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.

ABSTRACTcDNA for yieldin of Vigna unguiculata L. was cloned with reverse transcriptase-polymerase chain reaction using synthetic oligonucleotides as primers. The primers were designed on the basis of the N-terminal amino acid sequence of the yieldins isolated from the wall preparation of cowpea hypocotyls. The 1·2 kbp cDNA for yieldin contained an open reading frame of 981 base pairs, encoding 327 amino acids including 23 amino acids as a putative signal sequence. An homology search of the deduced amino acid sequence revealed that the yieldin was homologous to acidic class III endochitinases (EC 3·2.1·14) and concanavalin B. A cDNA fragment containing the yieldincoding region was introduced to Escherichia coli cells using an expression vector to express the recombinant protein. The recombinant yieldin was obtained from the recombinant E. coli and its effect on the wall mechanical properties was examined by reconstitution experiments. The recombinant yieldin fully restored the acid-induced change of the yield threshold tension (y) of heat denatured glycerinated hollow cylinders (GHCs) of cowpea hypocotyls. Northern hybridization analysis revealed that the yieldin mRNA was expressed mainly in the rapid and moderate elongation region of the etiolated hypocotyl of Vigna unguiculata L.