Abstract. Hemidesmosomes (HDs) mediate cell adhesion to the extracellular matrix and have morphological association with intermediate-sized filaments (IFs) through cytoplasmic plaques . Though several proteins have been located in HDs, most of them have not been well characterized, with the exception of the 230-kD antigen of bullous pemphigoid (BP), an autoimmune skin blistering disease. Only recently we have succeeded in isolating HDs from bovine corneal epithelial cells and in identifying five major components on SDS-PAGE (Owaribe K., Y. Nishizawa, and W. W. Franke . 1991 . Exp. Cell Res . 192 :622-630) . In this study we report on immunological characterization of one of the major components, termed HDl, with an H EMIDESMOSOMES (HDs)' and focal adhesions are cell junctions that mediate adhesion to the extracellular matrix (ECM) . These two plasma membrane junction types have common ultrastructural features ; e.g., both are associated with cytoskeeeool elements through electron-dense cytoplasmic plaques. They thus provide us with models to explore the mechanisms of transmembrane connection between the ECM and the cytoskeleton. In focal adhesions associated with actin-containing microfilaments (for reviews see references 2 and 50), a number ofproteins are known to be involved, including ECM receptors of the integrin family (5,12,17,33) and plaque components such as talin (1) and vinculin (29) . Yet our knowledge about the molecular architecture of the ECM-to-microfilaments junctions is still incomplete.The HD, primarily found in the basement membrane zone (BMZ) of stratified and complex epithelia, has a complex structure: a cytoplasmic plaque associated with intermediate filaments (IFs), a subbasal dense plate with anchoring filaments in the basal lamina, and anchoring fibrils in collagenous connective tissues (6,16,39). The morphological appearance is essentially that of half of the desmosome of intercellular junctions. However, although several des- apparent molecular mass of 500 kD. Immunofluorescence microscopy showed colocalization of HDl with BP antigen at the basement membrane zone of those tissues that have typical HDs, including skin epidermis, corneal and tracheal epithelia, and myoepithelium . In cultured keratinocytes, HDl demonstrated colocalization with BP antigen in the precise way, while being absent from focal adhesions . Immunoelectron microscopy revealed that an epitope of HDl was located on the cytoplasmic side of HDs . Taking all these results together, we conclude that HDl is a new hemidesmosomal component . Interestingly, HDl also exists in endothelial and glial cells, which lack typical HDs . mosomal proteins have been identified (e.g., references 4, 26; for review see reference 35) since the establishment of appropriate isolation procedures (e.g., reference 36), none of them have been found in HDs (13, 35) . Thus, in marked contrast to other celljunctions, little information is available about the molecular components of HDs, with the exception ofthe well-characterized 230-kD antigen r...

The hemidesmosome (HD) is a cell-to-substrate adhesion apparatus found in stratified and complex epithelia. One of the putative cell-matrix adhesion molecules present in the HD is the 180-kDa bullous pemphigoid antigen (BP180), also termed type XVII collagen. In our previous study, using a monoclonal antibody (mAb) 1337, we have detected a 120-kDa collagenase-sensitive polypeptide in the HD fraction (Uematsu, J. and Owaribe, K. (1993) Cell Struct. Funct. 18, 588 (abstr.)). The present study was undertaken to assess the relation of the 120-kDa polypeptide to this BP180. Immunofluorescence microscopy of bovine skin revealed the basement membrane zone of skin to be stained clearly with mAb 1337, whereas the lateral surfaces of basal cells, which were decorated by typical antibodies against BP180, were not. The antibody did not detect HDs in cultured cells but rather in the culture medium. These results indicate a localization of mAb 1337 antigen distinct from BP180. However, the same polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Therefore, the polypeptide was identified as an extracellular fragment of BP180. mAb 1337 immunoprecipitated the 120-kDa fragment from the medium, but not the 180-kDa molecule of BP180 extracted from cultured cells, indicating that the antibody specifically recognizes the fragment. The mAb 1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Hence, the staining pattern observed for bovine skin demonstrated the presence of the 120-kDa extracellular fragment. Rotary shadow electron microscopy of affinity-purified 120-kDa fragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb 1337 shows unique epitope specificity, recognizing only the 120-kDa extracellular fragment of BP180, which is constitutively cleaved on the cell surface as a 120-kDa fragment both in in vivo and in vitro.

