Molecular genetics of the 180-kDa bullous pemphigoid antigen (BPAG2/COL17A1), a transmembrane protein of the basement membrane zone of the stratifying squamous epithelia
Cloning of the human and mouse 180-kD bullous pemphigoid antigen (BPAG2) cDNAs has provided amino acid sequence information, suggesting that this protein is a collagen. To test for the triple-helical conformation of native BPAG2, membrane proteins were isolated from BALB/k mouse keratinocytes and from human HaCaT107 cells. The proteins were biotinylated, and BPAG2 protein was immunoprecipitated with specific antibodies. Proteins obtained were subjected to limited pepsin digestion using conditions under which triple-helical collagenous domains resist proteolysis. The results indicated the presence of a 25-kD polypeptide, corresponding to the size of the COLI segment in BPAG2 polypeptide consisting of 242 amino acids. The proteins were also subjected to collagenase digestion, and results indicated that there is a fragment of 45-kDa corresponding to the NCl domain. In support of the collagenous nature of BPAG2 was the observation that incubation of the cells with ascorbic acid, a necessary co-factor for hydroxylation of prolyl residues in collagen, significantly enhanced the level of expression as determined by indirect immunofluorescence and flow cytometry. Finally, evaluation of the distribution of the transfected protein in HaCaT cells by immunofluorescence confocal laser scanning microscopy revealed that the C-terminus of the protein resided on the outer surface of the cells. Thus, BPAG2 is a transmembrane collagen XVII, in type II topography. We further developed deletion constructs that were transfected into HaCaT cells. It was found that the deletions of the 27 amino acid NC extracellular domain, which is located immediately following the transmembrane domain, and the COLI domain do not have an impact on membrane polarization. Deletion of a stretch of 12 amino acids at the N-terminus induces apical surface polarization, while deletion of the transmembrane domain prevents the protein from translocating to the extracellular matrix. Understanding the functional role of the protein domains is critical for evaluation of the consequences of the mutations in the COL17A1/BPAG2 gene, demonstrated in a patient with a dominant glycine substitution that results in junctional epidermolysis bullosa and abnormal dentition. This led to the generation of a minigene where 135 amino acids of the COLI domain were removed to test for the functional role of the triple helical domains of BPAG2. Five lines of mice were generated, where the protein was expressed but no dominant negative effect was observed. This indicated that a more subtle change, such as a mutation, in the triple helix could generate a more severe phenotype by having a greater impact in the triple helical stability as seen in GABEB patients.
Cellular biology|Genetics|Molecular biology
Limardo, Maria Isabel, "Molecular genetics of the 180-kDa bullous pemphigoid antigen (BPAG2/COL17A1), a transmembrane protein of the basement membrane zone of the stratifying squamous epithelia" (1997). ETD Collection for Thomas Jefferson University. AAI9829086.