Bullous pemphigoid antigens (BPAGs): identification of RFLPs in human BPAG1 and BPAG2, and exclusion as candidate genes in a large kindred with dominant epidermolysis bullosa simplex.

Bullous pemphigoid antigens (BPAGs) are defined as autoantigens in a blistering skin disease, bullous pemphigoid. Two of the BPAGs, a 230-kD (BPAG1) and a 180-kD (BPAG2) protein, have been localized to hemidesmosomes, attachment structures at the basal keratinocyte-basement membrane interphase. We have recently cloned cDNAs corresponding to human BPAG1 and BPAG2, and mapped the corresponding genes to human chromosomes 6p and 10q, respectively. These cDNAs have now been used in a search for RFLPs in the corresponding genes. Southern hybridizations of genomic DNA from normal unrelated individuals with a BPAG1 cDNA detected an informative MspI RFLP, and similar hybridizations with a BPAG2 cDNA revealed an informative TaqI RFLP. These RFLPs were applied to a large kindred with epidermolysis bullosa simplex (EBS), generalized (Koebner) type, consisting of 14 affected and 12 unaffected individuals in three generations. Linkage analysis excluded the EBS locus in this pedigree approximately 9 cM and approximately 5 cM on either side of the BPAG1 and BPAG2 loci, respectively, when a lod score of -2.0 was taken as the limit of exclusion. This study demonstrates that mutations in the BPAG1 or BPAG2 genes are not the primary genetic defect in this family with EBS.     

Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains.

Bullous pemphigoid antigens are defined as the autoantigens in a blistering skin disease, bullous pemphigoid. One of them, a 230-kDa protein (BPAG1), is associated with hemidesmosomes, attachment complexes at the basal keratinocyte-lamina lucida interface within the dermal-epidermal basement membrane zone. The precise functions and cellular compartmentalization of BPAG1 are unknown. In this study, a human keratinocyte lambda gt11 cDNA library was screened for clones corresponding to BPAG1. The composite of overlapping cDNAs delineated 8,930 base pairs of nucleotide sequences that contained an open reading frame encoding 2,649 amino acids. Analysis of the deduced amino acid sequences predicted a putative signal peptide of 43 amino acids and the presence of a membrane-associated sequence of 17 amino acids. Several potential sites for N-glycosylation, as well as for protein kinase C or cAMP- and cGMP-dependent protein kinase-mediated phosphorylation were identified. Three peptide segments were predicted to be highly antigenic, potentially serving as epitopes for the formation of autoantibodies. Eight repeat segments of 38 residues each with a high degree of homology with sequences in desmoplakin I, a component of desmosomal cytoplasmic plaques, were detected in the carboxyl-terminal end of the molecule. In addition, the presence of three subdomains characterized by heptad repeats predicted an alpha-helical coiled coil dimer structure in the central portion of the protein. These data suggest that BPAG1 may be a membrane-associated protein that plays a role in the attachment of basal keratinocytes to the underlying basement membrane.     

Human laminin a chain (LAMA) gene: Chromosomal mapping to locus 18p11.3

Laminin, an integral component of basement membranes, consists of three subunit polypeptides, A, B1, and B2 chains. We have recently isolated cDNAs corresponding to human laminin A chain. These cDNAs were utilized for chromosomal in situ hybridizations to establish the genomic location of the laminin A chain gene. Metaphase chromosomes of PHA-stimulated human peripheral blood leukocytes were examined by in situ hybridization with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human laminin A chain is at locus 18p11.3. Since human laminin B1 and B2 chain genes have been previously mapped to chromosomes 7 and 1, respectively, the results indicate that genes encoding human laminin chains reside in separate chromosomes.     

