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(Investigative Ophthalmology and Visual Science. 2002;43:656-661.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Molecular Properties of Wild-Type and Mutant ßIG-H3 Proteins

Jung-Eun Kim1, Rang-Woon Park1, Je-Yong Choi1, Yong-Chul Bae2, Ki-San Kim3, Choun-Ki Joo4 and In-San Kim1

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ßIG-H3 gene, also known as TGFBI, was first identified by Skonier et al.1 who isolated it by screening a cDNA library made from a human lung adenocarcinoma cell line (A549) that had been treated with TGF-ß. The ßIG-H3 protein is composed of 683 amino acids containing short amino acid regions homologous to similar motifs in Drosophila fasciclin-I and four homologous internal domains. We have reported that ßIG-H3 mediates corneal epithelial cell adhesion through {alpha}3ß1 integrin, and we have identified two motifs interacting with {alpha}3ß1 integrin within repeat domains of ßIG-H3.2 Mutations of ßIG-H3 were demonstrated to be responsible for 5q31-linked human autosomal dominant corneal dystrophies, such as granular (GCD), Reis-Bückler (RBCD), lattice type I (LCD-1), and Avellino (ACD) corneal dystrophies.3 These diseases are characterized by progressive accumulation of protein deposits in the cornea, leading to severe visual impairment. Depending on the mutation, the accumulations form rod-shaped crystalloid structures, amyloid, a combination of rod-shaped bodies with amyloid, or curly fibers.4 The appearance of the opacities depends on the location and nature of the corneal deposits, and this is presumably influenced by the three-dimensional structure of the mutant proteins. Although the immunohistochemical studies5 6 demonstrated that ßIG-H3 is strongly stained in the pathologic deposits in all ßIG-H3-related corneal dystrophies, the role of the different mutations in the formation of different types of deposits is largely unknown. Even, the structure of wild-type ßIG-H3 and its interaction with other extracellular matrix (ECM) proteins are not known. To gain insight into the mechanism of how mutations of ßIG-H3 lead to the accumulation of pathologic deposits in 5q31-linked corneal dystrophies, we first studied the molecular properties of ßIG-H3, including the structure and interactions with other ECM proteins, and then the effects of mutations on these properties.

In the present study, we demonstrated that the recombinant human ßIG-H3 protein polymerized to form a fibrillar structure and strongly interacted with type I collagen, fibronectin, and laminin. 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 ECM proteins, and cell adhesion activity. In addition, they apparently produced degradation products similar to those of wild-type ßIG-H3. Although mutations of ßIG-H3 found in 5q31-linked corneal dystrophies are systemic, other tissues, excepting the cornea, do not seem to be affected, which raises the question of whether other corneal components contribute to the aggregates. Taking all evidence together, we suggest that mutant forms of ßIG-H3 may require other cornea-specific factors for abnormal accumulations to develop in 5q31-linked corneal dystrophies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of Recombinant Wild-Type and Mutant ßIG-H3 Proteins
For making wild-type and mutant recombinant ßIG-H3 proteins, the cDNA coding amino acids 69-653, which encompass four fasciclin domains of ßIG-H3 was subcloned into the pET-29b(+) vector (pßN68; Novagen, Madison, WI). For mammalian cell transfection experiments, full-length ßIG-H3 cDNA was subcloned into pcDNA3.1/Myc-His A (pMycß-WT; Invitrogen, Carlsbad, CA). Substitution mutations were introduced into both pßN68 and pMycß-WT by two-step PCR, as described previously.7 We generated five substitution mutants commonly found in the 5q31-linked corneal dystrophies: three mutations of 124 arginine to histidine (R124H), cysteine (R124C), and leucine (R124L) and two mutations of 555 arginine to tryptophan (R555W) and glutamine (R555Q). The mutations were confirmed by DNA sequencing. The wild-type and substitution mutant recombinant proteins were purified as described previously.2 These recombinant proteins were analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and nondenaturing (ND)-PAGE. Western blot analysis was performed with an anti-ßIG-H3 antibody, which has been previously described by us.8

Cross-linking
Recombinant human ßIG-H3 proteins were incubated in glutaraldehyde solutions, with concentrations ranging from 0.01% to 1%, at 20°C for 5 minutes. Reactions were stopped by the addition of SDS-PAGE sample loading buffer. Mixtures were electrophoresed on a 10% SDS-polyacrylamide gel.

Cell Culture
Human corneal epithelial (HCE) cells were cultured in Dulbecco’s modified Eagle’s medium with nutrient mixture F-12 (DMEM/F-12; Gibco BRL, Gaithersburg, MD) supplemented with 15% FBS, 5 µg/mL insulin, 0.1 µg/mL cholera toxin, and 10 ng/mL of human epidermal growth factor (hEGF) at 37°C in 5% CO2. Cultured HCE cells have been characterized as showing properties similar to those of in vivo cells.9

Immobilization Assay
Flat-bottomed, 96-well, enzyme-linked immunosorbent assay (ELISA) plates were precoated with various ECM proteins at a concentration of 0.5 µg in 100 µL of 20 mM carbonate buffer (pH 9.6) overnight at 4°C. The coated ECM proteins used were as follows: purified human plasma fibronectin (pFN), chicken collagen types I and II (Chemicon International Inc. Temecula, CA), bovine collagen types IV and VI (Chemicon), mouse laminin (Chemicon), and bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO). Nonspecific binding sites were blocked using phosphate-buffered saline (PBS)-0.05% Tween 20 for 1 hour at 37°C. Different concentrations of recombinant ßIG-H3 proteins, ranging from 0.1 to 10 µg in 100 µL PBS-0.05% Tween 20, were added and incubated for 2 hours at 37°C. All wells were washed with PBS-0.05% Tween 20, incubated with anti-His-HRP antibody (Invitrogen) for 2 hours at 37°C, and rewashed. A 200-µL solution of 0.1 mg/mL o-phenylenediamine (Sigma) with 0.003% H2O2 and 1% methanol was added to each well and incubated for 1 hour at 37°C in a dark place. The reaction was stopped by the addition of 50 µL of 3 M H2SO4, and the absorbance was measured at 492 nm in a microplate reader (Multiskan MCC/340; Titertek, Huntsville, AL).

Cell Adhesion Assay
The cell adhesion assay was performed as described previously.2 Briefly, 96-well microculture plates (Falcon Labware; BD Biosciences, Mountain View, CA) were incubated with recombinant ßIG-H3 proteins or plasma fibronectin at 37°C for 1 hour and then blocked with PBS containing 0.2% BSA for 1 hour at the same temperature. HCE cells were trypsinized and suspended in the culture media at a density of 2 x 105 cells/mL, and 0.1 mL of the cell suspension was then added to each well of the plates. Cell attachment was analyzed as follows: After incubation for 1 hour at 37°C, unattached cells were removed by rinsing with PBS. Attached cells were incubated for 1 hour at 37°C in 50 mM citrate buffer (pH 5.0) containing 3.75 mM p-nitrophenol-N-acetyl 1-ß-D-glycosaminide (hexosaminidase substrate) and 0.25% Triton X-100. Enzyme activity was blocked by the addition of 50 mM glycine buffer (pH 10.4) containing 5 mM EDTA, and the absorbance was measured at 405 nm in the microplate reader.

Electron Microscopic Method
Negative staining was performed at a neutral pH to avoid the dissociation of aggregates. Ten microliters of a protein solution was placed on a coated (Formvar; SPI, West Chester, PA) single-slot grid, and 5 µL of a 2% sodium phosphotungstate solution of pH 7 was added. After removal of the first stain, incubation was repeated for 2 minutes. The stained grids were viewed in an electron microscope (model H-600; Hitachi Ltd., Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fibrillar Structure of ßIG-H3 and the Effects of Mutations
That ßIG-H3 has four internal repeated domains, which has been suggested to fold into a bivalent tetrameric structure,10 and that several bands with higher molecular mass than expected were detected on Western blot analysis of the recombinant ßIG-H3 protein (data not shown), prompted us to test whether ßIG-H3 forms multimeric structures. To answer this question, the recombinant ßIG-H3 protein was analyzed by ND-PAGE. As is shown in Figure 1A , ßIG-H3 showed multiple bands forming a ladder. Glutaraldehyde cross-linking analysis was then used to examine the oligomeric state of ßIG-H3. ßIG-H3 proteins were incubated with several concentrations of glutaraldehyde, ranging from 0.01% to 1% and then analyzed by SDS-PAGE (Fig. 1B) . After treatment with 0.01% glutaraldehyde, distinct cross-linked forms were observed with sizes corresponding to those of dimer, trimer, and tetramer. At higher concentrations of glutaraldehyde, ßIG-H3 proteins were quantitatively cross-linked to produce the corresponding high-molecular-mass forms. Overexpressed ßIG-H3 in HCE cells also showed monomeric, dimeric, and trimeric bands by Western blot analysis of SDS-PAGE when the film was exposed for a long time (Fig. 1C) . Multimeric bands of the overexpressed ßIG-H3 in HCE cells were observed by ND-PAGE followed by immunoblot analysis (Fig. 1C) .



