HYBRIDOMA Volume 11, Number 3, 1992 Mary Ann Liebert, Inc., Publishers

Characterisation of Eight Monoclonal Antibodies to Involucrin DAVID L.

HUDSON,1

KATHERINE L. WEILAND,2 THOMAS P. MARCIA SIMON,3 and FIONA M. WATT1

DOOLEY,2

'Keratinocyte Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London 2Upjohn Company, 7235-25-12, Kalamazoo, MI 49001, USA 3Department of Cellular and Molecular Physiology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA

ABSTRACT Involucrin is a precursor of the insoluble protein envelope that is assembled in the outermost layers of the epidermis. The coding sequence of the protein contains a number of short tandem repeats that have been greatly altered during mammalian evolution. We have characterised eight mouse monoclonal antibodies raised against human involucrin, all of which bind to the protein in immunoprecipitation, immunoblot and immunohistochemical preparations. Each antibody was screened for cross-reactivity with gorilla, owl monkey, dog and pig involucrin and with a fragment of the human protein, expressed in Xgt 11, that includes the entire early region of the modern segment of repeats. Three antibodies

recognised involucrin in all of these assays. Four antibodies recognised primate involucrins and the Xgt 11 fragment. One antibody, which showed cross-reactivity with lower molecular weight proteins, only recognised primate involucrins and therefore bound outside the early region of the modern segment. Since the antibodies can be used to detect involucrin both biochemically and histologically, in a range of species, they will have applications in further studies of the expression, function and evolution of the protein. INTRODUCTION Involucrin is a soluble cytoplasmic protein precursor of the epidermal cornified that becomes cross-linked by transglutaminase during envelope assembly (1-3). Involucrin is expressed in a range of stratified squamous epithelia, including the cornea, which lacks a distinct cornified layer (4). The protein is a useful marker of terminal differentiation: in normal epidermis it is first expressed in the upper spinous layers, and in keratinocyte cultures it is expressed by all cells that have left the basal layer (1,4). In pathological conditions, involucrin expression is altered: in psoriasis and other benign epidermal hyperplasias involucrin expression begins closer to the basal layer than normal (5-10); expression Is abnormal in squamous cell carcinomas (11-15) and premalignant lesions (16,17), and is reduced in severe dysplasias of the larynx and cervix (18-21). Involucrin genes of humans (22-24) and a range of anthropoid primates (25-30), prosimian and nonprimate mammals (31,32), and tarsioids (33) have been cloned and sequenced (Fig. 1). The involucrin genes contain two distinct sites which have expanded and contain tandem repeats. Nonprimate mammals and prosimian primates contain repeats at a site close to the 5' end of the coding region. Anthropoid primates contain repeats at a site M close to the 3' end of the gene and have deleted repeats at (31,34). Tarsioids contain repeats at and M (33). The human protein contains a modern segment of 38-41 tandem repeats of a 10 amino acid sequence at site M. The modern segment can be subdivided into early, middle

envelope

367

DOG INVOLUCRIN

HUMAN INVOLUCRIN

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154

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M

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78

540

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middle

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I—clone 1-3 -J FIGURE 1

The structure of human and dog involucrin is shown with each segment delineated by codon numbers. The region formerly designated as the 5' ancestral segment has been divided into an A and region separated by those codons giving rise to the modern segment of the prosimian primates and subprimates ( ). The region formerly designated as the 3' ancestral and C are separated by those codons segment is herein called C; forming the modern segment of the anthropoid primates (M). The position of the clone 1-3 fragment of human involucrin is shown.

and late regions by comparisons with the other anthropoid involucrin sequences. The early region has the highest % of sequence identity in the modern segment of repeats. The repeat pattern of the middle region is conserved only in the hominoids, suggesting that it formed after the divergence of the hominoids from the ceboids (29,30). The late region is species-specific and is the site of polymorphisms (23,24,27). In this paper we describe a series of eight mouse monoclonal antibodies raised against human involucrin. We show that the antibodies detect involucrin in a range of primate and nonprimate mammals. In addition we demonstrate that the antibodies can be used to detect involucrin by a variety of biochemical and cytochemical techniques. The antibodies will therefore have widespread applications in studies of the expression, function and evolution of the protein.

