Identification of the hemidesmosomal 500 kDa protein (HD1) as plectin.

HD1 is a 500 kDa hemidesmosomal plaque protein recognized by monoclonal antibody mAb-121. Recent research on inherited skin disease has suggested that it might be identical to plectin or an isoform. To cast light on this question, we have prepared several monoclonal antibodies that recognize a 500 kDa protein in the hemidesmosome fraction. Unexpectedly, some staining pattern heterogeneity was observed on immunofluorescence microscopy. Attention was focused on two monoclonal antibodies which gave different localization in bovine skin and retinal pigment epithelial cells. Determination of the amino-terminal sequence of an antigenic 100 kDa polypeptide fragment derived from the 500 kDa component of an insoluble fraction of bovine hepatocytes revealed it was identical to that of plectin. Using the two antibodies, we screened a cDNA library derived from BMGE+H, a bovine mammary gland epithelial cell line. The isolated cDNA clones corresponded to the rod domain of bovine plectin, with two separate epitope regions for each of the antibodies. From these results we conclude that the hemidesmosomal 500 kDa component HD1 is identical to plectin. As judged on rough estimation of molar ratios on this basis, hemidesmosomes are composed of plectin, BP230, the integrin beta4 subunit, and alpha6 in a 1:1:1:1 ratio.

Cleavage of BP180, a 180-kDa bullous pemphigoid antigen, yields a 120-kDa collagenous extracellular polypeptide.

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.

[Developmental plasticity of corticospinal projections in the spinal cord gray matter of normal and hemicortectomized rat].

In the previous study we demonstrated in rats that aberrant ipsilateral CST fibers were increased when the cerebral cortex was surgically ablated unilaterally during the neonatal period. Origin of these aberrant fibers was confirmed to involve collateral axons, ramified from their parent axons. In this study, possible plastic change and the critical period for the outgrowth of CST fibers into the spinal cord gray matter in the rat after unilateral (right side) cortical damage at different ages measured in days were examined using anterograde horseradish peroxidase (HRP). HRP was injected into the left sensorimotor cortex in both normal and experimental rats. In the normal control rats, the outgrowth of HRP positive axons into the spinal gray matter was first noticed at 7 day of age in the vicinity of the right dorsal funiculus, and then reached maximal density and extension at 10 and 14 days of age, respectively. From 21 days onwards, the density and extension of HRP positive axons in the gray matter decreased rapidly except in the medial part close to the dorsal funiculus. In rats whose right cerebral cortex were damaged at day 1 of age, the pattern of the outgrowth of HRP positive axons into the right gray matter was much the same as that in age-matched controls. However, significantly different from the control, many HRP positive axons were noted even in the contralateral gray matter. HRP-positive axons in the contralateral left gray matter were also abundant in the rats who sustained cortical damage at 7 and 14 days, but were decreased considerably when the cerebral cortex was damaged at 28 days of age. When damage occurred at 56 days of age, HRP-positive axons did not increase in the left gray matter, indicating that the critical period of axonal plasticity after localized damage was before 4 weeks of age in rats.

Development of corticospinal tract fibers and their plasticity. II. Neonatal unilateral cortical damage and subsequent development of the corticospinal tract in mice.

In this study, the right cerebral cortices of mice on postnatal day 0 (P0) were cryocoagulated with dry ice. Subsequent development of the corticospinal tract (CST) was studied morphologically and quantitatively, and was compared with that in age-matched controls. When the pyramidal tract was traced anterogradely by injecting HRP into the sensorimotor area of the left cerebral cortex of adult operated mice, the right CST originating from the healthy left hemisphere showed remarkable hypertrophy. The number of axons in the CST at the C4-C6 level became maximum on P14 in the control mice and rapidly decreased thereafter. In the operated mice, the axonal number in the right CST also was maximal on P14 and then rapidly decreased. However, the decrease in axonal number after P21 was less in the operated mice than in the controls. Moreover, the number of axons showed a slight increase after P56. These results indicate that the physiological elimination of the parent axons and their collaterals is much lower in the operated mice than in the controls, and that the increase in axon collaterals from parent axons in the hypertrophic right CST persists a long time in the operated mice.

