Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.     

Corneal Dystrophy Mutations Drive Pathogenesis by Targeting TGFBIp Stability and Solubility in a Latent Amyloid-forming Domain

Numerous mutations in the corneal protein TGFBIp lead to opaque extracellular deposits and corneal dystrophies (CDs). Here we elucidate the molecular origins underlying TGFBIp's mutation-induced increase in aggregation propensity through comprehensive biophysical and bioinformatic analyses of mutations associated with every major subtype of TGFBIp-linked CDs including lattice corneal dystrophy (LCD) and three subtypes of granular corneal dystrophy (GCD 1–3). LCD mutations at buried positions in the C-terminal Fas1–4 domain lead to decreased stability. GCD variants show biophysical profiles distinct from those of LCD mutations. GCD 1 and 3 mutations reduce solubility rather than stability. Half of the 50 positions within Fas1–4 most sensitive to mutation are associated with at least one known disease-causing mutation, including 10 of the top 11 positions. Thus, TGFBIp aggregation is driven by mutations that despite their physico-chemical diversity target either the stability or solubility of Fas1–4 in predictable ways, suggesting straightforward general therapeutic strategies.     

SAXS models of TGFBIp reveal a trimeric structure and show that the overall shape is not affected by the Arg124His mutation

Human transforming growth factor β induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple “beads-on-a-string” arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.     

Mutation effects on FAS1 domain 4 based on structure and solubility

Mutations in the fasciclin 1 domain 4 (FAS1–4) of transforming growth factor β-induced protein (TGFBIp) are associated with insoluble extracellular deposits and corneal dystrophies (CDs). The decrease in solubility upon mutation has been implicated in CD; however, the exact molecular mechanisms are not well understood. Here, we performed molecular dynamics simulations followed by solvation thermodynamic analyses of the FAS1–4 domain and its three mutants—R555W, R555Q, and A546T—linked to granular corneal dystrophy type 1, Thiel-Behnke corneal dystrophy and lattice corneal dystrophy, respectively. We found that both R555W and R555Q mutants have less affinity toward solvent water relative to the wild-type protein. In the R555W mutant, a remarkable increase in solvation free energy was observed because of the structural changes near the mutation site. The mutation site W555 is buried in other hydrophobic residues, and R557 simultaneously forms salt bridges with E554 and D561. In the R555Q mutant, the increase in solvation free energy is caused by structural rearrangements far from the mutation site. R558 separately forms salt bridges with D575, E576, and E598. Thus, we thus identified the relationship between the decrease in solubility and conformational changes caused by mutations, which may be useful in designing potential therapeutics and in blocking FAS1 aggregation related to CD.     

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.     

Lysophosphatidic acid activates TGFBIp expression in human corneal fibroblasts through a TGF-β1-dependent pathway

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease caused by a R124H point mutation in the transforming growth factor-β-induced gene (TGFBI). However, the cellular role of TGFBI and the regulatory mechanisms underlying corneal dystrophy pathogenesis are still poorly understood. Lysophosphatidic acid (LPA) refers to a small bioactive phospholipid mediator produced in various cell types, and binds G protein-coupled receptors to enhance numerous biological responses, including cell growth, inflammation, and differentiation. LPA levels are elevated in injured cornea and LPA is involved in proliferation and wound healing of cornea epithelial cells. Accumulating evidence has indicated a crucial role for LPA-induced expression of TGFBI protein (TGFBIp) through secretion of transforming growth factor-beta1 (TGF-β1). In the current study, we demonstrate that LPA induces TGFBIp expression in corneal fibroblasts derived from normal or GCD2 patients. LPA-induced TGFBIp expression was completely inhibited upon pretreatment with the LPA(1/3) receptor antagonists, VPC32183 and Ki16425, as well as by silencing LPA(1) receptor expression with small hairpin RNA (shRNA) in corneal fibroblasts. LPA induced secretion of TGF-β1 in corneal fibroblasts, and pretreatment with the TGF-β type I receptor kinase inhibitor SB431542 or an anti-TGF-β1 neutralizing antibody also inhibited LPA-induced TGFBIp expression. Furthermore, we show that LPA requires Smad2/3 proteins for the induction of TGFBIp expression. LPA elicited phosphorylation of Smad2/3, and Smad3 specific inhibitor SIS3 or siRNA-mediated depletion of endogenous Smad2/3 abrogates LPA-induced TGFBIp expression. Finally, we demonstrate that LPA-mediated TGFBIp induction requires JNK activation, but not ERK signaling pathways. These results suggest that LPA stimulates TGFBIp expression through JNK-dependent activation of autocrine TGF-β1 signaling pathways and provide important information for understanding the role of phospholipids involved in cornea related diseases.     

Lysosomal Trafficking of TGFBIp via Caveolae-Mediated Endocytosis

Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin α_Vβ₃. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

Mutation hot spots in 5q31-linked corneal dystrophies.

Mutations in the BIGH3 gene on chromosome 5q31 cause four distinct autosomal dominant diseases of the human cornea: granular (Groenouw type I), Reis-Bücklers, lattice type I, and Avellino corneal dystrophies. All four diseases are characterized by both progressive accumulation of corneal deposits and eventual loss of vision. We have identified a specific recurrent missense mutation for each type of dystrophy, in 10 independently ascertained families. Genotype analysis with microsatellite markers surrounding the BIGH3 locus was performed in these 10 families and in 5 families reported previously. The affected haplotype could be determined in 10 of the 15 families and was different in each family. These data indicate that R555W, R124C, and R124H mutations occurred independently in several ethnic groups and that these mutations do not reflect a putative founder effect. Furthermore, this study confirms the specific importance of the R124 and R555 amino acids in the pathogenesis of autosomal dominant corneal dystrophies linked to 5q.     

Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas

Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.     

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.     

A new L527R mutation of the βIGH3 gene in patients with lattice corneal dystrophy with deep stromal opacities

Mutations in the βIGH3 gene on chromosome 5q31 cause five distinct autosomal dominant corneal dystrophies: granular Groenouw type I, Reis-Bücklers’, lattice type I and IIIA, and Avellino corneal dystrophies. We present here a new mutation of the βIGH3 gene in patients with late-onset lattice corneal dystrophy manifest as a deep stromal opacity. To test the previously reported R124C, R124H, P501T, R555W, and R555Q mutations of the βIGH3 gene, 30 patients and 11 normal relatives from 16 independently ascertained families with lattice corneal dystrophy, 49 patients and 12 normal relatives from 40 independently ascertained families with other corneal dystrophies, and 40 unrelated normal volunteers, were analyzed. A L527R (CTG/CGG) mutation of the βIGH3 gene was found in 6 unrelated patients with lattice corneal dystrophy. A retrospective review of the patients’ records showed that the opacities were deep in the stromal layer and of late onset. The mutation was a heterozygous single base-pair transversion from T to G of the second nucleotide position of codon 527. This caused the substitution of arginine for leucine. These six patients did not have mutations in codons 124, 501, or 555. The L527R mutation was not detected in the other corneal dystrophies or 40 normal volunteers. Although phenotypic variations in the size and shape of the deposits were found, all patients with the L527R mutation showed deposits deep in the stromal layer. We conclude that there are now at least six different mutations that have been detected in the βIGH3 gene on chromosome 5q31 and that lead to corneal dystrophy. Received: 14 April 1998 / Accepted: 10 June 1998     

Molecular basis for dimer formation of TRbeta variant D355R

Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.     

A survey of hemoglobin quaternary structures

We perform an analysis of the quaternary structure and dimer/dimer interface in the crystal structures of 165 human hemoglobin tetramers; 112 are in the T, 17 the R, 14 the Y (or R2) state; 11 are high-affinity T state mutants, and 11 may either be intermediates between the states, or off the allosteric transition pathway. The tertiary structure is fixed within each state, in spite of the different ligands, mutations, and chemical modifications present in individual entries. The geometry of the tetramer assembly is essentially the same in all the R or the Y state entries; it is slightly different in high salt and low salt crystals of T state hemoglobins. The dimer/dimer interface differs in terms of size, chemical composition and polar interactions, between the states. It is loosely packed, like crystal packing contacts or the subunit interface of weakly associated homodimers, and unlike most oligomeric proteins, which have close-packed interfaces. The loose packing is most obvious in the liganded forms, where the tetramer is known to dissociate at low concentration. We identify cavities that contribute to the loose packing of the α1β2 and α2β1 contacts. Two pairs of cavities occur recurrently in both the T and the R state tetramers. They may contribute to the allosteric mechanism by facilitating the subunit movements and the tertiary structure changes that accompany the transition from T to R to Y.     

TGFBI gene mutations in the Ukrainian patients with inherited corneal stromal dystrophies

Mutations Arg124Cys, Thr538Arg, Arg555Thr, Arg555Gln, Leu558Pro, and His626Arg in TGFBI gene were analyzed by polymerase chain reaction and restriction in 84 patients with various forms of corneal stromal dystrophies from 49 unrelated families and 29 clinically healthy relatives of these patients. A new mutation in TGFBI gene, Leu558Pro, was identified in the patients with atypical lattice dystrophy. The haplotypes of four microsatellite markers surrounding TGFBI gene region were analyzed in 22 families. The data on association of genotype and phenotype suggest that the analysis of TGFBI gene mutations is important for differential diagnostics of corneal dystrophies.     

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 "secondary bridge" between the copper- and zinc-binding sites is disrupted, and the "electrostatic loop" and "zinc loop" elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1-SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.     

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

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.     

p.Ala546 > Asp and p.Arg555 > Trp mutations of TGFBI gene and their clinical manifestations in two large Chinese families with granular corneal dystrophy type I

The aim of this study was to conduct clinical, genetic, and molecular analysis of Chinese patients with granular corneal dystrophy type I (CDGG1). Two large unrelated Chinese families with CDGG1 were clinically and genetically evaluated. Molecular genetic analysis was performed on DNA extracted from peripheral blood. Exons 4, 11, 12, and 14 of the human transforming growth factor beta-induced gene (TGFBI, formerly designated BIGH3) were amplified by PCR, scanned for mutations using the single-strand conformation polymorphism method, and the mutations identified by nucleotide sequencing. One family segregated the p.Ala546 > Asp mutation, and the other family had a p.Arg555 > Trp mutation. These missense mutations were not found in 53 unrelated, healthy individuals analyzed as controls. Clinical and genetic evaluations revealed the variable severity, symmetry, and age of onset in visual impairment in these families for different mutations. Penetrance of visual impairment in these families was 100% and 75%, respectively. This study confirms that the p.Arg555 > Trp mutation is a frequent cause of CDGG1, and that the p.Ala546 > Asp mutation is also associated with this disease.