Materials and Methods

Clinical features

The proband was a newborn male from a con-sanguineous union of unaffected Emirate Arabian parents. Diagnosis of PA-JEB was established on the basis of clinical observations. At birth, the patient presented aplasia cutis congenita and microtia. Aplasia involved the legs, feet, nose, the right jaw, and both pinnae (Figure 1). Blistering of the skin and mucosa consequent to friction occurred shortly after birth. PA was detected at radiology, and a pyloric ablation was performed at the age of 4 d. At day 10 of age, aspiration pneumonia, consequent to nasojejunal feeding, caused deterioration of the general conditions. Widespread skin and oral mucosa blistering concomitant with thrombocytopenia and neutropenia resulted in septicemia and death 26 d after birth. Ultrastructural examination of PA-JEB skin biopsies revealed that the cleft plane of the blisters localized within the lamina lucida of the dermal–epidermal junction (not shown).

Figure 1.

Clinical features of the newborn with PA-JEB. Extensive blistering and erosions of the integument are the hallmarks of the condition. Note the malformation of the external ears and areas with absence of epithelium.

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Immunohistochemistry

Immunohistochemical studies were perfor-med on 4 m thick sections from skin biopsies (^{Verrando et al, 1991}). Immunofluorescence of cultured keratinocytes was performed on cell cultures grown for 36 h on glass coverslips, fixed using the aqueous mounting medium Gel/Mount (Biomeda, Foster City, California) and then treated as described (^{Gache et al, 1996}). Tissue sections and cell cultures were analyzed under an epifluorescence Zeiss Axiophot microscope.

Cell cultures

Epidermis from skin biopsies was dissociated in 0.25% trypsin–ethylenediamine tetraacetic acid at 37°C and plated on a feeder of irradiated mouse 3T3-J2 cells (^{Rheinwald and Green, 1975}). Keratinocytes were grown in a 3:1 mixture of Dulbecco minimal Eagle's medium/Ham's F12 medium (Gibco BRL, Life Technologies, Cergy Pontoise, France) supplemented with serum (Hyclone, Perbio Sciences, Bezons, France) and growth factors (^{Rheinwald and Green, 1975}). HaCaT and epithelial 293 cells were grown in Dulbecco minimal Eagle's medium containing 10% calf serum (Gibco BRL, Life Technologies). Subconfluent keratinocyte cultures were transfected using 3 g of plasmid DNA and the polyca-tionic lipid Fugene (Stratagene, Amsterdam, the Netherlands), as detailed elsewhere (^{Gagnoux-Palacios et al, 2001}). Epithelial 293 cells were transfected using 3 g of DNA and the calcium-phosphate technique (^{Chen and Okayama, 1987}).

Antibodies

The antibodies used were: anti-4 integrin monoclonal antibody (MoAb) 439–9B (^{Kennel et al, 1990}), MoAb 3E1 (Gibco BRL, Life Technologies), and polyclonal antibody (PoAb) anti-4 integrin (^{Niessen et al, 1994}); anti-6 integrin MoAb GoH3 (^{Sonnenberg et al, 1987}), MoAb 1A10 (^{Hogervorst et al, 1993a}), MoAb 4E9G8 (Immunotech, Marseille, France), MoAb SC-6597 (Santa-Cruz, Tebu, Le Perray en Yvelines, France); the anti-plectin HD121 (^{Hieda et al, 1992}) and the anti-3 integrin MoAb VM2 (Novocastra Laboratories, Tebu). Secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Dako S.A., Trappes, France) and Texas red-conjugated goat anti-mouse immunoglobulin (Dako S.A.).

