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

Hemidesmosomes, including anchoring filaments and fibrils, are adhesion complexes mediating firm attachment of epithelial basal cells to the underlying basement membrane, thereby mechanically linking the cytoplasmic keratin filament network to collagen fibrils and other extracellular matrix components of the connective tissue (1)(2)(3). In the past decade, several hemidesmosomal constituents have been identified and it has been possible to elucidate the cellular regulation of hemidesmosomes at the molecular level.
In the process of epidermal organization and differentiation, cells dynamically regulate their hemidesmosomes during attachment to and detachment from the basement membrane. In most cases, this involves new formation of hemidesmosomes, but, during cell movement in wound healing and differentiation in stratification, hemidesmosomes must become detached from the basement membrane, with separation of hemidesmosomal transmembrane proteins from their ligands being an important step. However, the underlying mechanism is still poorly understood.
BP180, a 180-kDa bullous pemphigoid antigen, is a type II transmembrane glycoprotein with a collagenous carboxyl-terminal extracellular domain and a noncollagenous amino-terminal cytoplasmic domain (7,8,20,21). The extracellular domain is interrupted by non-triple-helical sequences, consisting of 15 separate triple-helical stretches in the human form and 13 in the mouse. Autoantibodies to BP180 are thought to play a crucial role in skin blistering in patients with bullous pemphigoid, herpes gestationis, and cicatricial pemphigoid (22)(23)(24). This is supported by findings with a passive transfer model, featuring injection of rabbit polyclonal antibodies against a pathogenic epitope of BP180 into neonatal mice (25). In addition, mutations in the BP180 gene have been found in cases of generalized atrophic benign epidermolysis bullosa, which is characterized by universal alopecia and atrophy of the skin (26 -29). However, while these results suggest an important role for BP180 in cell to extracellular matrix adhesion, in contrast to integrin ␣ 6 ␤ 4 , our understanding of the molecular biology of BP180 is quite limited, and even its ligand(s) is unknown.
In our preliminary studies, we have detected a novel 120-kDa hemidesmosomal component, specifically recognized by a monoclonal antibody (mAb) 1 termed 1337 (30). The component was found to be present primarily in stratified epithelia by immunofluorescence microscopy. The fact that it was lost by collagenase treatment, suggesting a collagen nature is the rationale for the present examination of the relationship between the 120-kDa polypeptide and BP180. We present evidence here that the 120-kDa polypeptide is indeed derived from BP180 and that, both in tissue and in culture, cells can sever this extracellular portion of the molecule from their surfaces. The biological and clinical significance of this finding is also considered.

MATERIALS AND METHODS
Antibodies-Mouse mAbs against hemidesmosomal proteins were prepared by immunizing mice with the hemidesmosome fraction isolated from bovine corneal epithelial cells, as described previously (31). mAb 233, mAb 1D1, mAb D20, and mAb R223 are against the extracellular part, and mAb 1A8c and mAb 1A6 are against the cytoplasmic part of BP180 ( Fig. 1) (21). Epitopes of mAb 233 and mAb 1D1 are mapped on the carboxyl-terminal half, and that of mAb D20 is mapped on the amino-terminal half of the extracellular part. 2 mAb 1E5 binds specifically to BP230. mAb 855, mAb 310, and mAb 617 target the extracellular part and mAb 1A3 targets the cytoplasmic part of the integrin ␤ 4 subunit. For immunofluorescence microscopy, hybridoma supernatants of each mAb were diluted to 50 times their antibody titers, i.e. 50-fold the critical concentration to detect the antigen in skin basement membrane zone (BMZ) by immunofluorescence microscopy.
Purified ascites fluid of mAb 233 was biotinylated for some experiments. The purified immunoglobulin fraction of mAb 233 was mixed with sulfosuccinimidobiotin in 0.1 M HEPES buffer (pH 8.0) containing 50 mM NaCl to a biotin/mAb 233 molar ratio of 20. After incubation for 60 min at room temperature, the reaction was stopped by the addition of 1 M Tris-HCl (pH 7.5), and the fraction containing biotinylated antibody was dialyzed against Tris-buffered saline (TBS).
