Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

Abstract

The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue.In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin.The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture.Anchoring fibrils are well-known suprastructures of skin. They presumably mediate the attachment of the epidermis to the underlying dermal connective tissue [l -31. At their upper end they are inserted into the basement membrane zone of the dermo-epidermal junction. Their lower end can be localized among the connective tissue fibers in the most superficial layer of dermis, i.e. the papillary dermis. The lateral density of anchoring fibrils varies significantly from one location to another in normal skin [2]; and in some genetic disorders, such as hereditary epidermolysis bullosa, the fibrils appear to be defective or absent [4 -61. There are various collagenous suprastructures in skin containing the interstitial collagens type I, 111, V and VI. The dermo-epidermal basement membrane consists in part of typeIV collagen [7, 81. Anchoring fibrils are also of collagenous nature. Sakai and coworkers recently demonstrated by immunoelectron microscopy that type VII is an important structural component of these fibrils [9].Type VII collagen was first isolated as peptic fragments from human fetal membranes. It contains a triple-helical domain 1.5 times the length of the interstitial collagens and, therefore, was first called long-chain collagen [lo]. It comprises three supposedly identical disulfide-linked chains and can be fragmented by pepsin to yield the characteristic polypeptides P1 and P2.

Proteinases of the Bone Morphogenetic Protein-1 Family Convert Procollagen VII to Mature Anchoring Fibril Collagen

“…NC1-F3 to the NC-1 domain, 3 VII-aff against the triple-helical domain (46), and NC2-7 and NC2-10 against the NC-2 domain of procollagen VII (32). The antibody NC2-7 recognizes epitopes within the propeptide, and therewith only procollagen VII but not mature collagen VII.…”

Some, but Not All, Glycine Substitution Mutations inCOL7A1 Result in Intracellular Accumulation of Collagen VII, Loss of Anchoring Fibrils, and Skin Blistering

“…Antibodies and Immunofluorescence Staining-The polyclonal antibodies to the triple helical and carboxyl-terminal (NC-2) domains of human collagen VII were produced as described (3,30). The monoclonal antibody LH-7.2 to the amino-terminal (NC-1) domain of collagen VII was a kind gift from Dr.…”

Tissue-specific expression and long-term deposition of human collagen VII in the skin of transgenic mice: implications for gene therapy

“…For the human fetal skin and E16.5 mouse fetuses, the cryosections were air dried, rinsed with PBS and incubated at 4°C for 16 h with 1.3a-VII (diluted 1:2 in PBS/BSA), a rabbit-anti-human, polyclonal antibody against human collagen VII. 57 After washing in PBS, goat anti-rabbit fluorescein-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA), diluted 1:50 in PBS/BSA, was applied for 30 min at room temperature. After further washing in PBS, the sections were mounted with Vector Shield (Vector Laboratories) and photographed under a fluorescence microscope (Zeiss Axioskop; Zeiss, Jena, Germany) using Kodak Ektachrome ASA400 (Kodak, Rochester, New York, USA).…”