Plasmids

Plasmids p6WT and p4WT are derived from vector pRC-CMV (Invitrogen, Life Technologies, Cergy Pontoise, France) and contain the full-length 6 and 4 integrin cDNA, respectively (^{Hogervorst et al, 1993b;Niessen et al, 1994}). Plasmid p6M, encoding the mutated S47L 6 integrin, was gene-rated by polymerase chain reaction (PCR) amplification using the QuickChange site-directed mutagenesis kit (Stratagene), primers (left) 5'-AGCCTCTTCGGCTTCTTGCTGGCCATGCAC-3' and (right) 5'-G CCAGTGCATGGCCAGCAAGAAGCCGAAGA-3' and plasmid p6WT as a template. Plasmid pGFP encoding the Green Fluorescent Protein was purchased from Invitrogen, Life Technologies.

Immunoprecipitation and western blot analysis

Exponentially growing keratinocytes were maintained for 24 h in methionine- and cysteine-deficient culture medium (Gibco BRL, Life Technologies) supplemented with 110 Ci per mL ³⁵S-methionine and ³⁵S-cysteine, 3% fetal calf serum (Invitrogen, Life Technologies), and 2 mM glutamine (Invitrogen, Life Technologies). Samples of the cell lysates (10⁷ cpm) were cleared using rabbit anti-mouse IgG (Sigma, St Louis, Missouri) conjugated to protein A-Sepharose (Amersham Pharmacia Biotech, Orsay, France), and then processed as previously described (^{Gagnoux-Palacios et al, 1996}). For western analysis, 48 h after transfection, 293 cells were treated with either 50 or 150 M chloroquine (Sigma), 0.5 or 20 M MG132 (Calbiochem, Meudon, France), or with 10 M lactacystin (Calbiochem) during 6 h. PA-JEB keratinocytes were treated for 24 h with either 5 or 10 M lactacystin or either 50 or 150 M chloroquine. Cells were lyzed using 0.5% sodium deoxycholate, 1% nonidet P-40, and 20 mM Tris–HCl pH 6.8, in the presence of proteases inhibitors (^{Gagnoux-Palacios et al, 1996}). The cell extracts were fractionated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions. Immunoblotting was performed according to standard procedure and the hybridization bands were visualized using the ECL detection system (Amersham Pharmacia Biotech).

Northern analysis

Total RNA was purified from cultured keratinocytes using the RNable extraction kit (Eurobio, Les Ullis, France) and 10 g were fractionated in a denaturing 1% agarose-formaldehyde gel (^{Chavanas et al, 1996}). ³²P-labeled random-primed cDNA probes ph4 (^{Hogervorst et al, 1990}), ph6 (^{Hogervorst et al, 1991}), and GAPDH were used to detect the 6, 4 integrin, and GAPDH RNA transcripts, respectively.

Screening for genetic mutations

Twenty micrograms of total RNA purified from the PA-JEB keratinocytes were reverse transcribed in a volume of 50 L using avian myeloblastosis virus reverse transcriptase (Promega, Charbonnières, France). One microliter of the reaction mixture was used to amplify overlapping 500 bp cDNA fragments spanning the open reading frame of integrins 4 (5.6 kb) and 6 (6.1 kb) RNA transcripts. The amplified cDNA fragments were directly sequenced using an ABI Prism 310 automated sequencing system (Applied Biosystems, Foster City, California). Genomic DNA was purified from peripheral blood following standard techniques and used (100 ng) as a template for PCR amplification and direct sequencing.

In vitro translation

One microgram of plasmid DNA was incubated with 40 L of the coupled Transcription/Translation TnT Quick Mater Mix Systems (Promega) and 20 Ci of ³⁵S methionine (Amersham Pharmacia Biotech) for 90 min at 30°C. Two microliters of the incubation mixture were analyzed by 7.5% SDS–PAGE. The translation products were visualized by autoradiography of the polyacrylamide gel.

