Characterization of the collagen in the hexagonal lattice of Descemet's membrane: its relation to type VIII collagen.

Abstract

Abstract. To investigate the nature of the hexagonal lattice structure in Descemet's membrane, monoclonal antibodies were raised against a homogenate of bovine Descemet's membranes. They were screened by immunofluorescence microscopy to obtain antibodies that label Descemet's membrane. Some monoclonal antibodies labeled both Descemet's membrane and fine filaments within the stroma. In electron microscopy, with immunogold labeling on a critical point dried specimen, the antibodies labeled the hexagonal lattices and long-spacing structures produced by the bovine corneal endothelial cells in culture; 6A2 antibodies labeled the nodes of the lattice and 9H3 antibodies labeled the sides of the lattice. These antibodies also labeled the hexagonal lattice of Descemet's membrane in situ in ultrathin frozen sectioning. In immunofluorescence, these antibodies stained the sclera, choroid, and optic nerve sheath and its septum. They also labeled the dura mater of the spinal cord, and the perichondrium of the tracheal cartilage. In immunoblotting, the antibodies recognized 64-kD collagenous peptides both in tissue culture and in Descemet's membrane in vivo. They also recognized 50-kD pepsin-resistant fragments from Descemet's membranes that are related to type VIII collagen. However, they did not react either in immunoblotting or in immunoprecipitation with medium of subconfluent cultures from which type VIII collagen had been obtained. The results are discussed with reference to the nature of type VIII collagen, which is currently under dispute. This lattice collagen may be a member of a novel class of long-spacing fibrils.ESCEMET'S membrane (DM) ~ is the basement membrane of corneal endothelial cells. DMs of some animal species contain characteristic stacks of hexagonal lattices that are arranged parallel to the surface of the membrane (16). The lattice is composed of electron-dense nodes at the vertexes and rodlike structures that connect the nodes. This highly regular arrangement has aroused the interests of many researchers. Linearly-arranged long-spacing (LS) structures with similar periodicities are often found in the vicinity of various basement membranes (for review, see reference 11). In an in vitro culture experiment, it was shown that the lattice consists of dumbbell-shaped unit structures 160 nm in length (33). They adhere to each other at their round ends to form hexagonal lattices or linear LS structures of ,x,150 nm in periodicity. The component of the lattice has been regarded as collagenous (16,32). Type 11 collagen (15), type IV collagen (41), type VI collagen (6, 7), and type VIII collagen (18) have been postulated as candidates for the component of the lattice. However, in our previous immunoelecDr. Sawada's present address is the

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