HCl (20 mM) and urea (8 M). After centrifugation (30 min, 400 rpm), the pellet was extracted with a buffer (5 mL; pH 7.4) containing Tris.HCl (20 mM), urea (8 M), and β-mercaptoethanol (0.1 M, ref 17). The crude keratin was purified on a G 75 column (100 × 2 cm) with a buffer (pH 7.6) containing sodium dodecyl sulfate (SDS, 0.5%), Tris‚HCl (10 mM), and dithiothreitol (DTT, 10 mM); flow, 0.25 mL/min. Appropriate fractions were collected and dialyzed against water. The remaining solution was lyophilized. Representative yield: 20 mg of keratin/100 mg of callus. The amino acid composition of the isolated keratin was in reasonable agreement with literature data (18). Quantification of Binding. A suspension of human callus (0.5 g/mL) in 0.9% NaCl/2-propanol (1/1, v/v) was exposed to various concentrations (0.1 µM to 10 mM) of [14C]sulfur mustard for 6 h at 37 °C. The radioactivity in the extracted keratin fractions was measured. Next, the keratin was purified on a Sephadex G-75 column by elution with a Tris buffer (10 mM Tris‚HCl, 10 mM DTT, 0.5% SDS, pH 7.6) to separate it from low molecular weight material. Synthesis of Nr-Fmoc-Nω-HETE-Asparagine and NrFmoc-Nω-HETE-Glutamine. These compounds were synthesized by PyBop-mediated condensation of Boc-Asp-OtBu or BocGlu-OtBu with 2-(2-aminoethylthio)ethanol, followed by acidcatalyzed removal of protecting groups and subsequent introduction of the Fmoc group (19). Solid-Phase Synthesis of Peptides Containing an NωHETE-Glutamine or Nω-HETE-Asparagine Residue. Peptides were synthesized as described elsewhere (19). The peptides with the native glutamic or aspartic acid residue were also synthesized. Immunization of Mice with Synthesized Antigens. Mice were immunized with antigens consisting of one peptide or a mixture of two or three peptides. For each antigen three mice were immunized (ip) with 50 µg of antigen to which spekol was added (5-10 mL/kg). The mice received a second immunization with the same antigens at 4 weeks after the first immunization. Subsequently, a booster with the antigens (volume up to 0.2 mL) was administered 12 weeks after the second immunization. After 3 days one animal from each group was killed with CO2 anesthesia and the blood was collected by heart puncture for a final test of the serum for antibody response. A cell suspension of the spleen of each killed animal was prepared for the production of hybrid cell strains. Blood samples were taken from each mouse at 7 days after the first and second immunization to test the serum for antibody response against keratin isolated from human callus treated with 0, 50, or 100 µM sulfur mustard, in an enzyme-linked immunosorbent assay (ELISA, 5). Production of Hybrid Cell Strains. Hybrid cell strains were produced as described previously (5) with two modifications: at 24 h after fusion, the cells were centrifuged (20 min at 10g) and resuspended in complete RPMI-medium (38 instead of 30 mL); during the whole procedure no feeder layer of macrophages was applied. Immunoassays (ELISA) with the Polyclonal Antisera and Hybridoma-Supernatants. The polyclonal antisera and hybridoma-supernatants were tested in a direct ELISA (5) against keratin isolated from human callus treated with sulfur mustard (0, 50, or 100 µM). The ELISA was performed as