Results

Expression of the 6 and 4 integrin in the proband's keratinocytes

Frozen sections from the proband's skin in nonlesional epidermis were performed using antibodies reacting to the major basement membrane components. Staining of the epidermal basement membrane with MoAb GoH3 directed against 6 integrin was negative, and was strongly reduced with MoAb 439-9B specific to the 4 integrin subunit (Figure 2a,c). Staining of the dermal–epidermal junction was also negative with MoAb 135-13C specific to the N-terminal region of 6 integrin (not shown). In contrast, MoAb HD121 to plectin reacted with the dermal–epidermal junction and the staining pattern was comparable with that noted in healthy controls (Figure 2ae). A similar continuous staining of the dermal–epidermal junction was obtained with antibodies specific to the other hemidesmosome components, the 230 kDa bullous pemphigoid antigen-1 (BPAG1) and collagen type XVII (data not shown). Strong reactivity was also observed with antibodies specific to the basement membrane components, including laminin-5 and types IV and VII collagens (data not shown). In contrast, labeling with the anti-3 integrin antibody MoAb VM2 revealed relocalization of 31 integrin at the dermal–epidermal junction (Figure 2ag). Relocalization of 3 integrin was previously observed in other JEB patients, and is thought to compensate absence of 64 integrin in cell adhesion (^{Niessen et al, 1996;Gagnoux-Palacios et al, 1997;Ruzzi et al, 1997}). These observations indicated that 6 or 4 integrin were the candidate genes for PA-JEB in this proband.

Figure 2.

Expression of 64 integrin in the proband's keratinocytes. (A) Immunofluorescence analysis of noninvolved skin of the PA-JEB patient (a,c,e,g) and a healthy control (b,d,f,h) treated with MoAb GoH3 to 6 integrin (a,b), MoAb 439-9B to 4 integrin (c,d), MoAb HD121 to plectin (e,f) and MoAb VM2 to 3 integrin (g,h). Compared with the control, the patient's skin was not reactive to MoAb GoH3 and was weakly stained by MoAb 439-9B. Immunostaining with HD121 is comparable in the patient's and the control's skin. In the proband, labeling with VM2 is relocalized at the ventral side of the basal keratinocytes. Arrows point out the dermal–epidermal junction. (B) Immunoprecipitation analysis of keratinocytes extracts obtained from cultures of wild-type (C) and PA-JEB keratinocytes (P) using MoAb GoH3 to 6 integrin and MoAb 3E1 to 4 integrin. Expression of 4 integrin is detected in the patient's keratinocytes, whereas 6 integrin is absent. The apparent molecular mass markers are indicated on the left (kDa).

Full figure and legend (145K)

To confirm that loss of immunoreactivity to 64 integrin correlated with an altered expression of this cellular receptor, immunoprecipitation analysis was performed on cell extracts obtained from the proband's keratinocytes. MoAb GoH3 immunoprecipitated the 64 integrin complex from wild-type human keratinocytes, but not from the proband's cells (Figure 2b). Consistent with this result, MoAb 3E1, specific to the 4 integrin subunit, immunoprecipitated the heterodimer 64 from the control cells, but only the 4 integrin subunit from the PA-JEB keratinocytes (Figure 2b). Altogether, these results and the immunofluorescence studies suggest that in this patient 6 integrin is not expressed, and expression of 4 integrin is drastically reduced. Decreased expression of the integrin partner of a mutated integrin chain has been reported both in PA-JEB patients and mice with null mutations in either 6 or 4 subunits (^{Vidal et al, 1995;Dowling et al, 1996;Georges-Labouesse et al, 1996;van der Neut et al, 1996;Ruzzi et al, 1997}).

Identification of mutation S47L in the 6 cDNA

In a pathologic context, absence of a mutated polypeptide is usually caused by enhanced decay of the corresponding aberrant mRNA consequent to the presence of a premature termination codon generated by a genetic mutation. To assess this possibility, we analyzed the expression level of the RNA transcripts encoding the 64 integrin in the proband's keratinocytes. Total RNA isolated from primary cultures of the proband's keratinocytes was analyzed by northern blot using a ³²P-labeled cDNA probes specific to the integrin 6 and 4 subunits. The steady-state level of 6 or 4 subunit mRNA in the PA-JEB keratinocytes was found to be comparable with that of the control wild-type keratinocytes (Figure 3a). We therefore concluded that absence of the 6 integrin in the patient's keratinocytes is not associated with a transcriptional defect or enhanced decay of 6 mRNA.