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  		Figure 1. Analysis of recombinant and overexpressed ßIG-H3 proteins by electrophoresis. (A) ND-PAGE analysis of purified recombinant ßIG-H3 protein. The purified recombinant ßIG-H3 protein was subjected to 8% ND-PAGE. Molecular mass standard (MW) for ND-PAGE urease indicates 272 kDa and 545 kDa. (B) Analysis of ßIG-H3 oligomeric forms by cross-linking with glutaraldehyde. ßIG-H3 protein was incubated with various concentrations of glutaraldehyde, as indicated. Cross-linked ßIG-H3 proteins were resolved by 10% SDS-PAGE. Arrowheads: distinct cross-linked forms corresponding to dimer, trimer, tetramer, and multimer. (C) Analysis of overexpressed native ßIG-H3. CHO cells were transiently transfected with pMycß-WT cDNA. ßIG-H3 protein secreted from transfected cells was then resolved by SDS-PAGE (lane 1) and ND-PAGE (lane 2), followed by immunoblot analysis with anti-ßIG-H3 antibody. Arrowheads: dimeric and trimeric bands on SDS-PAGE.

 
The nature of these multimeric structures was revealed by electron microscopy (Fig. 2) . Many thin fibrils aggregated in parallel to form thick fiberlike structures, with an apparent size distribution. They were assembled into approximately 8- to 10-nm wide fibrils and then clustered into thick fibers with different sizes ranging from 50 to 800 nm. These results suggest that ßIG-H3 self-assembles to form a fibrillar structure.



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  		Figure 2. The fibrillar structures of ßIG-H3 as visualized by electron microscopy after negative staining. Many thin fibrils assembled in parallel to form thick fiberlike structures.

 
To test whether protein aggregates in the pathologic deposits may be due to alterations in polymerization and structure caused by mutations, we constructed five mutant forms of ßIG-H3, which are commonly found in 5q31-linked corneal dystrophies. Arginine at 124 was replaced with cysteine, histidine, or leucine and arginine at 555 mutated into tryptophan or glutamine (Fig. 3A ). To test this hypothesis, recombinant proteins were analyzed by SDS-PAGE and ND-PAGE (Fig. 3B) . The results demonstrated that all five mutants showed multiple bands, as did the wild-type on ND-PAGE. Overexpressed mutant ßIG-H3 proteins in HCE cells (Fig. 3C) also showed patterns similar to those of wild-type ßIG-H3. In addition, electron microscopic examination showed that all mutant ßIG-H3 proteins had a fibrillar structure that was basically not different from that of the wild-type (data not shown). These results suggest that mutations of ßIG-H3 found in 5q31-linked corneal dystrophies may not elicit significant changes in the structure. They may affect the interactions with other ECM proteins, the susceptibility to proteases, or other biologic activities.



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  		Figure 3. Analysis of corneal dystrophy mutant ßIG-H3 proteins. (A) Diagram of corneal dystrophy mutant recombinant ßIG-H3 proteins. Hatched and gray boxes: highly conserved sequences of each repeat domains, and two mutation sites at 124 and 555 amino acids are indicated. (B) SDS-PAGE (left) and ND-PAGE (right) analysis of recombinant mutant ßIG-H3 proteins. (C) ND-PAGE analysis of mutant ßIG-H3 proteins overexpressed in CHO cells.