Development of corticospinal tract fibers and their plasticity I: quantitative analysis of the developing corticospinal tract in mice.

This study was undertaken to elucidate ultrastructurally and quantitatively the development of the corticospinal tract (CST) axons of mouse at the intumescence level of the cervical cord. An anterograde HRP study showed that the CST was located at the ventral one-third of the dorsal funiculus, and a few HRP-positive fibers were noted at the medialmost part of the ipsilateral anterior funiculus. Ultrastructurally, the CST was composed of unmyelinated axons, growth cones and a few degenerating axons until postnatal day 10 (P10), then the axons in CST gradually increased in size. The number of axons constituting the right CST was calculated at different days of age. The total numbers of axons at P0, P4, P14, P21 and P56 were 2.3 x 10(4), 6.2 x 10(4), 10.4 x 10(4), 7.1 x 10(4) and 3.5 x 10(4), respectively. These results indicate that the number of CST axons at the cervical intumescence of mouse becomes maximum at P14, and then decreases rapidly to reach the adult level of 3.5 x 10(4) (at P56), about 68% of them thus being lost.

[Measurement of total body and leg bone mineral densities by dual energy X-ray absorptiometry in severely handicapped children and adults].

Total body and leg bone mineral densities (BMD) were measured in 42 severely handicapped children and adults using a dual energy X-ray absorptiometry (DEXA) (LUNAR Radiation Corp., DPX). Despite the differences in motor ability and nursing history, about 95% of patients, except for 2 cases, were diagnosed as having osteopenia. The degree of osteopenia was dependent on the motor disabilities of their original disease. Therefore we should bear in mind a precaution and therapy from early period. Because BMD of the legs in females were less than -3 SD of the age-matched control values, we should be careful for a possible fracture of femur. Since X-ray hazard of DEXA for patients is considerable to be negligible, this will provide an effective means for quantifying bone mineral in severely handicapped patients.

[Mechanism on formation of new ipsilateral corticospinal tract following neonatal unilateral cortical ablation in rats].

The corticospinal tract in the rat after neonatal ablation of the unilateral cerebral cortex was studied morphologically using the antegrade horse-radish peroxidase (HRP) tracing method. An aberrant ipsilateral tract was observed 7 days after the operation. Formation of the aberrant neuronal pathway has been confirmed to be contributed mainly by new axons, which ramified at the level of the pyramidal decussation from healthy corticospinal fibers. This ramified axon ran toward the ipsilateral dorsal funiculus. A few fibers also contributed to the aberrant pathway by changing their direction on the way of extension at the level of the pyramidal decussation. These results indicate that the ramification and change of the direction of extending axons play an important role for formation of a new ipsilateral corticospinal tract.

Axon ramification following unilateral cortical ablation in neonatal rats.

We confirmed the formation of an aberrant ipsilateral corticospinal tract after unilateral cerebral cortical ablation during the neonatal period in rat. This tract was studied using anterograde horseradish peroxidase (HRP) tracing. Ramifications of the axons in the pyramidal tract were found to contribute to the ipsilateral tract at the level of the pyramidal decussation, suggesting that ramification of immature axons play an important role in the formation of the ipsilateral corticospinal tract.

Demonstration of type II hemidesmosomes in a mammary gland epithelial cell line, BMGE-H.

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.

HD4, a 180 kDa bullous pemphigoid antigen, is a major transmembrane glycoprotein of the hemidesmosome.

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.

Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes.

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 HD1, with an apparent molecular mass of 500 kD. Immunofluorescence microscopy showed colocalization of HD1 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, HD1 demonstrated colocalization with BP antigen in the precise way, while being absent from focal adhesions. Immunoelectron microscopy revealed that an epitope of HD1 was located on the cytoplasmic side of HDs. Taking all these results together, we conclude that HD1 is a new hemidesmosomal component. Interestingly, HD1 also exists in endothelial and glial cells, which lack typical HDs.