van der Schans et al. follows. Polystyrene “high binding” 96-well microtiter plates were coated with adducted and nonadducted keratin (50 µL of 10 µg/mL in water/well) as described previously (5). Excess binding sites were blocked with PBS containing 1% FCS (fetal calf serum) for 60 min at 37 °C and again washed three times with PBS containing 0.05% Tween 20. The polyclonal antisera and the hybridoma supernatants were diluted 10-1000 times and 5-100 times, respectively, in PBS with 0.05% Tween 20 and 0.1% FCS. Of these dilutions 50 µL was added per well and incubated for 60 min at 37 °C. After washing, the second antibody, goat-anti-mouse-Ig(total)-alkaline phosphatase, diluted 1:1,000 in PBS containing 0.05% Tween 20, 0.5% gelatin, and 5% FCS, was added (50 µL/well) and the plates were incubated for 60 min at 37 °C. The subsequent procedure was the same as described previously (5). After addition of substrate, the plates were incubated at 37 °C for 1 h. Product fluorescence (excitation, 355 nm; emission, 480 nm) was recorded with a Cytofluor II (PerSeptive Biosystems, Framingham, MA). Antibody saturated supernatant of the hybridoma 1H10 was also tested in a competitive ELISA (5). The supernatant was diluted 1:25 in PBS containing 0.05% Tween 20 and 1% fetal calf serum and various concentrations of inhibitors. Ex Vivo Exposure of Human Skin to Sulfur Mustard. Pieces of human skin were exposed to saturated sulfur mustard vapor as described by Mol et al. (20) at 27 °C, which corresponds to a sulfur mustard concentration of 1040 mg m-3, for 10 s, 30 s, or 1 min. In addition, pieces of skin (0.5 × 0.5 cm2) were covered with a solution of sulfur mustard (1 mL of 0, 50, or 100 µM) in 0.14 M NaCl, 2.6 mM KCl, 8.1 mM Na2HPO4, and 15 mM KH2PO4, pH 7.4 (PBS) containing 1% acetonitrile, for 30 min at 27 °C. Preparation of Skin Cryostat Sections. After exposure, a piece of the skin was cut from the central part of the treated area, stretched between microscope slides and, without fixation, stored at -20 °C. For the preparation of cryostat sections, a small piece of skin was embedded in Tissue Tek (O. C. T. compound, Miles Inc., Elkhart, IN). Subsequently, cryostat sections (5 µm thickness) were prepared at -35 °C with a cryostat microtome (2800 Frigocut, Rechert-Jung, Leica, Rijswijk, The Netherlands) on slides precoated with a solution of 3-aminopropyl tri-ethoxysilane (2% in acetone). The cross-sections were stored at room remperature. Immunofluorescence Microscopy. Immunofluorescence microscopy to analyze the formation of sulfur mustard-keratin adducts was carried out on skin cryostat sections. Briefly, the following procedure was applied: fixation with 70% ethanol on aminoalkylsilane-precoated slides; washing with 20 mM Tris HCl, 150 mM NaCl, pH 7.4 (TBS); precoating with TBS + 5% milkpowder (30 min at room temperature); treatment with antibody specific for sulfur mustard-modified keratin (culture supernatants in a 1:1 dilution of up to 32 selected monoclonal antibodies in TBS containing 0.05% Tween 20 and 0.5% gelatin, overnight at 4 °C); treatment with a second antibody, fluorescein isothiocyanate labeled (FITC) “goat-anti-mouse” (GAM-FITC), 75-fold diluted in TBS containing 0.05% Tween 20 and 0.5% gelatin (2 h at 37 °C) and counterstaining with propidium iodide (100 ng/mL, 10 min at room temperature). Twin images were obtained with a laser scanning microscope (LSM-41, Zeiss, Oberkochen, Germany). The fluorescence of the fluoresceine group and of the propidium iodide were measured consecutively to visualize the presence of sulfur mustard-keratin adducts in the skin and the DNA in the nuclei, respectively. Adduct levels were estimated from the brightness of the fluorescence above the stratum corneum.