Mouse polyclonal anti-NC16A antibody was prepared by immunizing a mouse with the human NC16A domain fused with glutathione Stransferase (32).
Cells and Cell Cultures-BMGEϩH and BMGEϪH cells, mammary gland epithelial cell lines derived from a lactating bovine udder, were the kind gift of Dr. W. W. Franke of the German Cancer Research Center (Heidelberg, Germany) (33). The DJM-1 cell is an isolated cell line from human skin squamous cell carcinoma (34). A431 cells of an epidermal cell carcinoma cell line derived from human vulva were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Cell culture conditions were as described previously (35).
Immunofluorescence Microscopy-Freshly prepared tissues were snap-frozen in isopentane precooled in liquid nitrogen. Sections from frozen specimens were cut at 5-6 m with a cryostat, mounted on glass slides, air-dried, and fixed in 100% acetone at Ϫ20°C for 10 min. In some cases the sections were treated with 0.5% Triton X-100 or 1.5 M KCl in phosphate-buffered saline before fixation. Cells grown on glass coverslips were fixed with acetone. These fixed sections and cells were processed for immunofluorescence staining as described previously (35).
Conditioned medium collected from 3-day cultures was centrifuged at 1,000 rpm for 10 min to remove unattached cells and 5 mM EDTA, 1 mM PMSF, 5 g/ml leupeptin, and 0.1 mM N-ethylmaleimide were added. Proteins were precipitated by the addition of saturated ammonium sulfate solution (half the volume of the medium). Precipitates were resuspended in TBS containing 1 mM EDTA as the medium fractions. In some experiments, the spent medium was precipitated first with 50% ammonium sulfate for concentration, and subsequently the precipitant dissolved in TBS containing 1 mM EDTA was precipitated with 33% ammonium sulfate.
For immunoprecipitation, the resuspension buffer for the medium fractions was changed to a low salt buffer containing 0.5% Triton X-100, to equalize the buffer conditions for the Triton X-100-soluble membrane-bound fractions.
Collagenase Digestion-Collagenase from Clostridium histolyticum (Amano Pharmaceutical, Nagoya, Japan) was added at 50 Mandle units/ml final concentration to the HD fraction isolated from corneas of 30 bovine eyes. After incubation for 60 min at 37°C, the enzymatic reaction was stopped by the addition of 2ϫ SDS sample buffer.
Electrophoresis and Immunoblotting-SDS-PAGE was performed according to the method of Laemmli with a slight modification (36). Immunoblotting was performed using SDS-PAGE and subsequent electrophoretic transfer onto nitrocellulose sheets using a semidry system as described previously (37).
Immunoprecipitation-12 ml of Triton X-100-soluble fraction and 1.2 ml of medium fraction were prepared from three 15-cm dishes of BMGEϩH cells as described above. The prepared fractions were divided into four aliquots. Each aliquot was supplemented with 3 ml of hybridoma supernatant, incubated for 1 h at room temperature, mixed with 400 l of second antibodies conjugated to agarose beads (American Qualex International Inc., La Mirada, CA), and incubated for an additional 1 h. The agarose beads were washed with the same washing buffers used for the immunoblotting. After removal of the supernatant by centrifugation, aliquots were added to 150 l of 4ϫ SDS sample buffer (4% SDS, 125 mM Tris-HCl (pH 6.8), 20% glycerin). The agarose beads were then suspended and boiled for 2 min. After the addition of ␤-mercaptoethanol, the polypeptides were separated by SDS-PAGE and subsequently transferred electrophoretically onto nitrocellulose sheets. The sheets were incubated with 1% BSA in TBS (pH 8.0) containing 0.05% Tween 20 (Tween-TBS) and subsequently with biotinylated mAb 233 for 90 min. After washing with Tween-TBS, they were incubated with alkaline phosphatase-labeled avidin for 60 min, followed by visualization with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3indolyl phosphate.