Figure 3.

(A) Northern analysis of total RNA purified from control (C) and PA-JEB keratinocytes (P) using ³²P-labeled cDNA probes specific to 6 integrin, 4 integrin, and GAPDH. In the control and patient's keratinocytes the steady-state level of the 6 and 4 mRNA transcripts is comparable. (B) Identification of the sequence variation 286 C-to-T. Direct nucleotide sequencing of the PCR amplified fragments of exon 1 of the ITAG6 gene detected a C-to-T transition at position 286 homozygous in the proband (P) and heterozygous in the proband's parents. Father (F), unrelated control (C). (C) Pedigree of the nuclear PA-JEB family. Arab numbers identify the 10 family members genotyped in this study. The proband (filled symbol), carriers of the S47L mutation (half filled symbols) and wild-type members (empty symbols) are indicated. (D) Schematic representation of the -propeller domain of integrins. Position of the S47L mutation in the first -strand of repeat 1 (W1) is indicated (star).

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To verify whether absence of the 6 integrin polypeptide in the patient's skin is associated with the presence of a genetic mutation in the gene ITGA6, total RNA purified from cultured keratinocytes of the proband was amplified by reverse transcription–PCR. Direct sequencing of the amplified PCR products detected a homozygous base pair substitution that changes the cytosine at position 286 into a thymidine (286 C-to-T) (Figure 3b). Direct sequencing of exon 1 of gene ITGA6 amplified by PCR using genomic DNA of the proband and the proband's parents as templates demonstrated homozygosity of the patient for the mutation 286 C-to-T and heterozygosity of the healthy parents (Figure 3b). The Mendelian inheritance of the mutation was assessed by genotyping of the different members of the PA-JEB family (Figure 3c). Variation 286 C-to-T was not detected in alleles in a cohort of 100 healthy unrelated individuals (data not shown). This base pair transition substitutes the codon for serine 47 with a codon for a leucine residue (S47L) and results in a mis-sense mutation localized within a stretch of four amino acids highly conserved in integrins (Figure 4). Specifically, the S47L mutation localizes within the first anti-parallel -strand of the first repeat of the -propeller of 6 integrin (Figure 3d). No additional nucleotide sequence variation was detected in the coding sequence of 6 integrin (not shown).

Figure 4.

Nucleotide sequence homology of the 5' cDNA sequence in integrin chains. Alignment of human integrins a6, a3, a2b, a5, and a4 sequences for the first N-terminal repeat of the b-propeller was made using the ClustalW alignment software (^{Thompson et al, 1994}). Conserved amino acids are framed in black, semiconserved amino acids are boxed in gray and the nonconserved amino acids are indicated by light boxes. Assignment of the first b-strand within the first repeat in integrin a4 (underlined) is according to^{Springer (1997)}. GenBank accession numbers: NP000201 (6 integrin); NP002195 (3 integrin); A34269 (2b integrin); P08648 (5 integrin); P13612 (4 integrin).

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Mutation S47L hampers expression of 6 integrin

To demonstrate the causative role of the mis-sense S47L mutation in the absence of 6 integrin expression, we cotransfected the proband's keratinocytes using a eukaryotic vector expressing either the full-length wild-type 6 cDNA (p6WT) or a mutated counterpart carrying transition 286 C-to-T (p6M) with the GFP expression vector pGFP. Analysis of the transfected keratinocytes by immunofluorescence staining using MoAb GoH3 revealed lack of expression of the mutated recombinant 6 integrin and efficient expression of the wild-type 6 polypeptide (Figure 5ab,c). The keratinocytes expressing the wild-type 6 integrin also restored expression of 4 integrin, which conversely, was not detected in the PA-JEB keratinocytes transfected with the mutant 6 cDNA (Figure 5bb,c). These results demonstrate the negative effect of the S47L mutation on expression of 64 integrin.