Results and Discussion Quantitation of Binding and Tentative Identification of Binding Sites. To study the binding of sulfur mustard to keratin, a suspension of human callus was exposed to [14C]sulfur mustard (0.1 µM up to 10 mM) for

Antibodies against Sulfur Mustard Keratin Adducts

Chem. Res. Toxicol., Vol. 15, No. 1, 2002 23 Table 1. Antibody Specificities of Clones Obtained from a Fusion after Immunization with Peptide 1, from a Mixture of Peptides 2 and 3, or of the Three Peptides 1, 2, and 3, Presented in Figure 1

Figure 1. Three synthesized peptides, containing a sulfur mustard adduct on the amide of asparagine or glutamine and which are derived from partial sequences (except the N-terminal glycine and C-terminal lysine) of end domains (head or tail) in human keratin. Peptide 1, Val451-Glu464 of keratin K14; peptide 2, Ile140-Asn154 of keratin K5, this peptide also contains the sequence Ile151-Thr155 in keratin 1; peptide 3, Val459-Asn471 of keratin K14. HETE: 2-[(hydroxyethyl)thio]ethyl.

6 h at 37 °C. The extracted keratin fractions contained 15-22% of the added radioactivity, in each case. Since keratin contains a large number of glutamic and aspartic acid moieties (18), it can be expected that upon exposure to sulfur mustard these moieties are converted into esters of thiodiglycol which can be readily hydrolyzed with alkali. To check this hypothesis, a purified keratin sample, isolated from human callus exposed to [14C]sulfur mustard, was incubated with aqueous NaOH (0.5 M). After chromatography of the mixture on a Sephadex G-75 column, only 20% of total radioactivity coincided with keratin, whereas 80% of total radioactivity eluted as material with low molecular mass. TLC analysis with radiometric detection of the obtained extract (in ethyl acetate) showed that the radioactive component with low molecular mass coeluted with thiodiglycol. These results strongly suggest that most of the adducts formed with keratin are esters of thiodiglycol with glutamic and aspartic acid residues, which are readily hydrolyzed with alkali. Design and Synthesis of Haptens Containing a Glutamine- or Asparagine-Sulfur Mustard Adduct. Since evidence was obtained that glutamic acid and aspartic acid residues in keratins are efficiently alkylated by sulfur mustard, we intended to synthesize partial sequences of the end domains of keratins, containing a sulfur mustard adduct of these amino acids, which can be used as antigens for raising antibodies. These end domains are predominantly located on the surface of the intermediate filaments (21) and are consequently relatively readily accessible to both alkylating agents and to antibodies raised against the adducts of these agents. In Figure 1 the synthesized peptides are presented which are derived from partial sequences of tail or head end domains of the major keratins K5 and K14 from human basal keratinocytes and one of the major keratins from stratum corneum, i.e., K1 (22, 23). Because it can be expected that the thiodiglycol esters are not stable during immunization, we decided to employ the corresponding amides. Antibodies against the Peptides Containing NωHETE-Glutamine or Nω-HETE-Asparagine. Mice were immunized with peptide 1, with a mixture of peptide 2 and 3, or with a mixture of all three peptides presented in Figure 1. Three mice were immunized (ip) with each antigen, and immunization was repeated after 4 weeks and another 12 weeks. At 3 days after the last immunization, the spleen of one animal of each group was selected for three separate fusion experiments carried out simultaneously. With sera of two of the three mice, a modest dose response was observed in a direct ELISA on keratins isolated from human callus exposed to 0, 50, and 100 µM of sulfur mustard. Clones were

clone

peptides used for immunization

2.3D9 1.2B6 1.3C2 3.2G8

1 2+3 2+3 1+2+3

antibody response against keratin exposed to sulfur mustard at a concentration of (fluorescence in arbitrary units) 0 µM 50 µM 100 µM 300 1400 300 300

2300 3900 1500 1400

3400 4300 2700 2400

Supernatants of cultures in a 1:5 dilution were assayed in a direct ELISA on keratin from human callus treated with 0, 50, or 100 µM sulfur mustard. Fluorescence (in arbitrary units, with an estimated error of about 20%) is presented as a measure for antibody binding.

