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Molecular properties of wild-type and mutant betaIG-H3 proteins.

Abstract：

PURPOSE:BetaIG-H3 is a TGF-beta-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 betaIG-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 betaIG-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 betaIG-H3 with other matrix proteins. Wild-type and mutant betaIG-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 betaIG-H3. Cell adhesion was assayed by measuring enzyme activities of N-acetyl-beta-D-glucosaminidase. RESULTS:The recombinant betaIG-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 betaIG-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 betaIG-H3. CONCLUSIONS:BetaIG-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 betaIG-H3 may require other cornea-specific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Kim JE,Park RW,Choi JY,Bae YC,Kim KS,Joo CK,Kim IS

keywords:

subject

Has Abstract

pub_date

2002-03-01 00:00:00

pages

656-61

issue

eissn

0146-0404

issn

1552-5783

journal_volume

pub_type

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Molecular properties of wild-type and mutant betaIG-H3 proteins.