Figure 5.

(A,B) Immunofluorescence analysis of the proband's keratinocytes cotransfected with expression vector pGFP encoding the GFP reporter gene, and either vector p6WT expressing the wild-type 6 integrin or vector p6M expressing the mutant S47L 6. (A) PA-JEB keratinocytes untransfected (a), cotransfected with vectors pGFP and p6WT (b) or pGFP and p6M (c) were stained with MoAb GoH3 to 6 integrin. Transfection with vector p6WT results in expression of 6 integrin, whereas transfection of p6M fails to express 6 polypeptides. (B) PA-JEB keratinocytes untransfected (a) or transfected with vector pGFP either alone (d) or with vectors p6WT (b,e) and p6M (c,f) were stained with the anti-integrin 4 MoAb 3E1 (a–c). Among the transfected PA-JEB cells expressing GFP (d–f), only keratinocytes transfected with vector p6WT expressed 4 integrin (b), whereas those transfected with p6M did not react with MoAb 3E1 (c). (C) In vitro translation assay. Plasmids p6WT, p6M, and p4WT were used as templates in a coupled transcription/translation system in the presence of radioactive ³⁵S methionine. Analysis of the translated polypeptides by 7.5% SDS–PAGE shows synthesis of comparable amounts of wild-type and mutant S47L 6 polypeptides. Negative control is assay with no DNA central (C). The apparent molecular mass markers are indicated on the left (kDa).

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To assess whether lack of expression of the mutant 6 integrin was due to accelerated degradation of the aberrant neo-synthesized polypeptide or to a reduced translation rate of the mutant 6 RNA messenger, vectors p6M and p6WT were used as templates for an in vitro translation assay in the presence of radioactive ³⁵S-labeled methionine. Analysis of the translated polypeptides by 7.5% SDS–PAGE showed that both the wild-type and the mutated cDNA templates allow synthesis of a comparable amount of 6 polypeptide exhibiting the apparent molecular mass (140 kDa) of the precursor 6 integrin (Figure 5c). We then concluded that the mis-sense S47L mutation does not interfere with translation of the abnormal 6 mRNA, but rather affects the stability of the mutated polypeptide.

Mutation S47L causes high instability of 6 integrin polypeptide

To verify the idea that the S47L mutation triggers instability of the neo-synthesized 6 integrin in vivo, we followed the fate of the mutated 6 integrin using an overexpressing eukaryotic system. Epithelial 293 cells were cotransfected with vector p4WT expressing the wild-type 4 integrin and either plasmid p6M or plasmid p6WT. Forty-eight hours after transfection, western blot analysis of the cellular extracts obta-ined from the transfected cells and separated on a 7.5% SDS–PAGE was performed using MoAb 4E9G8 directed against the COOH-terminal domain of 6 integrin that detects the proteolytically uncleaved 6 precursor and the light chain of the proteolytically cleaved 6 integrin (Figure 6a). In 293 cells expressing plasmid p6WT, a strong hybridization signal was observed at the apparent molecular mass of 25 kDa, which corresponds to the migration position of the 6 light chain. A signal corresponding to the migration mass (140 kDa) of the 6 chain precursor was also detectable (Figure 6a, lane 7). In contrast, in 293 cells expressing the mutant S47L 6 integrin, only a faint hybridization double band with an apparent mass slightly inferior to the 6 integrin precursor was detected (Figure 6a, lane 10). These results confirm that the presence of the mis-sense S47L mutation dramatically reduces the expression level of 6 integrin. To assess whether the decreased expression level of the mutated S47L 6 integrin correlates with instability of the neo-synthesized 6 polypeptide, the transfected 293 cells were exposed to increasing concentrations of chloroquine, an inhibitor of the lysosomal proteolytic pathway. In the presence of both 50 and 150 M chloroquine, 293 cell cultures transfected with plasmid p6WT expressed amounts of 6 integrin comparable with those found in untreated 293 cells (Figure 6a, lanes 8 and 9). The proteolytic cleavage of the 6 integrin precursor appeared to be affected, however, as judged from the intensity of the 140 and 25 kDa hybridization bands in treated and untreated cells. The 293 cells transfected with plasmid p6M expressed reduced amounts of the mutant S47L 6 integrin, and only the precursor form was detectable. The expression level of the mutated 6 polypeptide was increased in the presence of chloroquine (Figure 6a, lanes 11 and 12) compared with the untreated counterpart (Figure 6a, lane 10). Western blot analysis of 293 cells transfected with vector p4WT using an anti-4 integrin PoAb showed that chloro-quine had no appreciable effect on the expression level of 4 integrin (Figure 6a, lanes 4–12). The hybridization signal was more intense in the presence of wild-type 6 integrin, however, due to 4 integrin interaction with 6 integrin and formation of stable 64 heterodimers (Figure 6a, lanes 7–9). Altogether, these results indicate that the S47L mutation triggers instability of 6 integrin, which is at least partially mediated by the lysosomal degradation pathway.