Immunoaffinity Purification of BP180 -Ascites fluid of mAb 233 and mAb 1A8c were purified with an Ampure™ PA kit (Amersham, Tokyo, Japan) and the immunoglobulin fractions were coupled to Affi-Gel 10 (Bio-Rad, Tokyo, Japan). The Triton X-100-soluble membrane-bound fraction of DJM-1 cells prepared from 10 dishes (15 cm diameter) was applied to an mAb 1A8c immunoaffinity column (2 ml) and washed with a washing buffer (20 mM Tris-HCl (pH 7.5), 0.6 M NaCl, 5 mM MgCl 2 , 1 mM ATP, 0.1% Triton X-100). The medium fraction prepared from 500 ml of conditioned cultured medium of DJM-1 cells was applied to an mAb 233 immunoaffinity column (2 ml) and washed with a washing buffer (20 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.1% Triton X-100). Bound antigens were eluted with 3 M NaSCN containing 0.1% Triton X-100, and the eluted fractions were dialyzed against a low salt buffer containing 0.1% Triton X-100. The purity of the eluted fractions was assessed by SDS-PAGE using silver staining and immunoblotting.
Low Angle Rotary-shadowing Electron Microscopy-Affinity-purified samples were mixed with 50% glycerol, and samples were sprayed, using an air brush, onto freshly cleaved mica. The droplets on the mica were dried at room temperature in a vacuum at 10 Ϫ8 mmHg in newly developed freeze-etch equipment (Hitachi HR7000) for 10 min. Dried specimens were rotary-shadowed with platinum using an electron gun positioned at 2.5°to the mica surface, and then coated with a film of carbon generated by an electron gun positioned at 90°to the mica surface. The replica was floated on distilled water and collected on a grid covered with Formvar film. The specimens were examined with a JEM 100CX electron microscope.

Identification of the mAb 1337 Antigen-
The mAb 1337 is a monoclonal antibody obtained by immunizing a mouse with the HD fraction, and stained BMZ of bovine epidermis in immunofluorescence microscopy. Immunoblot analysis of the HD fraction showed that the antibody recognized a 120-kDa polypeptide as described previously in our preliminary report (Fig. 2) (30). To confirm the previous results and further characterize the antigen, the HD fraction was treated with collagenase and examined by immunoblotting (Fig. 2). The 120-kDa band was completely lost by collagenase digestion as reported previously, showing that the polypeptide has a collagenous domain(s) as BP180 do. Hence, we investigated the 120-kDa polypeptide with special reference to BP180, a hemidesmosomal transmembrane collagen. As shown in Fig. 2, immunoblotting with mAbs against extracellular parts of BP180 showed the electrophoretic mobility of the antigen to be equal to that of the smallest polypeptide among a group of proteolytic fragments appearing in the HD fraction. On two-dimensional SDS-PAGE and subsequent immunoblotting, the antigen and the BP180 fragment showed an identical spot (data not shown). From these results, we conclude that mAb 1337 recognizes primarily the 120-kDa fragment of BP180 on immunoblotting.
Immunofluorescence Microscopy of Bovine Skin and Cultured Cells-To examine whether the mAb 1337 specifically recognizes the 120-kDa fragment of BP180 by immunofluorescence microscopy, the staining pattern of mAb 1337 was compared with those of mAbs against BP180. The titers among the mAbs were equalized to allow comparative observations. The mAb 1337 clearly stained the BMZ of bovine skin, as did the other mAbs to BP180, but, in contrast, not the lateral surfaces of the basal cells (Fig. 3, A-D). Thus, mAb 1337 did not recognize Triton X-100-soluble BP180, which is free from the cytoskeleton and not incorporated into HDs (35). Immunofluorescence microscopy of BMGEϩH cells and primary cultured bovine conjunctival epithelial cells (data not shown) showed that mAb 1337 did not detect HDs (Fig. 4), pointing to a lack of binding to insoluble BP180. These results are quite different from those with other typical anti-BP180 mAbs, and suggest that mAb 1337 does not recognize intact BP180 in either soluble or insoluble states. Therefore, its staining pattern observed in bovine skin indicates the presence of the 120-kDa fragment. When frozen sections of bovine skin were treated with 0.5% Triton X-100 or 1.5 M KCl and stained with mAb 1337, the immunofluorescent staining was essentially the same as in control sections, showing that the 120-kDa fragments are largely insolubilized in the tissue (Fig. 3, E-H).