 
Interactions of ßIG-H3 with ECM Proteins and the Effects of Mutations
Because ßIG-H3 has been suggested to interact with some collagen types and other ECM proteins,11 some mutations may affect these interactions, resulting in abnormal accumulations. A solid-phase assay was conducted to examine the binding potential of various immobilized extracellular matrix proteins to ßIG-H3, when used as a soluble ligand. A dose-dependent curve was obtained when each immobilized ECM protein was incubated with increasing concentrations of ßIG-H3 (Fig. 4) . A plateau was obtained at a concentration of 50 µg/mL. A distinct high level of binding was observed in the case of type I collagen, fibronectin, and laminin, whereas moderate binding with type II and type VI collagen was observed. The interaction with collagen type IV was minimal (Fig. 4A) . We then tested the effects of mutations on these interactions. The interactions of all mutant ßIG-H3 proteins with ECM proteins were basically not different from those of the wild-type. The representative result of mutant R124C is demonstrated in Figure 4B . These results indicate that the interaction of ßIG-H3 with major ECM proteins may not play a critical role in forming abnormal aggregates in the cornea of 5q31-linked corneal dystrophies.



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  		Figure 4. Solid-phase interaction assay of wild-type (upper) and mutant proteins with several ECM proteins (lower). Flat-bottomed, 96-well ELISA plates were precoated with 0.5 µg of each ECM protein: human plasma fibronectin (FN), chicken collagen types I and II, bovine collagen types IV and VI, mouse laminin, and BSA. After blocking with PBS–0.05% Tween 20, wells were incubated with the indicated concentrations from 0.1 to 20 µg of recombinant ßIG-H3 proteins and then incubated with anti-ßIG-H3 antibody. All interactions were detected with 0.1 mg/mL o-phenylenediamine solution. The absorbance was measured at 492 nm in a microplate reader.

 
Degradation Products in Wild-Type and Mutant ßIG-H3 Proteins
That abnormal turnover of ßIG-H3 was found in some mutant forms12 prompted us to test whether mutant ßIG-H3 proteins have degradation products different from those of the wild-type ßIG-H3. Overexpressed mutant ßIG-H3s in HCE cells were immunoprecipitated by anti-ßIG-H3 antibody and then examined by immunoblot analysis. Unexpectedly, the degradation patterns of all mutant forms were not significantly different from those of the wild-type (Fig. 5A ). Similar patterns of degradation were observed between wild-type and mutant forms in the Western blot analysis of recombinant proteins (Fig. 5B) .



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  		Figure 5. Comparison of degradation products of wild-type and mutant ßIG-H3 proteins. (A) CHO cells were transfected with wild-type (WT) and five mutant ßIG-H3 proteins. Media from overexpressed cells were immunoprecipitated by anti-ßIG-H3 antiserum and detected by the same antibody. (B) Western blot analysis of recombinant wild-type and mutant proteins. Arrows: degradation products.

 
Effect of Mutations of ßIG-H3 on Cell Adhesion Activity
ßIG-H3 has been known to be synthesized in the corneal epithelium and has been suggested to play a role in maintaining the integrity of the corneal epithelium.13 Thus, abnormal ßIG-H3 proteins synthesized by the affected corneal epithelial cells may impair the integrity of the corneal epithelium. We2 have reported that ßIG-H3 supports corneal epithelial cell adhesion through {alpha}3ß1 integrin. Although {alpha}3ß1 integrin-interacting motifs of ßIG-H3 are different from the mutation sites, its cell adhesion activity may be affected by the conformational changes caused by mutations. All mutant ßIG-H3 proteins, however, mediated cell adhesion as efficiently as the wild-type ßIG-H3 (Fig. 6) .