Figure 6.

Western analysis of epithelial cell cultures expressing the mutant S47L 6 integrin after treatment with protease inhibitors. (A) Untransfected (NT) 293 epithelial cells (lanes 1–3), transfected with vector p4WT (lanes 4–6), or cotransfected with vectors p4WT and p6WT (lanes 7–9), or vectors p4WT and p6M (lanes 10–12) were treated with 50 M (lanes 2, 5, 8, 11) or 150 M (lanes 3, 6, 9, 12) chloroquine. Untreated cells: lanes 1, 4, 7, 10. Cells extracts (40 g) were analyzed by 7.5% SDS–PAGE and immunoblotting using MoAb 4E9G8 specific to 6 integrin C-terminal domain or PoAb anti-4 integrin. The migration bands identifying the 6 precursor (140 kDa), the 6 light chain polypeptide (25 kDa) and 4 integrin (200 kDa) are indicated. The molecular weight markers are reported on the left (kDa). (B) PA-JEB keratinocytes were treated for 24 h with 10 M (lane 2) or 5 M (lane 3) lactacystin, or with 150 M (lane 4) or 50 M (lane 5) chloroquine. Untreated PA-JEB keratinocytes (lane 1) and HaCaT cells (C). Cell extracts (100 g) were analyzed in 10% SDS–PAGE and Western blot analysis was done using MoAb SC-6597 to the NH₂-terminus of 6 integrin. The migration bands identifying the 6 precursor (140 kDa) and the 6 heavy chain polypeptide (120 kDa) are indicated.

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To assess whether proteasomal proteolysis could also be implicated in the degradation of the mutated S47L 6 integrin, the transfected 293 cells were treated with MG132 or lactacystin, two specific inhibitors of the proteasomal degradation pathway. Western blot analysis of transfected cells treated with these drugs failed to demonstrate the implication of the proteasomal degradation pathway in the degradation of the mutant S47L 6 integrin (data not shown).

Because the chloroquine treatment increased the stability of the mutated 6 integrin in 293 cells, we wanted to verify this observation in PA-JEB keratinocytes. Cell extracts of PA-JEB keratinocytes grown in the presence of chloroquine were analyzed by 10% SDS–PAGE and immunoblotting using PoAb SC-6597, specific to the N-terminal domains of 6 integrin. Extract from untreated PA-JEB keratinocytes showed a unique weak signal corresponding to the 140 kDa unprocessed 6 integrin (Figure 6b, lane 1). A stronger hybridization signal with an identical electrophoretic mobility was detected in extracts of the proband's keratinocytes treated with chloroquine (Figure 6b, lanes 4 and 5). These findings confirmed the inhibitory action of chloroquine on degradation of the mutant 6 polypeptide. Treatment with lactacystin failed to stabilize the mutant 6 integrin in the PA-JEB cell cultures (Figure 6b, lanes 2 and 3). Altogether, these results demonstrate that the mutant S47L 6 integrin is detected at extremely low levels in the proband's keratinocytes as an unstable precursor 6 polypeptide rapidly degraded by the cell.