Detection of 120-kDa Fragments in Culture Medium-Immunofluorescence microscopy with mAb 1337 showed the anti-  7-12) or without (lanes 1-6) collagenase were stained with Coomassie Blue (lanes 1 and  7) or immunoblotted with mAb 1337 (lanes 2 and 8), mAb 233 (lanes 3 and 9), mAb 1D1 (lanes 4 and 10), mAb 1A8c (lanes 5 and 11), and anti-integrin ␤ 4 mAb 1A3 (lanes 6 and 12). genic fragment to be lacking or present at only a very low level on BMGEϩH cells (Fig. 4). If the fragment does not have a transmembrane domain nor cytoplasmic part, diffusion into the culture medium would be expected. To examine this possibility, spent media from BMGEϩH cells were analyzed by immunoblotting using mAb 1D1, a monoclonal antibody against the extracellular part of BP180, and a specific band of 120 kDa was detected (Fig. 5A). This polypeptide was concentrated in the fraction precipitated with 33% ammonium sulfate, and was also recognized by mAb 1337 and mAb 233, but not by mAb 1A8c, which recognizes the cytoplasmic part of BP180 (Fig. 5A). The electrophoretic mobility of the 120-kDa polypeptide proved equal to that of the fragment from the HD fraction. Therefore, the polypeptide in the spent medium was concluded to be the 120-kDa fragment of the HD fraction. The 120-kDa polypeptide appeared prone to degradation to a 100-kDa polypeptide when the spent medium was precipitated first with 50% ammonium sulfate for concentration, and consequently the precipitant dissolved in TBS containing 1 mM EDTA was precipitated with 33% ammonium sulfate. The 100-kDa polypeptide was recognized by mAb 1337 and 233, but not by mAb 1D1, the epitope of which appeared to be removed (Fig.  5B).
In accordance with these results, no polypeptide was recognized in the Triton X-100-insoluble fraction by immunoblot analysis with mAb 1337 (Fig. 6). Both mAb 233 and 1D1 recognized the 120-kDa polypeptide of the medium fraction as well as the 180-kDa polypeptide in the Triton X-100-insoluble fraction, while mAb 1A8c did not recognize the 120-kDa polypeptide. The molar ratio of the 120-kDa polypeptide, Triton X-100soluble BP180, and Triton X-100-insoluble BP180 was roughly 1:6:3 in BMGEϩH cells. The approximately 60-kDa component recognized by mAb 1A8c should be noted (Fig. 6E). This 60-kDa polypeptide was not recognized by either mAb 233 or 1D1, and its apparent molecular mass and specific recognition by mAb 1A8c indicate that it mainly comprises the cytoplasmic part of BP180. Since the 60-kDa polypeptide was not detected in the cytosolic fraction but in both Triton X-100-soluble membranebound and insoluble fractions, it would appear to have a transmembrane domain. On the other hand, the soluble form of BP230, a hemidesmosomal plaque component, was detected in the cytosolic fraction but not in Triton X-100-soluble mem-brane-bound fraction (Fig. 6F). It is reasonable to assume that the 60-kDa polypeptide is the fragment that remains after cleavage of the 120-kDa extracellular part from BP180. There-   fore, considering the apparent molecular mass of the two fragments, the cleavage site(s) is probably on the extracellular side near the cell membrane.
120-kDa Fragment Is Present as a Trimer Form-Trimer formation by the 120-kDa fragment was confirmed by immunoblotting (Fig. 7). When the membrane-bound fraction in SDS-PAGE sample solution was not boiled, a 600-kDa component was detected for BP180. The apparent molecular mass is equal to that of the chemically cross-linked trimers of BP180 described previously (35). Therefore, in the non-boiled condition, BP180 molecules exist in a trimer form, probably due to the collagen links. In the non-boiled condition, the 120-kDa fragment in the medium fraction was detected as a 350-kDa component. Since this molecular mass is consistent with a trimer of the fragment, it is concluded that the fragment exists as trimer form. mAb 1337 recognized the fragment in both monomer and trimer forms, but did not recognize the intact 180-kDa polypeptide.