Molecular complexity of the cutaneous basement membrane zone

Ultrastructural examination of the cutaneous basement membrane zone (BMZ) reveals the presence of several attachment structures, which are critical for integrity of the stable association of epidermis and dermis. These include hemidesmosomes which extend from the intracellular compartment of the basal keratinocyte to the underlying basement membrane where they complex with anchoring filaments, thread-like structures traversing the lamina lucida. At the lower portion of dermal-epidermal attachment zone, anchoring fibrils extend from the lamina densa to the papillary dermis, where they associate with basement membrane-like structures, known as anchoring plaques. Molecular cloning of the cutaneous BMZ components has allowed elucidation of the structural features of the proteins which constitute these attachment structures. Specifically, hemidesmosomes have been shown to consist of at least four distinct proteins. The intracellular hemidesmosomal inner plaque is comprised of the 230-kD bullous pemphigoid antigen (BPAG1), and plectin, a high-molecular weight cytomatrix protein, encoded by the corresponding gene, PLEC1. The transmembrane component of the hemidesmosomes consists of the 180-kD bullous pemphigoid antigen (BPAG2), a collagenous protein also known as type XVII collagen (COL17A1), as well as of the basal keratinocyte-specific integrin 64. The anchoring filaments consist predominantly of laminin 5 with three constitutive subunit polypeptides, the 3, 3 and 2 chains, which is associated with laminin 6 with the chain composition 3, 1 and 1. Also associated with anchoring filaments is a novel protein, ladinin, which serves as autoantigen in the linear IgA disease, and the corresponding gene, LAD1, has been mapped to human chromosome 1. Finally, the major, if not the exclusive, component of anchoring fibrils is type VII collagen, encoded by the gene (COL7A1) which consists of 118 distinct exons, the largest number of exons in any gene published thus far. Collectively, the cutaneous basement membrane zone is a complex continuum of macromolecules which form a network providing the stable association of the epidermis to the underlying dermis. Thus, genetic lesions resulting in abnormalities in any part of this network could result in a blistering skin disease, such as epidermolysis bullosa.Abbreviations BMZ basement membrane zone - EB epidermolysis bullosa - JEB junctional EB - GABEB generalized atrophic benign EB - EB-MD epidermolysis bullosa with muscular dystrophy - EB-PA epidermolysis bullosa with pyloric atresia     

Localization of the human gene for the type I cyclic GMP-dependent protein kinase to chromosome 10.

We have recently characterized cDNAs and genomic DNA fragments for human type I cGMP-dependent protein kinase (cGK). By probing human x hamster hybrid cell lines with a 1.2-kb intron fragment from the human type I cGK gene, we identified a 5.9-kb BglII restriction fragment and localized it to human chromosome 10. In situ hybridization analyses using 3H-labeled cDNA and genomic DNA probes for the human type I cGK to human metaphase chromosomes supported the somatic cell hybrid data and indicated that the gene (PRKG1B; protein kinase, cGMP-dependent) maps to 10p11.2----q11.2.     

Chromosomal Localisation of the Human Envoplakin Gene (EVPL) to the Region of the Tylosis Oesophageal Cancer Gene (TOCG) on 17q25

Envoplakin is a membrane-associated precursor of the epidermal cornified envelope. Envoplakin is homologous to desmoplakin I and desmoplakin II (DPI/II), bullous pemphigoid antigen 1 (BPAG1), and plectin and is proposed to link desmosomes and keratin filaments to the cornified envelope. We describe the isolation of cosmids and yeast artificial chromosomes containing the complete human envoplakin gene (EVPL) and show, by analysis of somatic cell hybrids and chromosomalin situhybridisation, that the envoplakin gene, unlike the genes encoding BPAG1 and DPI/II, maps to 17q25 and is physically linked to D17S1603. This sequence-tagged site segregates with the autosomal dominant human disease focal nonepidermolytic palmoplantar keratosis (NEPKK; “tylosis”), which is associated with an increased risk of oesophageal cancer. The chromosomal localisation of the envoplakin gene, the homology of the encoded protein to keratin-binding proteins, and its expression in epidermal and oesophageal keratinocytes all raise the possibility that loss of envoplakin function could be responsible for this form of palmoplantar keratoderma.     

BPAG1e Maintains Keratinocyte Polarity through β4 Integrin–mediated Modulation of Rac 1 and Cofilin Activities

α6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.     

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1

The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cβ3 and PYGM on human chromosome 11q13. The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome. A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs). Five different EST cDNA clones were identified and mapped on the contig. One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis. The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13. Received: 22 July 1998 / Accepted: 24 August 1998     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome

delta-Aminolevulinate synthase (ALAS) catalyzes the first committed step of heme biosynthesis. Previous studies suggested that there were erythroid and nonerythroid ALAS isozymes. We have isolated cDNAs encoding the ubiquitously expressed housekeeping ALAS isozyme and a related, but distinct, erythroid-specific isozyme. Using these different cDNAs, the human ALAS housekeeping gene (ALAS1) and the human erythroid-specific (ALAS2) gene have been localized to chromosomes 3p21 and X, respectively, by somatic cell hybrid and in situ hybridization techniques. The ALAS1 gene was concordant with chromosome 3 in all 26 human fibroblast/murine(RAG) somatic cell hybrid clones analyzed and was discordant with all other chromosomes in at least 6 of 26 clones. The regional localization of ALAS1 to 3p21 was accomplished by in situ hybridization using the 125I-labeled human ALAS1 cDNA. Of the 43 grains observed over chromosome 3, 63% were localized to the region 3p21. The gene encoding ALAS2 was assigned by examination of a DNA panel of 30 somatic cell hybrid lines hybridized with the ALAS2 cDNA. The ALAS2 gene segregated with the human X chromosome in all 30 hybrid cell lines analyzed and was discordant with all other chromosomes in at least 8 of the 30 hybrids. These results confirm the existence of two independent, but related, genes encoding human ALAS. Furthermore, the mapping of the ALAS2 gene to the X chromosome and the observed reduction in ALAS activity in X-linked sideroblastic anemia suggest that this disorder may be due to a mutation in the erythroid-specific gene.     

Laminin-5 Inhibits Human Keratinocyte Migration

Laminin-5 (previously known as kalinin, epiligrin, and nicein) is an adhesive protein localized to the anchoring filaments within the lamina lucida space of the basement membrane zone lying between the epidermis and dermis of human skin. Anchoring filaments are structures within the lamina lucida and lie immediately beneath the hemidesmosomes of the overlying basal keratinocytes apposed to the basement membrane zone. Human keratinocytes synthesize and deposit laminin-5. Laminin-5 is present at the wound edge during reepithelialization. In this study, we demonstrate that laminin-5, a powerful matrix attachment factor for keratinocytes, inhibits human keratinocyte migration. We found that the inhibitory effect of laminin-5 on keratinocyte motility can be reversed by blocking the α3 integrin receptor. Laminin-5 inhibits keratinocyte motility driven by a collagen matrix in a concentration-dependent fashion. Using antisense oligonucleotides to the α3 chain of laminin-5 and an antibody that inhibits the cell binding function of secreted laminin-5, we demonstrated that the endogenous laminin-5 secreted by the keratinocyte also inhibits the keratinocyte's own migration on matrix. These findings explain the hypermotility that characterizes keratinocytes from patients who have forms of junctional epidermolysis bullosa associated with defects in one of the genes encoding for laminin-5 chains, resulting in low expression and/or functional inadequacy of laminin-5 in these patients. These studies also suggest that during reepithelialization of human skin wounds, the secreted laminin-5 stabilizes the migrating keratinocyte to establish the new basement membrane zone.     

14-3-3 sigma isoform interacts with the cytoplasmic domain of the transmembrane BP180 in keratinocytes

The protein bullous pemphigoid antigen-2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin alpha6, integrin beta4, or bullous pemphigoid antigen-1 (BPAG1/BP230) is critical for this attachment in skin. The protein 14-3-3 sigma, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14-3-3 protein family naturally found in all eukaryotic cells. Here, we have used a 14-3-3sigma GST pull-down screening assay and showed that sigma (sigma) isoform of the 14-3-3 protein family interacts with the cytoplasmic N-terminal domain of BP180. Analysis of a series of truncated or deleted 14-3-3sigma revealed that only intact 14-3-3sigma molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14-3-3 sigma is essential for this interaction. Further, a BP180 co-immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14-3-3 sigma interacts with cytoplasmic domain, but not ecto-domain of the BP180. In conclusion, the finding of this study provides evidence that 14-3-3sigma isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored.     

Human elastin gene: new evidence for localization to the long arm of chromosome 7.

In this study we have utilized human elastin cDNAs in molecular hybridizations to establish the chromosomal location of the human elastin gene. First, in situ hybridizations were performed with metaphase chromosomes from phytohemagglutinin-stimulated human peripheral blood lymphocytes. In three separate experiments using two different regions of human elastin cDNAs, the distribution of grains was found to be concentrated on the long arm of chromosome 7 within the [q11.1-21.1] region, and the peak number of grains coincided with the locus 7q11.2. Second, hybridizations with a panel of human-rodent cell hybrids showed concordance with human chromosome 7. Third, PCR analyses with elastin-specific primers of DNA from a hybrid cell line containing chromosome 7 as the only human chromosome yielded a product of the expected size, while DNA containing human chromosome 2, but not chromosome 7, did not result in a product. The results indicate that the human elastin gene is located in the proximal region of the long arm of chromosome 7. The precise localization of the elastin gene in the human genome is useful in establishing genetic linkage between inheritance of an allele with a mutated elastin gene and a heritable disorder.     