Hemidesmosomes (HDs) constitute a major cellular apparatus for substratum adhesion in stratified and complex epithelia. A large number of components participate in their construction. HD4, a 180 kDa polypeptide, which is one of the major constituents of the isolated HD fraction, has been suggested to be a glycoprotein, is probably identical to the 180 kDa bullous pemphigoid (BP) antigen [Owaribe, K., Nishizawa, Y., & Franke, W.W. (1991) Exp. Cell Res. 192, 622-630]. By using a sensitive method for detection of glycoproteins, HD4 was confirmed to be a major glycoprotein in cytoskeletal fractions of certain cultured epithelial cells as well as in the HD fraction. To further characterize HD4, we prepared two groups of monoclonal antibodies (mAbs), one recognizing extracellular parts of the HD4 molecule (group I) and the other recognizing intracellular ones (group II). In cultured keratinocytes, type I mAbs, as well as BP autoantibodies that recognize both 230 and 180 kDa polypeptides, stained living cells while type II mAbs did not. The two mAbs exhibited identical staining patterns in fixed cells. HD4 molecules proved partially susceptible to collagenase and Dispase digestion, which removed epitopes of type I mAbs but not those of type II. Immunoelectron microscopy revealed the epitopes of group I mAbs to be localized in the extracellular region of HDs, whereas those of group II were on the cytoplasmic side. These results indicate that the HD4 (BP180) molecule is a major transmembrane glycoprotein with collagen domains in its extracellular portion.

Hemidesmosomes (HDs) are specialized cell-substrate junctions with distinct cytoplasmic plaques where intermediate filaments (IFs) are anchored. In our previous work, we described two types of HDs in terms of their molecular constituents, i.e. type I HD and type II HD [Hieda, Y., Nishizawa, Y., Uematsu, J.,& Owaribe, K. (1992) J. Cell Biol. 116, 1497-1506]. In the present study we further characterized type II HDs in cultured cells, comparing their composition and function with those of conventional type I HDs. Although the two bovine mammary gland epithelial cell lines, BMGE+H and BMGE-H, were derived from the same tissue, their cell-substrate adhesion properties are markedly different. Immunological examination showed that BMGE-H cells express HD1 and the integrin alpha 6 beta 4 complex but not bullous pemphigoid antigens, while BMGE+H cells express all these components, i.e. the former have type II HDs and the latter have type I HDs. GoH3, a monoclonal antibody to the integrin alpha 6 subunit, inhibited BMGE-H cell adhesion to laminin as a substrate, as also observed for BMGE+H cells. These and electron microscopic results indicate that BMGE-H cells form type II HD-like structures containing HD1 and alpha 6 beta 4 which are associated with IFs. This structure mediates adhesion of the cell to laminin. This is the first demonstration of an adhesion function for type II HDs in cultured cells.

Three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type 2 (hPIV-2). The lectins, concanavalin A (Con A), lens culinaris agglutinin (LCA) and peanut agglutinin (PNA), inhibited cell fusion and hemadsorption induced by hPIV-2. Virus nucleoprotein (NP) gene synthesis was largely inhibited, but fusion (F) and hemagglutinin-neuraminidase (HN) gene syntheses were not. An indirect immunofluorescence study showed that Con A inhibited virus NP, F and HN protein syntheses, but LCA did not completely inhibit them, and that PNA inhibited only NP protein synthesis. Using a recombinant green fluorescence protein-expressing hPIV-2, without matrix protein (rghPIV-2ΔM), it was found that virus entry into the cells was not completely prevented. The lectins considerably reduced the number of viruses released compared with that of virus infected cells. The lectins bound to cell surface within 10 min, and many aggregates were observed at 30 min. Con A and LCA slightly disrupted actin microfilaments and microtubules, but PNA had almost no effect on them. These results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors.