Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide (1
There are at least three hemidesmosomal transmembrane polypeptides, i.e. integrin alpha 6 and beta 4 subunits and BP180, which is also called type XVII collagen. The integrin alpha 6 beta 4, an adhesion molecule whose ligands are laminins, especially laminin-5, plays an essential role in assembly and functioning of hemidesmosomes. 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. 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.
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. 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. mAb 1337 recognizes primarily the 120-kDa fragment of BP180 on immunoblotting.
Immunofluorescence microscopy with mAb 1337 showed the antigenic fragment to be lacking or present at only a very low level on BMGE+H cells.
Media from BMGE+H 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. 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. 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.
The molar ratio of the 120-kDa polypeptide, Triton X-100-soluble BP180, and Triton X-100-insoluble BP180 was roughly 1:6:3 in BMGE+H cells. The approximately 60-kDa component recognized by mAb 1A8c should be noted. 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. the soluble form of BP230, a hemidesmosomal plaque component, was detected in the cytosolic fraction but not in Triton X-100-soluble membrane-bound fraction.
Trimer formation by the 120-kDa fragment was confirmed by immunoblotting. In the non-boiled condition, the 120-kDa fragment in the medium fraction was detected as a 350-kDa component. mAb 1337 recognized the fragment in both monomer and trimer forms, but did not recognize the intact 180-kDa polypeptide.
While mAb 233 precipitated both the 120-kDa fragment and the intact BP180 molecule, mAb 1337 precipitated only the 120-kDa fragment. The BMGE−H cell line lacking BP antigens was also found to be only one without the 120-kDa fragment in its medium fraction. 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. The eluted fractions from the mAb 233 column showed the 120-kDa band, recognized by mAb 233 and other monoclonal antibodies (mAb 1D1, mAb D20, and mAb R223) that binding to the extracellular part of BP180. 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. The 120-kDa fragment was recognized by the polyclonal antibody, while the 60-kDa fragment was hardly detected. 100-kDa fragments are produced by cleavage at the carboxyl-terminal side of the 120-kDa fragment. The 100-kDa fragment was recognized by mAbs 233, D20, and R223, but not by mAb 1D
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. 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. 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. Linear IgA bullous dermatosis (LAD) is an autoimmune subepidermal blistering disease characterized by linear deposits of IgA autoantibodies at the dermal-epidermal basement membrane.