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.     

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-induced dimerization of transforming growth factor-β–induced protein may drive protein aggregation in granular corneal dystrophy

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-β–induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography–small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.     

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.     

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.     

Expression, purification and characterization of fourth FAS1 domain of TGFβIp-associated corneal dystrophic mutants

Corneal dystrophies (CDs) are a group of inherited bilateral disorders affecting the corneal tissue of the eye. Most of these CDs in the stromal layer of the cornea have been associated with mutations found on the TGFBI gene that codes for a 683-amino acid transforming growth factor induced protein (TGFβIp). These mutations have been found to induce atypical aggregation and progressive accumulation of protein aggregates in the cornea that leads to loss of corneal transparency and hence blindness. At present, 65 distinct pathogenic mutations have been identified in TGFBI that are associated with different clinical phenotypes. More than 80% of these missense mutations occur in the 4th FAS (fasciclin-like) 1 domain. Current treatment includes surgical intervention, which often involves high recurrence rates. Hence, it is imperative to examine the properties of the TGFβIp and the pathogenic mutant proteins to understand the pathology of the disease mechanism and to develop potent therapeutics. Here, we report the recombinant expression, purification, characterization and the effect of four clinically significant pathogenic TGFβIp mutants - R555W, H572R, A620D, and H626R on the biophysical properties of the wild type (WT) 4th FAS1 domain of TGFβIp. While a higher proportion of the R555W, H572R and H626R mutants of the 4th FAS1 domains remained stable, the A620D mutant largely existed as inclusion bodies in native state and aggregates under physiological conditions. These mutants present a unique platform to examine protein aggregation-prone diseases wherein single amino-acid mutations present distinct pathogenic phenotypes. Though pathogenically and phenotypically diverse, these mutants do not exhibit variations in secondary structure and stability, except for the A620D mutant, when examined by CD and UV spectroscopy.     

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.     

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.     

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.     

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1–157) and α-rich C-terminal (residues 158–316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50–100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.     

FAS1 Domain Protein Inhibits VEGF165-Induced Angiogenesis by Targeting the Interaction between VEGFR-2 and αvβ3 Integrin

It is known that VEGF receptors (VEGFR) and integrins interact with each other to regulate angiogenesis. We reported previously that the fasciclin 1 (FAS1) domain-containing protein, TGFBIp/βig-h3 (TGF-β-induced protein) is an angiogenesis regulator that inhibits both endothelial cell migration and growth via αvβ3 integrin. In an attempt to target the interaction between VEGFR-2 and αvβ3 integrin, we determined whether the FAS1 domain region of TGFBIp/βig-h3 (FAS1 domain protein) can block the interaction between the two receptors, leading to the suppression of angiogenesis. In this study, we showed that FAS1 domain protein inhibits VEGF(165)-induced endothelial cell proliferation and migration via αvβ3 integrin, resulting in the inhibition of VEGF(165)-induced angiogenesis. We also defined a molecular mechanism by which FAS1 domain protein blocks the association between αvβ3 integrin and VEGFR-2, showing that it binds to αvβ3 integrin but not to VEGFR-2. Blocking the association of these major angiogenic receptors with FAS1 domain protein inhibits signaling pathways downstream of VEGFR-2. Collectively, our results indicate that FAS1 domain protein, in addition to its inhibitory effect on αvβ3 integrin-mediated angiogenesis, also inhibits VEGF(165)-induced angiogenesis. Thus, FAS1 domain protein can be further developed into a potent anticancer drug that targets two principal angiogenic pathways. Mol Cancer Res; 10(8); 1010-20. ?2012 AACR.     

Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg(336) site were replaced with those surrounding Arg(562), were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 9-16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.     

The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1.

The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.     

The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.     

A missense mutation in the Na(+)/glucose cotransporter gene SGLT1 in a patient with congenital glucose-galactose malabsorption: normal trafficking but inactivation of the mutant protein

The Na(+)/glucose cotransporter gene SGLT1 was analyzed in a Japanese patient with congenital glucose-galactose malabsorption. Genomic DNA was used as a template for amplification by the polymerase chain reaction of each of the 15 exons of SGLT1. The amplification products were cloned and sequenced. About half of the exon 5 clones of the patient contained a C-->T transition, resulting in an Arg(135)-->Trp mutation, whereas the remaining clones contained the normal exon 5 sequence. In addition, whereas some exon 12 clones exhibited the normal sequence, others showed a CAgtaggtatcatc-->CAgacc mutation at the splice donor site of intron 12 that may result either in the skipping of exon 12 or in read-through of intron 12. Neither the Arg(135)-->Trp mutant nor either of the possible intron 12 mutant proteins exhibited Na(+)-dependent glucose transport activity when expressed in Xenopus oocytes. Immunocytochemical analysis indicated, however, that the Arg(135)-->Trp mutant was localized to the oocyte plasma membrane. DNA sequence analysis revealed that the missense mutation in exon 5 and the splice site mutation in intron 12 were inherited from the proband's father and mother, respectively. These results indicate that the patient is a compound heterozygote for this disease, and that the Arg(135)-->Trp mutant of SGLT1 undergoes normal trafficking to the plasma membrane but is non-functional.     

Structures of tetrahydrobiopterin binding-site mutants of inducible nitric oxide synthase oxygenase dimer and implicated roles of Trp457.

To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOS(ox); residues 1-498) in maintaining the structural integrity of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) binding site located at the dimer interface and in supporting H(4)B redox activity, we determined crystallographic structures of W457F and W457A mutant iNOS(ox) dimers (residues 66-498). In W457F iNOS(ox), all the important hydrogen-bonding and aromatic stacking interactions that constitute the H(4)B binding site and that bridge the H(4)B and heme sites are preserved. In contrast, the W457A mutation results in rearrangement of the Arg193 side chain, orienting its terminal guanidinium group almost perpendicular to the ring plane of H(4)B. Although Trp457 is not required for dimerization, both Trp457 mutations led to the increased mobility of the N-terminal H(4)B binding segment (Ser112-Met114), which might indicate reduced stability of the Trp457 mutant dimers. The Trp457 mutant structures show decreased pi-stacking with bound pterin when the wild-type pi-stacking Trp457 position is occupied with the smaller Phe457 in W457F or positive Arg193 in W457A. The reduced pterin pi-stacking in these mutant structures, relative to that in the wild-type, implies stabilization of reduced H(4)B and destabilization of the pterin radical, consequently slowing electron transfer to the heme ferrous-dioxy (Fe(II)O(2)) species during catalysis. These crystal structures therefore aid elucidation of the roles and importance of conserved Trp457 in maintaining the structural integrity of the H(4)B binding site and of H(4)B-bound dimers, and in influencing the rate of electron transfer between H(4)B and heme in NOS catalysis.     

Insight to pyrazinamide resistance inMycobacterium tuberculosisby molecular docking

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.