Discussion

The widespread disadhesion of the stratified epithelia and PA characteristic of lethal PA-JEB have so far been associated with absent expression of 64 integrin consequent to genetic mutations that generate premature termination codons mainly in the 4 but also for a minor part in the 6 integrin messenger RNA (^{Vidal et al, 1995;Ruzzi et al, 1997}). Premature termination codons are known to cause rapid decay of the mutated RNA transcripts and reduction of their steady-state levels (^{McIntosh et al, 1993}). In this study, we examined the genetic background of a severe form of PA-JEB associated with lack of skin immunoreactivity to 6 integrin antibodies despite synthesis of stable 6 integrin molecules at levels comparable with those detected in healthy controls. Screening for genetic mutations associated the PA-JEB phenotype with a homozygous point mutation in exon 1 of the gene ITGA6 resulting in the substitution of a serine residue with a leucine (S47L) in 6 integrin. Lines of evidence suggested the causative role of the mis-sense S47L mutation in PA-JEB: (1) besides transition 286 C-to-T in the proband's genomic DNA that leads to substitution S47L, no additional sequence variation was detected in the cDNA for 6 integrin and the regulatory DNA sequences of the ITGA6 gene compared with wild-type controls; (2) the mutated ITGA6 allele was exclusively found in a heterozygous state in healthy carriers of the proband's family, and was not detected in cohorts of unrelated healthy individuals; (3) transition 286 C-to-T affected neither expression nor stability of the mutated integrin 6 messenger RNA; (4) transfer of the 6 cDNA carrying transition 286 C-to-T into integrin 6-null PA-JEB keratinocytes failed to restore detectable expression of 6 integrin; and (5) the S47L 6 integrin failed to be normally expressed in 293 cells.

The deleterious effect of mis-sense mutations on functionality and/or stability of proteins is a well-known cause of genetic disorders (^{Bross et al, 1999;Soto, 2001}). The aberrant proteins are retained in the cell cytoplasm and degraded. It has been previously reported that mis-sense mutations in the extracellular domain of 4 integrin may be responsible for a few cases of lethal PA-JEB (^{Pulkkinen et al, 1998;Nakano et al, 2001}). In this study, we report one of the first cases of lethal PA-JEB caused by a mis-sense mutation in the 6 integrin and for the first time we explain the molecular mechanisms underlying the pathogenesis of the patient. Indeed, in this PA-JEB patient the polypeptide is virtually undetectable by immunohistochemistry and immunoblot analyses, both in the patient's keratinocytes in vivo and in 6-null keratinocytes in vitro after transient transfection with the mutant 6 cDNA. Nevertheless, the extremely rapid degradation rate of the aberrant 6 polypeptide by the cell machinery was inferred from the fact that the mutated integrin was expressed in in vitro translation assays at levels comparable with those obtained with a wild-type control, and that its presence was detected by western blot analysis in extracts of 293 cells transfected with a mutated S47L 6 cDNA and used as an eukaryotic overexpression system. The rapid degradation of the mutant 6 is intriguing, because several functional studies on integrin chains have been performed by directed mutagenesis of the N-terminal domains of the polypeptides, and, to our knowledge, instability of the mutant recombinant integrin chains has never been observed. For instance, mutagenesis of the 4 subunit residues Gly130 and Gly190 conserved in all the integrin -propeller domains has been shown to induce conformational changes that affect association of 4 integrin with the integrin 1 subunit and binding to ligands without altering the stability of the mutated 4 chain (^{Guerrero-Esteo et al, 1998}). In this study, the rapid decay of the abnormal 6 polypeptide may be explained by the particular localization of the S47L mutation, within a stretch of four amino acids (FGFS), which is part of the first -strand of the first amino-terminal repeat of the seven that form the seven-bladed -propeller structure of the extracellular head of integrin units (^{Springer, 1997}). The achievement of the crystal structure of the integrin V3 extracellular portion has confirmed that the toroidal arrangement of the seven amino-terminal -sheets mediates the interaction of subunits with the subunits partners and regulates binding of the integrin heterodimers with their extracellular ligands (^{Xiong et al, 2001}). The -propeller, which is found in numerous proteins, was first identified in the subunits of heterotrimeric guanosine triphosphate-binding proteins (G proteins), where it mediates the / subunit interactions (^{Sondek et al, 1996}). The correct conformation of the -propeller structure is therefore expected to undergo strict control by the post-translational machinery of the cell. This hypothesis is confirmed by mis-sense mutations affecting the 4 subunit. As a general rule, mis-sense mutations within 4 integrin are associated with the nonlethal form of PA-JEB. For a few exceptions, however, mis-sense mutations may cause a lethal phenotype. In these cases, mis-sense mutations affect conserved amino acids of the putative 4 integrin ligand binding domain (^{Nakano et al, 2001}).