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  		Figure 6. Comparison of cell adhesion activities of wild-type and mutant ßIG-H3 proteins. HCE cells were seeded onto 96-well microculture plates coated with increasing concentrations of recombinant wild-type or mutant proteins and incubated for 1 hour at 37°C. HCE cells attached to the surfaces were quantified by hexosaminidase assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To gain insight into the mechanism leading to abnormal deposits in 5q31-linked corneal dystrophies, we first investigated the structure of ßIG-H3 and its interactions with ECM proteins. In this study, we demonstrated that ßIG-H3 polymerizes to form a fibrillar structure. This is the first evidence showing the fibrillar structure of ßIG-H3. It was previously thought that the four repeat domains of ßIG-H3 are folded into a potential bivalent tetrameric structure.10 However, our results showed multiple bands, rather than a single tetrameric band. Although it showed dimeric, trimeric, or tetrameric bands, most proteins were incorporated into polymers. The polymeric nature was revealed by the electron microscopic examination. ßIG-H3 assembled into fibrillar aggregates and clustered into thick fibers with different sizes. Currently, it is unclear how ßIG-H3 assembles into polymers and which motifs are necessary for mediating assembly. We found that a single repeat domain itself did not show well-organized oligomeric bands. In addition, our study using artificial proteins having tandem repeats of one repeat domain showed that at least three repeat domains were required to have organized multiple oligomeric bands (data not shown). This suggests that a single repeat domain is not able to form polymeric structures. However, the exact mechanism for the fibrillar structure of ßIG-H3 remains to be further studied. The fibrillar structure of ßIG-H3 may account for the abnormal accumulations in the affected cornea. In our results, however, mutations did not significantly affect the fibrillar structure. Arginine at 124 is not located within one of the repeat domains. Although arginine at 555 exists in the fourth repeat domain, it is not well conserved in either of the other three repeat domains of ßIG-H3 or in fas-1 domains found in other proteins. Therefore, assuming that four repeat domains participate to form the fibrillar structure of ßIG-H3, it is unlikely that a single point mutation significantly affects the whole structure of ßIG-H3. Recently, Schmitt-Bernard et al.14 reported that a 22-amino-acid peptide containing the A124C mutation linked to LCD-1 forms the amyloid fibrils, whereas the native one merely forms fibrils. These findings suggest, together with our results, that amyloid deposits may be associated with a degradation product of ßIG-H3 containing a mutant amino acid, rather with the alterations of whole structure of ßIG-H3. The degradation products themselves, however, may not be sufficient to form amyloid deposits in vivo, because patients with LCD-1 do not display deposits in the skin,15 suggesting that other factors, probably locally determined in the cornea, are involved in the formation of amyloid deposits.

Although ßIG-H3 is prominent in aggregates in the 5q31-linked corneal dystrophies,6 the evidence is not conclusive of whether ßIG-H3 is the major component of the deposits. These aggregates may in part be due to interactions of ßIG-H3 with other matrix proteins, such as collagens, fibronectin, and laminin. In our solid-phase interaction assays, relatively strong interactions were observed with type I collagen, laminin, and fibronectin and weak interactions with type II and VI collagens. In contrast, the protein interacted minimally with type IV collagen. Type I collagen is the major collagen component of the corneal stroma and its ordered arrangement with other matrix components is important for corneal transparency. In fact, the major localization of ßIG-H3 in the corneal stroma is at the interfaces between collagen lamellae and at junctions of collagen bundles attached to disparate types of collagen, such as in the Descemet membrane.6 This suggests that the interaction between type I collagen and ßIG-H3 confers a bridging function on the ßIG-H3 protein. There is increasing evidence13 16 that ßIG-H3 colocalizes with type VI collagen in the cornea, and thus ßIG-H3 together with this collagen is suggested to have an anchoring function between the corneal stroma and the adjacent Descemet membrane and subepithelial tissues.6

It has been reported that ßIG-H3 is seen just beneath detached corneal epithelium in the subepithelial matrix13 and serves as an adhesion matrix for the corneal epithelial cells.2 In addition, the major source for ßIG-H3 in the normal cornea is thought to be the epithelium.16 Therefore, our results showing the interactions of ßIG-H3 with laminin and fibronectin suggest that ßIG-H3 is associated with components of basement membrane and functions to support the maintenance of corneal epithelial integrity. These interactions, however, do not seem to be affected significantly by mutations, because our mutational studies failed to show any marked alterations in interaction activities with matrix proteins. Because a single repeat domain did not interact at all with any of the tested ECM proteins (data not shown), multiple repeat domains may be required for the interactions of ßIG-H3 with matrix proteins. As is the case in the structural study, a single mutation of ßIG-H3 does not have any significant effect on its interaction activity. Alternatively, the variety of structural forms resulting from accumulation of ßIG-H3 in the dystrophic aggregates is considerable, raising the question of whether other corneal components other than matrix proteins contribute to the aggregates. Substances may include lectin-positive carbohydrate in LCD-1 and GCD17 and phospholipids in GCD.18 Recently, apolipoproteins J and E also have been suggested to be associated with amyloid deposits in LCD-1.19 Mutant ßIG-H3 proteins or their degradation products may bind these lipid carbohydrate moieties and other proteins aberrantly so they become incorporated in the deposits.