Specificity of mAb 1337-The immunoreactivity of mAb 1337 against the 120-kDa fragment and the intact BP180 molecule in their native soluble forms was examined by immunoprecipitation. Immunoprecipitants were also analyzed by immunoblotting with mAb 233. While mAb 233 precipitated both the intact molecule and the fragment, mAb 1337 precipitated only the latter (Fig. 8A). The result demonstrated that mAb 1337 specifically recognizes the fragment, offering an explanation for the unique staining pattern observed on immunofluorescence, and supports the idea that the fragment actually exists in tissues. The recognition of the mAb 1337 immunoprecipitant with mAb 233 clearly demonstrated that the 120-kDa fragment is identical to the extracellular part of BP180.
The 120-kDa fragment was also detected in several cultured cells derived from epithelial tissues. When medium fractions of BMGEϩH, DJM-1, A431, and BMGEϪH cells were examined by immunoblotting using mAb 233 (Fig. 8B), the BMGEϪH cell line lacking BP antigens was also found to be only one without the 120-kDa fragment in its medium fraction. Thus, cleavage of BP180 to give the 120-kDa fragment is a common phenomenon in cells expressing the bullous pemphigoid antigen.
Purification of the 120-kDa Fragment and Determination of Its Morphology-120-kDa fragments were purified from the medium fraction by immunoaffinity column chromatography using mAb 233 (Fig. 9). Intact BP180 molecules were purified from the Triton X-100-soluble membrane-bound fraction by immunoaffinity column chromatography using mAb 1A8c. Fractions were prepared from DJM-1 cells, a human skin squamous carcinoma cell line. The purity of the eluted fractions was assessed by SDS-PAGE using silver staining (Fig. 9, lanes 1  and 4). The eluted fractions from the mAb 233 column showed the 120-kDa band, recognized by mAb 233 (Fig. 9, lane 2) and other monoclonal antibodies (mAb 1D1, mAb D20, and mAb R223) that binding to the extracellular part of BP180 (data not shown). The eluted fraction of the mAb 1A8c column demonstrated the 180-kDa band, and, on immunoblotting with mAb 1A8c, an additional 60-kDa band was detected (Fig. 9, lane 5). As described above, the 60-kDa polypeptide appears to be the remnant BP180 after removal of its 120-kDa extracellular fragment. To identify the cleavage site more precisely, the 120-kDa fragment and the 60-kDa fragment were immunoblotted with the polyclonal antibody against the human NC16A domain comprising the 76-amino acid stretches positioned between the transmembrane domain and the first extracellular collagenous domain. The 120-kDa fragment was recognized by the polyclonal antibody, while the 60-kDa fragment was hardly detected (Fig. 9, lanes 3 and 6). This demonstrates most of the FIG. 7. Examination of trimer formation by the 120-kDa fragment. Medium (lanes 1 and 2) and Triton X-100-soluble membranebound (lanes 3 and 4) fractions from BMGEϩH cells were immunoblotted with mAb 1337 (A) and mAb 1D1 (B) under boiled (lanes 1 and  3) or non-boiled conditions (lanes 2 and 4).  8. Immunoprecipitation of the 120-kDa fragment and the  intact BP180 molecule. A, medium (lanes 1-3) and Triton X-100soluble membrane-bound (lanes 4 -6) fractions were immunoprecipitated with mAb R223 (lanes 1 and 4), mAb 1337 (lanes 2 and 5), and mAb 233 (lanes 3 and 6). Immunoprecipitants were immunoblotted with biotinylated mAb 233. mAb R223 does not recognize bovine BP180 and was used as a negative control. mAb 1337 immunoprecipitated the  9. Immunoaffinity purification of 120-and 100-kDa fragments from the medium fraction. Eluted fractions containing 120-kDa fragments (lanes 1-3), intact BP180 molecules (lanes 4 -6), and 100-kDa fragments (lanes 7-9) were silver-stained (lanes 1, 4, and 7) and immunoblotted with mAb 233 (lanes 2 and 8), mAb 1A8c (lane 5), and the anti-NC16A domain polyclonal antibody (lanes 3, 6, and 9). NC16A domain to be within the 120-kDa fragment. Therefore, the cleavage site(s) is localized within the NC16A domain near the cell membrane. 100-kDa fragments, degradation products of 120-kDa fragments, were also purified using the mAb 233 column and found to be recognized by the polyclonal antibody against the NC16A domain. Thus, this fragment is produced by cleavage at the carboxyl-terminal side of the 120-kDa fragment (Fig. 9, lane 9). The 100-kDa fragment was recognized by mAbs 233 (Fig. 9, lane 8), D20, and R223, but not by mAb 1D1 (data not shown). Amino-terminal sequencing of the purified 120-kDa fragment failed, probably due to the low amount of the protein.