Diverse diseases, including epidermolysis bullosa, arise from protein misfolding (^{Soto, 2001}). Aberrantly folded neo-synthesized polypeptides stably interact with molecular chaperones that drive proteolytic degradation by intracellular proteases. For instance, calnexin was shown to retain mutated ₁-anti-trypsin in the ER before degradation of the abnormal protein by the proteasome, with consequent ₁-anti-trypsin deficiency and onset of pathologies, such as infantile liver disease and adult emphysema (^{Qu et al, 1996;Liu et al, 1997}). Protein degradation also takes place in the lysosome after protein re-orientation from the trans-Golgi network (^{Ellgaard et al, 1999}). From our studies based on the use of proteasome-specific inhibitors, we could not demonstrate the implication of this protein degradation pathway in the removal of the mutated 6 polypeptide, because in our experimental conditions treatments with the proteasome inhibitors MG132 and lactacystin affect expression of the wild-type 6 integrin. Conversely, the involvement of the lysosomal pathway in degradation of the mutated 6 integrin was suggested by the positive effect exerted by chloroquine on the stability of the aberrant polypeptide. Considering, however, that the treatment by chloroquine does not fully preserve expression of the mutated 6 integrin, and also that degradation of the aberrant polypeptide is extremely rapid, the implication of different protein degradation pathways is likely. Treatment of the transfected 293 cells with chloroquine and the proteasome inhibitors also show that most of the mutated 6 polypeptide precursor displays an enhanced electrophoretic mobility, which possibly reflects an altered post-translational modification. Interestingly, our results also indicate that chloroquine interferes with the proteolytic maturation of the wild-type 140 kDa 6 integrin precursor by furin, which is in agreement with previous observations reporting that chloroquine hinders the movement of furin along the endocytic pathway by neutralizing endosomal acidification (^{Chapman and Munro, 1994}).

In conclusion, our results demonstrate that mutation S47L induces a rapid degradation of the neo-synthesized 6 polypeptide because substitution of the polar serine with the hydrophobic leucine residue alters folding in a key functional domain of the integrin chains. The extremely accelerated decay of the mutated 6 polypeptide resulting in absent expression of 64 integrin underlies the severity of the condition. Our studies also provide new insight into the pathogenic effect of mis-sense mutations and into functional domains of the integrin cell receptors.

References

References

Acknowledgments

We acknowledge A. Spadafora for technical assistance, R. Falcioni for helpful discussions and A. Charlesworth for critical reading of the manuscript. This work was supported by DEBRA U.K., Association Française contre les Myopathies (AFM) and Epidermolyze Bulleuse Association d'Entraide (EBAE). Maryline Allegra was recipient of a fellowship from the Fondation pour la Recherche Médicale (FRM).