selected in a direct ELISA on their specificity for keratins isolated from stratum corneum in human callus exposed to 0, 50, and 100 µM of sulfur mustard. The results of the four clones giving the best response in the direct ELISA are presented in Table 1. Nine clones were selected for further characterization and subcloning. So far, 32 monoclonal clones, all originating from clone 1.3C2, have been selected of which antibodies showed specificity against keratins isolated from human callus treated with 50 µM sulfur mustard. Antibodies of one clone, 1H10, were characterized further. Cross-reactivity toward the three peptides containing adduct and the corresponding nascent peptides was assessed in a competitive ELISA. With 1.4 nmol/well of peptide 2, 50% inhibition was observed, whereas with the corresponding nascent peptide, 6.5 nmol/well was required to achieve the same extent of inhibition. Peptide 1, which was not used for the immunization resulting in clone 1H10, also showed some cross-reactivity (50% inhibition with 7.6 nmol/well). All other peptides tested, including peptide 3 used for the immunization, did not show any cross-reactivity in the concentration range tested, i.e., 50% inhibition is not achieved at amounts less than 20 nmol/well. Immunofluorescence Microscopy on Human Skin Exposed to Sulfur Mustard. The selected 32 monoclonal antibodies were tested in an immunofluorescence experiment with human skin exposed to 0, 50, and 100 µM sulfur mustard (30 min at 27 °C) or to saturated sulfur mustard vapor (1 min at 27 °C; Ct ≈1040 mg min m-3). Cross-sections of the sulfur-mustard exposed skin samples were prepared and processed with the antibodies. The binding of the antibodies to the stratum corneum was detected by binding of a second antibody conjugated with FITC, which was directed against the first antibody. A large number of monoclonal antibodies (18 out of 32) showed some specificity to sulfur mustard adducts in the stratum corneum of sulfur mustard exposed skin as indicated by the higher fluorescence intensity in comparison with the background intensity found in nonexposed skin. In general, the vapor-exposed skin samples showed the highest response. One of the monoclonal antibodies, i.e., 1H10, was tested at higher dilutions in an attempt to lower the a-specific binding of antibodies to nonexposed skin and to increase the sensitivity of this immunofluorescence procedure in the stratum corneum and possibly in other keratincontaining parts of the epidermal layer. Exposure of

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mg min m-3), 30 s, or 1 min was demonstrated by using a 50-fold diluted supernatant. Photographs of skin crosssections are given in Figure 2. Sulfur mustard adducts are clearly detected in the stratum corneum whereas DNA counterstaining visualizes the presence of DNA in the nucleated cells. Hardly any fluorescence due to antibody treatment is measured over the nonexposed skin cross-section at the conditions used. Fluorescence from the basal cells, i.e., the layer of cells between the epidermis and the dermis, is not observed, although antibody 1H10 was raised against adducts present in end domains of keratins K5, K14, and K1, while K5 and K14 are two of the most important keratins in basal cells. Presumably, this is due to the low levels of adducts in these cells relative to that in stratum corneum which is primarily exposed. It cannot be excluded that other monoclonal antibodies obtained might be suitable for adduct detection in the basal cells. In conclusion, monoclonal antibodies have been obtained that recognize sulfur mustard keratin adducts in the stratum corneum of human skin. It should be emphasized that the antibodies, raised against sulfur mustard adducts to keratin, were directly applied to the human skin cross-sections without preconditioning of the samples. This opens the way for development of a detection kit that can be applied directly to skin of humans who have supposedly been exposed to sulfur mustard. In this way a simple, fast, and noninvasive assay for diagnosis of skin exposure to sulfur mustard will become available. To the best of our knowledge, this is the first example of immunochemical detection of adduct formation of toxic chemicals with skin proteins. A similar approach can be followed for skin exposure to environmental pollutants. In addition, the antibodies presented may be a useful tool in histochemical studies on sulfur mustard intoxication, protectants, and medical countermeasures.