Recently, abnormal turnover of ßIG-H3 protein in the corneal tissues was reported to be associated with the mutations at arginine 124.12 The investigators showed the disease-specific fragments found in affected corneas. Contrary to their results, our overexpression experiments of each mutant ßIG-H3 in human corneal epithelial cells failed to show any fragments markedly different from those of the wild-type ßIG-H3. Similar results were observed with the recombinant proteins. Even the sizes of fragments did not match well. The most unusual fragment that they showed was a fragment of 44 kDa in R124C cornea, which was not present in the normal cornea. We also found this 44-kDa fragment but it existed in both the wild-type ßIG-H3 protein and all mutant forms. Unfortunately, we could not find any mutant-specific fragment from our results. This discrepancy may be due to using different samples and different antibodies. Although we used human corneal epithelial cells, they may not reflect the in vivo situation. Indeed, Korvatska et al.12 also analyzed cultured primary skin fibroblasts from patients with the R124C mutation and found no production of disease-specific fragments in the affected corneas. Despite ubiquitous expression of ßIG-H3 in the organism, patients with 5q31-linked corneal dystrophy do not manifest any sign of systemic abnormality, suggesting that corneal tissue–specific factors may also contribute to the pathologic deposits. In addition, the corneal deposits are distributed more strongly in the central part of the corneal than in the peripheral part, suggesting that corneal environmental factors may also contribute to the formation of corneal deposits.

The frequency of degenerate epithelial cells in the dystrophies suggests that mutant ßIG-H3 proteins do not function well to support corneal epithelial cell adhesion. Although mutation sites are not directly related with the motif for cell adhesion, mutations may elicit conformational changes, resulting in the loss of cell adhesion activity. However, this is very unlikely, because ßIG-H3 has two motifs for corneal epithelial cell adhesion, and those motifs are independently active in mediating cell adhesion.2 Even if one motif is mutated, it may not abolish the adhesion activity of ßIG-H3, because the other intact motif is sufficient for mediating cell adhesion. Mutant ßIG-H3 proteins found in the corneal dystrophies, however, may change their structure in vivo, resulting in their functional motif becoming cryptic, leading to the loss of function. In this regard, more in vivo functional studies are needed to define how mutations of ßIG-H3 could lead to abnormalities found in the congenital corneal dystrophies.

In conclusion, we have demonstrated that ßIG-H3 protein polymerizes to form the fibrillar structure and that it interacts with several ECM proteins, including laminin, type I collagen, and fibronectin. Our mutation studies suggest that the pathogenic processes for different forms of abnormal accumulations found in 5q31-linked corneal dystrophies are mediated by multiple factors in a tissue-specific manner.


   Acknowledgements
 
The authors thank Masatsugu Nakamura (Santen Pharmaceutical Co., Osaka, Japan) for the use of human corneal epithelial cells.


   Footnotes
 
Supported by Program M10104000036-01J0000-01610 of the National Research Laboratory.

Submitted for publication June 25, 2001; revised October 12, 2001; accepted November 1, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: In-San Kim, Department of Biochemistry, School of Medicine, Kyungpook National University, 101 Dongin-dong, Jung-gu, Taegu, 700-422, Korea; iskim{at}knu.ac.kr