Molecular shapes of the 120-kDa fragment were examined by rotary shadowing electron microscopy and compared with those of intact BP180 (Fig. 10). The molecular dimensions of the human BP180 molecule were essentially as same as those of its bovine counterpart reported previously (Fig. 10B) (35). The rotary-shadowed images of the 120-kDa fragment showed the molecule to be composed of the central rod and the flexible tail, but lack the globular head of the intact BP180 (Fig. 10, compare C and B). Twelve selected images of the 120-kDa fragment were measured, and the averaged lengths of the rod and the tail were 68.2 nm and 116.3 nm, respectively. Since the dimensions of the central rod and the flexible tail of the fragment were found to be almost equal to those of the intact molecule, the amino-terminal side of the intact molecule consisting of the cytoplasmic and presumably the transmembrane domains must be within the globular head. The molecular shape of the 100-kDa fragment was similar to that of the 120-kDa fragment, having a little shorter tail (ϳ80 nm), consistent with the results of immunoblot analyses (Fig. 10D). DISCUSSION The present study demonstrated the existence of a 120-kDa extracellular fragment of BP180, which can be further degraded to a 100-kDa form, in culture medium of keratinocytes and skin BMZ, suggesting that the cells cleave the BP180 molecule at their surfaces. The mAb 1337 was found to be very useful to distinguish the fragment from intact BP180, apparently recognizing a unique epitope that is exposed or formed by the cleavage. We could not determine the cleavage site exactly, but it is located in the NC16A domain near to the cell membrane. The epitope may be present in the NC16A domain, which is probably more affected by the cleavage than other stable collagenous parts.
Rotary shadowing of intact BP180 molecules confirmed our previous observation, i.e. that the molecule consists of a globular head, a central rod and a flexible tail (35). It is clear from the rotary-shadowed images of the 120-kDa fragments together with biochemical results that they have no globular head, showing that the globular head corresponds to the aminoterminal cytoplasmic part of the molecule as we speculated previously.
The immunofluorescent staining pattern of mAb 1337, showing the distribution of the 120-kDa fragments, differed between tissues and cultured cells. The fragments were localized exclusively in the BMZ in skin, whereas, in cultured cells, they are detected in the culture medium. Since the staining of skin BMZ with mAb 1337 proved resistant to 0.5% Triton X-100 or 1.5 M KCl treatments, the fragments in the tissue appear to be insoluble, whereas the intact BP180 molecules present in the lateral surfaces of basal cells are solubilized by 0.5% Triton X-100 treatment (35). The insolubility of the fragments in the tissue is probably due to their anchorage to or interaction with other molecule(s). Since the fragments lack the transmembrane domain and seem to be no longer anchored to the cell body directly, they are probably fixed extracellularly. Hemidesmosomal transmembrane proteins such as integrin ␣ 6 ␤ 4 and BP180 are candidates for the extracellular anchorage of the fragment. In fact, Hopkinson et al. (18) have shown that truncated BP180 lacking the entire collagen domain, in other words consisting of only the cytoplasmic and non-collagenous extracellular domains, is incorporated into hemidesmosomes and associates with integrin ␣ 6 subunit extracellularly. However, these candidates cannot explain the absence of the 120-kDa fragment on the basal sides of cultured cells, because they do have hemidesmosomes including integrin ␣ 6 ␤ 4 and BP180. Another possibility is that an unidentified extracellular ligand(s) of BP180 exists in tissue but not in cultured cells.