The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.     

BPAG1n4 is essential for retrograde axonal transport in sensory neurons

Disruption of the BPAG1 (bullous pemphigoid antigen 1) gene results in progressive deterioration in motor function and devastating sensory neurodegeneration in the null mice. We have previously demonstrated that BPAG1n1 and BPAG1n3 play important roles in organizing cytoskeletal networks in vivo. Here, we characterize functions of a novel BPAG1 neuronal isoform, BPAG1n4. Results obtained from yeast two-hybrid screening, blot overlay binding assays, and coimmunoprecipitations demonstrate that BPAG1n4 interacts directly with dynactin p150Glued through its unique ezrin/radixin/moesin domain. Studies using double immunofluorescent microscopy and ultrastructural analysis reveal physiological colocalization of BPAG1n4 with dynactin/dynein. Disruption of the interaction between BPAG1n4 and dynactin results in severe defects in retrograde axonal transport. We conclude that BPAG1n4 plays an essential role in retrograde axonal transport in sensory neurons. These findings might advance our understanding of pathogenesis of axonal degeneration and neuronal death.     

Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts.

Ataxia-telangiectasia (A-T) is an inherited human disease of unknown etiology associated with neurologic degeneration, immune dysfunction, cancer risk, and genetic instability. A-T cells are sensitive to ionizing radiation and radiomimetic drugs, offering the possibility of cloning A-T genes by phenotypic complementation. We have used this sensitivity to isolate the first human cDNAs reported to complement A-T cells in culture. Complementation group D A-T fibroblasts were transfected with an episomal vector-based human cDNA library, approximately 610,000 resultant transformants were treated with the radiomimetic drug streptonigrin-resistant, and nine unrelated cDNAs were recovered from 29 surviving streptonigrin-resistant clones. Five cDNAs were mapped, but none localized to 11q23, the site of A-T complementation group A and C loci. Four of the mapped cDNAs conferred mutagen resistance to A-T D fibroblasts on secondary transfection. One cDNA was identified as a fragment of dek, a gene involved in acute myeloid leukemia. The dek cDNA fragment and pCAT4.5, a 4.5-kb cDNA that mapped to 17p11, independently complemented three different phenotypic abnormalities of A-T D fibroblasts (mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis). The pCAT4.5 cDNA did not complement the mutagen sensitivity of an A-T group C fibroblast line, suggesting that it represents a candidate disease gene for group D A-T. Our results indicate that phenotypic complementation alone is insufficient evidence to prove that a candidate cDNA is an A-T disease gene. The complementing cDNAs may represent previously uncharacterized genes that function in the same pathway as does the A-T gene product(s) in the regulation of cellular responses to DNA damage.     

FcR-independent effects of IgE and IgG autoantibodies in bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by IgE and IgG class autoantibodies specific for 180-kDa BP Ag 2 (BP180), a protein involved in cell-substrate attachment. Although some direct effects of BP IgG have been observed on keratinocytes, no study to date has examined direct effects of BP IgE. In this study, we use primary cultures of human keratinocytes to demonstrate Ag-specific binding and internalization of BP IgE. Moreover, when BP IgE and BP IgG were compared, both isotypes stimulated FcR- independent production of IL-6 and IL-8, cytokines critical for BP pathology, and elicited changes in culture confluence and viability. We then used a human skin organ culture model to test the direct effects of these Abs on the skin, whereas excluding the immune inflammatory processes that are triggered by these Abs. In these experiments, physiologic concentrations of BP IgE and BP IgG exerted similar effects on human skin by stimulating IL-6 and IL-8 production and decreasing the number of hemidesmosomes localized at the basement membrane zone. We propose that the Ab-mediated loss of hemidesmosomes could weaken attachment of basal keratinocytes to the basement membrane zone of affected skin, thereby contributing to blister formation. In this article, we identify a novel role for IgE class autoantibodies in BP mediated through an interaction with BP180 on the keratinocyte surface. In addition, we provide evidence for an FcR-independent mechanism for both IgE and IgG class autoantibodies that could contribute to BP pathogenesis.