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Skonier, J, Neubauer, M, Madisen, L, Bennett, K, Plowman, GD, Purchio, AF (1992) cDNA cloning and sequence analysis of ßig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-ß DNA Cell Biol 11,511-522[Medline][Order article via Infotrieve]
  2. Kim, J-E, Kim, S-J, Lee, B-H, Park, R-W, Kim, K-S, Kim, I-S. (2000) Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-ß-induced gene, ßig-h3 J Biol Chem 275,30907-30915[Abstract/Free Full Text]
  3. Munier, FL, Korvatska, E, Djemai, S, LePaslier, D, Zografos, L, Pescia, G, Schorderet, DF (1997) Kerato-epithelin mutations in four 5q31-linked corneal dystrophies Nat Genet 15,247-251[Medline][Order article via Infotrieve]
  4. Klintworth, GK (1999) Advances in the molecular genetics of corneal dystrophies Am J Ophthalmol 128,747-754[Medline][Order article via Infotrieve]
  5. Korvatska, E, Munier, FL, Chaubert, P, et al (1999) On the role of kerato-epithelin in the pathogenesis of 5q31-linked corneal dystrophies Invest Ophthalmol Vis Sci 40,2213-2219[Abstract/Free Full Text]
  6. Streeten, BW, Qi, Y, Klintworth, GK, Eagle, RC, Jr, Strauss, JA, Bennett, K. (1999) Immunolocalization of ßig-h3 protein in 5q31-linked corneal dystrophies and normal corneas Arch Ophthalmol 117,67-75[Abstract/Free Full Text]
  7. Kim, I-S, Sinha, S, deCrombrugghe, B, Maity, SN (1996) Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interact simultaneously with both the CBF-A and CBF-C subunit to form a heterotrimeric CBF molecule Mol Cell Biol 16,4003-4013[Abstract]
  8. Lee, HE, Seomun, Y, Hwang, K-H, et al (2000) Overexpression of the transforming growth factor-b-inducible gene ßig-h3 in anterior polar cataracts Invest Ophthalmol Vis Sci 41,1840-1845[Abstract/Free Full Text]
  9. Araki-Sasaki, K, Ohashi, Y, Sasabe, T, et al (1995) An SV40-immortalized human corneal epithelial cell line and its characterization Invest Ophthalmol Vis Sci 36,614-621[Abstract/Free Full Text]
  10. Zinn, K, McAllister, L, Goodman, CS (1988) Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila Cell 53,577-587[Medline][Order article via Infotrieve]
  11. Hashimoto, K, Noshiro, M, Ohno, S, et al (1997) Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/ßig-h3) that binds to collagen Biochim Biophys Acta 1355,303-314[Medline][Order article via Infotrieve]
  12. Korvatska, E, Henry, H, Mashima, Y, et al (2000) Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein J Biol Chem 275,11465-11469[Abstract/Free Full Text]
  13. Hirano, K, Klintworth, GK, Zhan, Q, Bennett, K, Cintron, C. (1996) ßig-h3 is synthesized by corneal epithelium and perhaps endothelium in Fuchs’ dystrophic corneas Curr Eye Res 15,965-972[Medline][Order article via Infotrieve]
  14. Schmitt-Bernard, C-F, Chavanieu, A, Derancourt, J, et al (2000) In vitro creation of amyloid fibrils from native and Arg124Cys mutated ßig-h3 peptides, and its relevance for lattice corneal amyloid dystrophy type I Biochem Biophys Res Commun 273,649-653[Medline][Order article via Infotrieve]
  15. Schmitt-Bernard, C-F, Guitard, C, Arnaud, B, et al (2000) BIGH3 exon 14 mutations lead to intermediate type I/IIIA of lattice corneal dystrophies Invest Ophthalmol Vis Sci 41,1302-1308[Abstract/Free Full Text]
  16. Rawe, IM, Zhan, Q, Burrows, R, Bennett, K, Cintron, C. (1997) Beta-ig, molecular cloning and in situ hybridization in corneal tissues Invest Ophthalmol Vis Sci 38,893-900[Abstract/Free Full Text]
  17. Rodrigues, MM, Krachmer, JH (1988) Recent advances in corneal stromal dystrophies Cornea 7,19-29[Medline][Order article via Infotrieve]
  18. Rodrigues, MM, Streeten, BW, Krachmer, JH, et al (1983) Microfibrillar protein and phospholipid in granular corneal dystrophy Arch Ophthalmol 101,802-810[Abstract/Free Full Text]
  19. Nishida, K, Quantock, AJ, Dota, A, Choi-Miura, N-H, Kinoshita, S. (1999) Apolipoproteins J and E co-localise with amyloid in gelatinous drop-like and lattice type I corneal dystrophies Br J Ophthalmol 83,1178-1182[Abstract/Free Full Text]



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