The presence of the 120-kDa fragment in skin suggests that the cleavage has some biological meaning. In wound healing or stratification, hemidesmosomes must be detached from the basement membrane, with transmembrane proteins becoming separated from their ligands.
Like other integrins, ␣ 6 ␤ 4 appears highly regulative and its recycling has been shown during cell locomotion (38). During epithelial wound healing of cornea, the integrin ␣ 6 ␤ 4 redistributes from their location within hemidesmosomes to more evenly in the basal cell membrane, and this event occurs without any measurable change in the synthesis of ␣ 6 ␤ 4 and with no indication of proteolytic cleavage of either ␣ 6 or ␤ 4 chain (39). Recent studies have demonstrated that the ligand ligation of integrin ␣ 6 ␤ 4 causes tyrosine phosphorylation on its cytoplasmic domain (40). Considering these results, it is reasonable to suppose that integrin ␣ 6 ␤ 4 controls ligand detachment by some structural change in the cytoplasmic domain. Our structural analyses of BP180 have demonstrated collagenous trimer formation (35). From the molecular dimension, we suggested that BP180 is a major component of anchoring filaments and warranting attention as a target molecule of cicatricial pemphigoid, responsible for an autoimmune subepidermal blistering disease with scar formation. Recently, Masunaga et al. (41) have demonstrated that the extracellular domain of BP180 extends to the lamina densa of skin BMZ by immunoelectron microscopy. Considering the stable collagenous extracellular rod of BP180, it might be expected that BP180 is not as regulative as integrin ␣ 6 ␤ 4 . Thus if structural change in the cytoplasmic domain cannot cause detachment of its extracellular domain from its ligand, a capacity for cleavage would be necessary to allow differentiation and movement away from the basement membrane. Kitajima et al. (42,43) observed disappearance of BP180 in DJM-1 cells during rearrangement of hemidesmosomes induced by the treatment with 12-O-tetradecanoylphorbol-13-acetate. However, the possibility that the fragment plays a more direct role in cell-matrix adhesion distinct from the intact molecule, cannot be excluded. The fact that significant amounts of the 60-kDa polypeptide could be detected on immunoblotting of cell extracts using mAb 1A8c but not 233 or 1D1, is suggestive of constitutive cleavage and removal of the 120-kDa extracellular part rather than generation of alternatively spliced products of BP180 gene.
Although this is highly speculative, cleavage of the extracellular part of BP180 might have some clinical, in addition to biological, significance. Thus alteration in BP180 fragments might cause autoimmune diseases like bullous pemphigoid and other skin blistering disorders. Linear IgA bullous dermatosis (LAD) is an autoimmune subepidermal blistering disease characterized by linear deposits of IgA autoantibodies at the dermal-epidermal basement membrane. Immunoblot analyses of patients' sera have demonstrated that the target antigens are heterogeneous, although a 97-kDa antigen is most frequently detected (44,45). Recent studies have shown that the autoantibodies recognize a 97-kDa and a 120-kDa polypeptide in skin extracts and a 120-kDa form in keratinocyte culture medium (46). The available data suggest that the 97-kDa peptide is a degradation rather than a specifically processed product. Immunoelectron microscopy has indicated that the 97-kDa polypeptide is localized to lamina lucida, suggesting a novel anchoring filament protein (47). Very recently, Pas et al. (48) have found that both BP IgG and LAD IgA recognize the same 120-kDa protein in keratinocyte conditioned medium and in cell extracts. They also showed that the 120-kDa protein is glycosylated and collagenous, and lacking in culture medium and in skin from patients with generalized atrophic benign epidermolysis bullosa. These results suggest close relationship between LAD antigen and BP180, though LAD sera have so far not been found to recognize BP180.
We have no direct evidence concerning the relationship between the LAD antigen(s) and the 120-kDa BP180 fragment at present. However, further characterization of the latter appears warranted to clarify the molecular nature and function of BP180, under physiological and pathological conditions.