Molecular properties of wild-type and mutant βIG-H3 proteins

Jung Eun Kim, Rang Woon Park, Je Yong Choi, Yong Chul Bae, Ki San Kim, Choun Ki Joo, In-San Kim

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

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 of βIG-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 of βIG-H3 may require other corneaspecific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies.

Original languageEnglish
Pages (from-to)656-661
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number3
Publication statusPublished - 2002 Mar 11
Externally publishedYes

Fingerprint

Mutation
Laminin
Collagen Type I
Proteins
Fibronectins
Epithelial Cells
Collagen Type VI
Hexosaminidases
Collagen Type IV
Collagen Type II
Extracellular Matrix Proteins
Cell Adhesion Molecules
Immunoprecipitation
Cell Adhesion
Transfection
Electrophoresis
Electron Microscopy
Complementary DNA
Gels
Escherichia coli

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Kim, J. E., Park, R. W., Choi, J. Y., Bae, Y. C., Kim, K. S., Joo, C. K., & Kim, I-S. (2002). Molecular properties of wild-type and mutant βIG-H3 proteins. Investigative Ophthalmology and Visual Science, 43(3), 656-661.

Molecular properties of wild-type and mutant βIG-H3 proteins. / Kim, Jung Eun; Park, Rang Woon; Choi, Je Yong; Bae, Yong Chul; Kim, Ki San; Joo, Choun Ki; Kim, In-San.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 3, 11.03.2002, p. 656-661.

Research output: Contribution to journalArticle

Kim, JE, Park, RW, Choi, JY, Bae, YC, Kim, KS, Joo, CK & Kim, I-S 2002, 'Molecular properties of wild-type and mutant βIG-H3 proteins', Investigative Ophthalmology and Visual Science, vol. 43, no. 3, pp. 656-661.
Kim JE, Park RW, Choi JY, Bae YC, Kim KS, Joo CK et al. Molecular properties of wild-type and mutant βIG-H3 proteins. Investigative Ophthalmology and Visual Science. 2002 Mar 11;43(3):656-661.
Kim, Jung Eun ; Park, Rang Woon ; Choi, Je Yong ; Bae, Yong Chul ; Kim, Ki San ; Joo, Choun Ki ; Kim, In-San. / Molecular properties of wild-type and mutant βIG-H3 proteins. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 3. pp. 656-661.
@article{3502fce8e7af4682ba043b0839144fc7,
title = "Molecular properties of wild-type and mutant βIG-H3 proteins",
abstract = "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 of βIG-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 of βIG-H3 may require other corneaspecific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies.",
author = "Kim, {Jung Eun} and Park, {Rang Woon} and Choi, {Je Yong} and Bae, {Yong Chul} and Kim, {Ki San} and Joo, {Choun Ki} and In-San Kim",
year = "2002",
month = "3",
day = "11",
language = "English",
volume = "43",
pages = "656--661",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "3",

}

TY - JOUR

T1 - Molecular properties of wild-type and mutant βIG-H3 proteins

AU - Kim, Jung Eun

AU - Park, Rang Woon

AU - Choi, Je Yong

AU - Bae, Yong Chul

AU - Kim, Ki San

AU - Joo, Choun Ki

AU - Kim, In-San

PY - 2002/3/11

Y1 - 2002/3/11

N2 - 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 of βIG-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 of βIG-H3 may require other corneaspecific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies.

AB - 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 of βIG-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 of βIG-H3 may require other corneaspecific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies.

UR - http://www.scopus.com/inward/record.url?scp=0036184212&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036184212&partnerID=8YFLogxK

M3 - Article

C2 - 11867580

AN - SCOPUS:0036184212

VL - 43

SP - 656

EP - 661

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 3

ER -