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Jung-Eun Kim, Rang-Woon Park, Je-Yong Choi, Yong-Chul Bae, Ki-San Kim, Choun-Ki Joo, In-San Kim; Molecular Properties of Wild-Type and Mutant βIG-H3 Proteins. Invest. Ophthalmol. Vis. Sci. 2002;43(3):656-661.
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purpose. βIG-H3 is a TGF-β–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 βIG-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 βIG-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 βIG-H3 with other matrix proteins. Wild-type and
mutant βIG-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 βIG-H3. Cell adhesion was
assayed by measuring enzyme activities of N-acetyl-β-d-glucosaminidase.
results. The recombinant βIG-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 ofβ
IG-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 βIG-H3.
conclusions. βIG-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 ofβ
IG-H3 may require other cornea-specific factors, to form the
abnormal accumulations in 5q31-linked corneal
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