Acknowledgment. This work was supported in part by the U.S. Army Medical Research and Materiel Command under Cooperative Agreement DAMD17-97-2-7002 and by the Directorate of Military Medical Services of the Ministry of Defense, The Netherlands. The authors are grateful to Dr. Marijke Mol for the stimulating discussions about keratin structures. Supporting Information Available: NMR data of NRFmoc-Nω-HETE-glutamine and NR-Fmoc-Nω-HETE-asparagine. Table S1, Binding of [14C]sulfur mustard to keratin upon treatment of human callus suspended in 0.9% NaCl (1 g/mL) with various concentrations of the agent in an equal volume of 2-propanol. Figure S1, Chemical structures of the thiodiglycol esters and the corresponding amides. This material is available free of charge via the Internet at http://pubs.acs.org.

References Figure 2. Immunofluorescence microscopy of a cross-section of human skin exposed to saturated sulfur mustard vapor (1 min at 27 °C; Ct ≈1040 mg min m-3; (A) or sulfur mustard (100 µM, 30 min at 27 °C); (B) and of unexposed skin (C), using monoclonal antibody 1H10, directed against sulfur mustard adducts to human keratin, in a 1/50 dilution. The photographs are composed from an image obtained for FITC fluorescence (mainly emanating from the stratum corneum; green) and from an image obtained for propidium iodide fluorescence representing DNA (red) in the same cross-section.

human skin to a solution of 100 µM of sulfur mustard or to saturated sulfur mustard vapor during 10 s (Ct ≈175

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Chem. Res. Toxicol., Vol. 15, No. 1, 2002 25 DAMD17-92-V-2005, NTIS AD-B191 190/8. (14) Lin, R. C., Shahidi, S., Kelly, T. J., Lumeng, C., and Lumeng, L. (1993) Measurement of hemoglobin-acetaldehyde adduct in alcoholic patients. Alcohol Clin. Exp. Res. 17, 669-674. (15) Wraith, M. J., Watson, W. P., Eadsforth, C. V., Van Sittert, N. J., and Wright, A. S. (1988) An immunoassay for monitoring human exposure to ethylene oxide. In Methods for detecting DNA damaging agents in humans: applications in cancer epidemiology and prevention (Bartsch, H., Hemminki, K., and O’Neill, I. K., Eds) Proceedings of a symposium held in Espoo, Finland, 2-4 September 1987, pp 271-274, IARC Scientific Publication No 89, Lyon. (16) Fidder, A., Noort, D., and Benschop, H. P. (1999) A convenient synthesis of [14C]1,1′-thiobis(2-chloroethane), [14C]sulfur mustard. J. Lab. Comput. Radiopharm. 42, 261-266. (17) Sun, T. T., and Green, H. (1978) Keratin filaments of cultured human epidermal cells. J. Biol. Chem. 253, 2053-2060. (18) Fuchs, E., and Green, H. (1978) The expression of keratin genes in epidermis and cultured epidermal cells. Cell 15, 887-897. (19) Noort, D., Jacobs, E. H., Fidder, A., De Jong, L. P. A., Drijfhout, J. W., and Benschop, H. P. (1995) Solid-phase synthesis of peptide haptens containing a cysteine-sulfur mustard adduct. Int. J. Pept. Protein Res. 45, 497-500. (20) Mol, M. A. E., De Vries, R., and Kluivers, A. W. (1991) Effects of nicotinamide on biochemical changes and microblistering induced by sulfur mustard in human skin organ cultures. Toxicol. Appl. Pharmacol. 107, 439-449. (21) Albers, K., and Fuchs, E. (1992) The molecular biology of intermediate filament proteins. Int. Rev. Cytol. 134, 243-279. (22) Morley, S. M., and Lane, E. B. (1994) The keratinocyte cytoskeleton. In The keratinocyte Handbook (Leigh, I., Lane, B., and Watt, F., Eds.) pp 273-321, Cambridge University Press, Cambridge. (23) Eichner, R., and Kahn, M. (1990) Differential extraction of keratin subunits and filaments from normal human epidermis. J. Cell Biol. 110, 1149-1168.

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