Two Forms of Collagen XVII in Keratinocytes(1)
Collagen XVII, also known as the 180-kDa bullous pemphigoid antigen or BP180, is a structural component of the hemidesmosomes in epithelial cells. Polyclonal antibodies raised against recombinant procaryotic fragments spanning the endodomain and the distal ectodomain of collagen XVII gave an intensive immune response in rabbits (antibody SA 3485) or in chicken (antibody Col17ecto-1).
The α1(XVII) chains from both cells and human epidermis showed similar migration on SDS-PAGE. The detergent was necessary for solubilization of collagen XVII from the cells, since no collagen XVII was extractable with phosphate-buffered saline or 0.5 M NaCl, 0.1 M Tris, pH 7.4, or 0.1 M acetic acid. Both antibodies SA 3485 and Col17ecto-1 precipitated a biotinylated 180-kDa band, demonstrating that the polypeptide recognized by them contained a biotinylated extracellular domain.
Treatment with highly purified bacterial collagenase yielded a band of approximately 65 kDa on SDS-PAGE under reducing conditions. Under non-reducing conditions, the collagenase-resistant fragment migrated with an apparent molecular mass of 170 kDa. The intact collagen XVII migrated as a large polymer under non-reducing conditions. The collagenase-resistant bands were recognized by the endodomain antibody SA 3485, but not by the ectodomain antibodies. Yielding several intermediate products with apparent sizes of 140, 120, and 105 kDa, and a final pepsin-resistant fragment of about 90 kDa, both under reducing and non-reducing conditions. Limited trypsin digestion also resulted in a 90-kDa fragment.
Antibody Col17ecto-1 raised against the 205 most COOH-terminal amino acid residues of collagen XVII reacted only with the 140-, 120-, and 105-kDa fragments, but not with the 90-kDa fragment. When collagenase digestion preceded deglycosylation, no difference in the molecular mass of the endodomain was noted. Differences emerged when the extracellular domain was deglycosylated: shifts in migration of the 140- and 120-kDa, but not of the 105- and 90-kDa pepsin fragments were observed.
To test for the presence of collagen XVII in cell culture media, concentrated keratinocyte medium was immunoblotted with collagen XVII antibodies. The antibodies Col17ecto-1 and IF 77/95 both showed reactivity with a 120-kDa polypeptide, but no signal was obtained with the antibody SA 3485. The immunoreactive molecule was sensitive to collagenase or to 1 μg/ml pepsin in a similar manner and extent as the extracellular domain of full-length collagen XVII. Deglycosylation with N-glycosidase F resulted in an apparent 3–5-kDa shift in migration on SDS-PAGE. The 120-kDa polypeptide was a very minor component in the cell extracts, and overloading on SDS-PAGE was required to demonstrate its presence. Sequential extraction of keratinocytes with 0.5 M NaCl in a neutral buffer, followed by Nonidet P-40 extraction, revealed the 120-kDa polypeptide in the 0.5 M NaCl extract, indicating that it did not require a detergent for solubilization. The estimated ratio of the 120-kDa polypeptides in the medium to the 180-kDa α1(XVII) chains in the cell layer was approximately 1:10–1:20. The amount of keratinocyte medium concentrate used for immunoblotting was derived from 10 ml of medium in a 50-cm2 monolayer culture.
Protein Extractions
For analysis of collagen XVII from cell cultures, proteins in the cell layer and the media were processed separately. The cell layers were extracted for 30 min on ice with 1 ml/75 cm2 of a neutral buffer containing 1% Nonidet P-40, 0.1 M NaCl, 25 mM Tris-HCl, pH 7.4, and 10 mM EDTA, 1 mM Pefabloc, and when appropriate,14 ug/ml chymostatin, 7 ug/ml antipain, 7 ug/ml leupeptin, and 14 ug/ml pepstatin as proteinase inhibitors. The cell lysate was then scraped with a rubber policeman, and the extract was centrifuged at 14,000 x g at 4 °C. The supernatant was used for further analyses. In some experiments, the above extraction was preceded by incubation of the cells with 0.5 M NaCl in 0.05 M Tris-HCl, pH 8.2, to release neutral salt soluble proteins. For analysis of medium proteins, proteinase inhibitors were added immediately after collecting the medium onto ice. After removing cellular debris by centrifugation 1000 rpm for 10 min, the proteins from 10 ml medium were precipitated with ammonium sulfate to 30% saturation for 4 h at 4 °C. After centrifugation at 15 000 x g for 60 min at 4 °C, the pellets were dissolved in 100 ml of a buffer containing 65 mM NaCl, 25 mM Tris-HCl, pH 7.4, 1 mM Pefabloc, and 1 mM EDTA. Fifty to 100 ul of the cell extracts or the medium concentrate were used for the enzyme digestions and 10–30 ul for immunoblotting.
For extraction of collagen XVII from the epidermis, normal human skin was subjected to artificial epidermolysis in a neutral buffer containing 20 mM EDTA and the above proteinase inhibitors at 4 °C overnight, and the epidermis and the dermis were mechanically separated. Both skin layers were extracted with 400 μl/cm2 of a buffer containing 8 M urea, 2% SDS, 0.05 M Tris-HCl, pH 6.8, and proteinase inhibitors for 2 min at 95 °C, followed by extensive dialysis against 0.8M urea, 2% SDS, 5% glycerol in 0.1 m Tris, pH 6.8, as described previously. Five to 15 μl of the extract was used for immunoblotting; application of significantly more than 15 μl of the epidermis extract onto SDS-PAGE1 was not possible, because the high content of keratins in the extract caused protein overloading of the lanes.