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1 From the Department of Biochemistry, School of Medicine and the 2 Department of Oral Anatomy, School of Dentistry, Kyungpook National University, Taegu, Korea; and 3 KIMKISAN Eye Center and the 4 Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
PURPOSE. ßIG-H3 is a TGF-ß–induced cell adhesion molecule, the mutations of which are responsible for a group of 5q31-linked corneal dystrophies. The characteristic findings in these diseases are accumulation of protein deposits of different ultrastructures. To understand the mechanisms of protein deposits in 5q31-linked corneal dystrophies, the molecular properties of ßIG-H3 and the effects of mutation on these properties were studied in vitro.
METHODS. Substitution mutations were generated by two-step PCR. Wild-type and mutant recombinant ßIG-H3 proteins were raised in Escherichia coli. For structural study, nondenaturing gel electrophoresis, cross-linking experiments, and electron microscopy examination were performed. A solid-phase interaction assay was performed for the interaction of ßIG-H3 with other matrix proteins. Wild-type and mutant ßIG-H3 cDNAs were cloned into a mammalian expression vector and overexpressed in the corneal epithelial cells by transient transfection. Immunoprecipitation and immunoblot analysis were performed with an antibody against human ßIG-H3. Cell adhesion was assayed by measuring enzyme activities of N-acetyl-ß-D-glucosaminidase.
RESULTS. The recombinant ßIG-H3 protein self-assembled to form multimeric bands and appeared to have a fibrillar structure. Solid-phase in vitro interaction assay showed that it bound strongly to type I collagen, fibronectin, and laminin; moderately to collagen type II and VI; and minimally to collagen type IV. Five recombinant mutant forms of ßIG-H3 (R124C, R124H, R124L, R555W, and R555Q) commonly found in 5q31-linked corneal dystrophies did not significantly affect the fibrillar structure, interactions with other extracellular matrix proteins, or adhesion activity in cultured corneal epithelial cells. In addition, the mutations apparently produced degradation products similar to those of wild-type ßIG-H3.
CONCLUSIONS. ßIG-H3 polymerizes to form a fibrillar structure and strongly interacts with type I collagen, laminin, and fibronectin. Mutations found in the 5q31-linked corneal dystrophies do not significantly affect these properties. The results suggest that mutant forms of ßIG-H3 may require other cornea-specific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies.
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