In the cornea, extracellular matrix (ECM) molecules provide structural support and the ability to transfer signal from the environment to cell surfaces. The cornea uses ECM molecules, in particular collagens, to achieve and maintain transparency. Our overall objective is to understand how the cornea regulates the synthesis, assembly, and differential expression of collagens during development. Regulation involves transcription of specific collagen type chains. Several collagens are being synthesized simultaneously, so the selection of correct alpha chains to assemble into trimeric molecules must be regulated. Collagens are then secreted and assembled within the extracellular environment and must be positioned near their interacting partners. We will investigate how the cornea regulates some of these events. At 5 days of embryonic development, the fibril associated collagens with interrupted triple helices (FACITs), types XII and XIV, are both synthesized by the corneal epithelium. Despite their similarity, they are assembled only as homotrimers, and never as heterotrimers.
Our first aim i s to identify the sequence within the non-collagenous carboxyl termini (NC1 domains) of each FACIT collagen that regulates trimer alpha chain selection. We will substitute regions of the collagen XIV NC1 domain with the corresponding region of collagen XII. We will then determine whether corneal epithelial cells assemble collagen XIV (containing a small collagen XII region) into heterotrimers with normal type XII alpha chains.
A second aim will investigate whether type XIV collagen, which sits on fibrils with its carboxyl end, might indirectly bind its amino terminal end to an adjacent fibril via a keratan sulfate proteoglycan. We will immobilize collagen XIV amino terminal domains on columns, then apply a mix of corneal keratan sulfate proteoglycans (KSPGs) to allow binding. The KSPGs will then be eluted and identified with specific antibodies by Western blots. The architecture of the cornea must be absolutely precise to be transparent. We hypothesize that the synthesis and placement of three newly discovered collagens are essential for development and function of the cornea, therefore our third aim is to examine their expression pattern during corneal development. One which we have cloned is type XX collagen, a new member of the FACITs. Another is type XXI collagen, a new fibrillar collagen, isolated at a time when it was believed that all the fibrillar collagens were known. The third is type XXIII collagen, a putative transmembrane molecule. Because the chick cornea is an ideal model for developmental studies that are not possible with the human cornea, we will try to correlate specific developmental events with possible functions of these molecules, based on expression pattern. This will be accomplished by immunofluorescence with specific antibodies and in situ hybridization with cDNA probes. We will also perform cell fractionations to prove collagen XXIII is a transmembrane molecule.
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