MATERIALS AND METHODS Cell culture

Human

keratinocytes (strains a, z, passages 3-12) were isolated from newborn epidermis. Gorilla keratinocytes (gorilla K) (27) were isolated from a vaginal biopsy. Owl monkey keratinocytes were derived from an oesophageal biopsy (35). Dog keratinocytes (strain gsd, passages 1-4) were isolated from skin removed from the edge of an incision in the ventral midline made during a spaying operation on an adult female German shepherd dog. All keratinocytes were isolated essentially as described by Rheinwald and Green (36). Keratinocytes were cultured in the presence of a feeder layer of mitomycin Cforeskin

treated 3T3 clone J2 cells (36,37). The culture medium consisted of 1 part Ham's F12 medium and 3 parts Dulbecco's MEM supplemented with 1.8 104M adenine, 10% foetal calf serum, O^g/ml hydrocortisone, 5µg/ml insulin, 10"10M cholera toxin and 10ng/ml epidermal growth factor (prepared by C. George-Nascimento and generously donated by Chiron Group, Emeryville, CA) (38). Keratinocytes cultured from pig skin were a generous gift of Harshad Navsaria (Royal London Hospital). Human involucrin

purification

One week post Involucrin was purified from cultured human keratinocytes. confluent keratinocytes were washed and released from the culture flask with phosphate buffered saline (PBS) containing 20mM EDTA. The epithelial sheet was then dispersed and the cells disrupted with a Bronsen sonifier at a setting of 6 for 3x30 sec. Particulate material was removed by centrifugation at 100,000 g for 30 min at 10°C. The supernatant (cytosol) was made 10% in glycerol and 62.5mM in Tris HCI, pH 6.8, and heated for 10 min at 100°C. Denatured proteins were removed by centrifugation at 15,000 g for 15 min. Involucrin in the supernatant was then electrophoretically separated from any contaminating proteins in an acrylamide gel and collected from the gel by electroelution according to the method of Etoh et al. (3). Immunisation and selection of

The antibodies

were

positive

prepared

clones

and selected

368

by

S.

Young (ICRF).

Female Balb/c mice

given three intraperitoneal injections, each of 15µg pure human involucrin, at intervals of three weeks. Complete Freund's adjuvant was used for the first injection; incomplete adjuvant was used for the second and third injections. An intravenous boost of 20µg involucrin in saline was given one week after the third injection. The spleen was removed three days after boosting. Spleen cells were fused with X-63 myeloma cells using polyethylene glycol 4000 (Merck) (39) and hybrids were selected in HAT medium. Culture supernatants were assayed for antibodies to involucrin by an ELISA assay in 96-well plates (Dynatech) coated with pure human involucrin. Wells containing antibody-producing cells were cloned twice by limiting dilution.

were

Immunofluorescence

unfixed frozen sections of newborn human foreskin were air-dried before Paraffin sections of human keratinocytes grafted subcutaneously onto the backs of nude mice, using the procedure of Barrandon et al. (40), were dewaxed through a graded series of ethanols before staining. Cultured keratinocytes were fixed for 8 -10min with 3.7% formaldehyde in PBS at room temperature and then permeabilised in absolute methanol for 5min on ice. Preparations were incubated with monoclonal antibodies in the form of culture supernatant or with rabbit antiserum to involucrin (41) for 45min at room temperature, rinsed thoroughly in PBS, then incubated with fluorescein-conjugated rabbit anti-mouse IgG or IgM (ICN) as before. After further washing in PBS, specimens were mounted in Gelvatol (Monsanto) and examined with a Zeiss Axiophot microscope.

5µ

staining.

Immunoprecipitation Keratinocytes were incubated overnight in culture medium containing 10µ / 35S-methionine (> 1000 Ci/mmol; Amersham). The feeders were removed by treatment with EDTA, then the keratinocytes were rinsed once with PBS and scraped into PBS containing 10mM EDTA. The extract was sonicated, then microfuged (10,000g, 5min) and the pellet discarded. Aliquots containing 30,000cpm of TCA-precipitable material were incubated with 100µ monoclonal antibody culture supernatant or control medium or with 5µ rabbit anti-involucrin or preimmune serum at 4°C for 2.5hr. 5µ rabbit anti-mouse IgG or IgM was added and incubated at 4°C for 1 hr. 40µ 50% (w/v) protein A-Sepharose (Pharmacia) in PBS containing 0.1% SDS, 0.5% Triton X-100 was added and mixed endover-end at 4°C for 1hr. The beads were washed extensively, as described by Adams and Watt (42), then resuspended in SDS-PAGE sample buffer and boiled for 3min. The protein A-Sepharose was removed by centrifugation (10,000g,