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1.  Identification and characterization of a novel TACSTD2 mutation in gelatinous drop-like corneal dystrophy 
Molecular Vision  2010;16:729-739.
Purpose
To study the clinical, histological, in vivo confocal microscopic, and molecular profile in a family with gelatinous drop-like corneal dystrophy (GDLD) from north India.
Methods
Two siblings from a consanguineous family presented with clinical features analogous to GDLD. Detailed clinical evaluations were performed for all the available affected and unaffected members of this family. In vivo confocal microscopy and histology was done wherever necessary. DNA isolated from peripheral blood samples was subjected to polymerase chain reaction (PCR) followed by direct sequencing to detect mutations in the tumor-associated calcium signal transducer 2 (TACSTD2) gene. Protein modeling studies were done to asses the effect of the mutation on the protein structure.
Results
The diagnosis of GDLD was established in the patient and the affected sibling on slit-lamp examinations, which revealed mulberry-like opacities in the subepithelium and anterior stroma that were confirmed on histopathology. The findings of the in vivo confocal microscopy were consistent with those reported in previous reports. Sequencing TACSTD2 revealed a novel homozygous missense mutation c.356G>A, leading to amino acid substitution C119Y in the two affected siblings. The mutation was found to be pathogenic on Sorting Intolerant From Tolerant (SIFT) analysis and was not found in normal controls and unaffected individuals of the family. A synonymous, previously reported, single nucleotide polymorphism (SNP; rs13267) was also seen in all the individuals of the family. Protein modeling studies involving wild-type and mutant protein indicated an exposed cysteine residue in the mutant protein.
Conclusions
A novel TACSTD2 C119Y mutation leading to an amino acid substitution was identified in two affected siblings of a family. Protein modeling studies revealed an exposed cysteine residue, which might cause interchain disulfide bond formation and protein aggregation leading to disturbed cell junctions of the corneal epithelium.
PMCID: PMC2862246  PMID: 20454699
2.  A novel TACSTD2 mutation identified in two Chinese brothers with gelatinous drop-like corneal dystrophy 
Molecular Vision  2009;15:1580-1588.
Purpose
To identify the molecular defect causing gelatinous drop-like corneal dystrophy (GDLD) in two Chinese brothers and report the morphological evaluation of GDLD by laser scanning confocal microscopy and Fourier-domain optical coherence tomography (OCT).
Methods
Genetic analysis included polymerase chain reaction (PCR) amplification and direct nucleotide sequencing of the coding region of the tumor-associated calcium signal transducer 2 gene (TACSTD2) in DNA from the two brothers and their relatives. Laser scanning confocal microscopy and Fourier-domain OCT were performed on the left cornea of the younger brother.
Results
We report a novel in-frame mutation of TACSTD2, c.526_576del 51, in the two brothers with GDLD. The identified molecular defect cosegregated with the disease and was not found in 50 unaffected individuals. The morphological evaluation on GDLD highlighted pathological observations at the level of epithelium and anterior stroma. The epithelial cells of GDLD cornea were irregular in shape and often elongated. Large accumulations of brightly reflective amyloid material was noted within or beneath the epithelium and within the anterior stroma.
Conclusions
The newly identified mutation expands the spectrum of mutations in TACSTD2 that may cause pathological corneal amyloidosis. Observations by in vivo confocal microscopy and Fourier-domain OCT were consistent with the histopathologic descriptions of GDLD.
PMCID: PMC2728569  PMID: 19693293
3.  Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis) 
Purpose:
To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene.
Methods:
A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD.
Results:
The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line.
Conclusions:
Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD.
PMCID: PMC3671362  PMID: 23818740
4.  Gelatinous drop-like corneal dystrophy with a novel mutation of TACSTD2 manifested in combination with spheroidal degeneration in a Chinese patient 
Molecular Vision  2010;16:1570-1575.
Purpose
To report the clinicopathological findings of a Chinese patient with an unusual phenotype of gelatinous drop-like corneal dystrophy (GDLD) combined with spheroidal degeneration and to detect molecular defect in the tumor-associated calcium signal transducer 2 (TACSTD2) gene.
Methods
Extensive physical and ophthalmologic examination of the patient was performed. Initially superficial keratectomy was performed for both eyes. Due to recurrence of the corneal opacity, penetrating keratoplasty for the right eye and deep lamellar keratoplasty for the left eye were performed. The obtained corneal tissues were examined by light microscopy. Molecular genetic analysis consisted of PCR amplification and direct automated sequencing of the complete coding region of TACSTD2.
Results
Slit-lamp biomicroscopy of the patient revealed bilateral band-like corneal opacities composed of brown-yellow, oily appearing droplets at the first visit. Two years after superficial keratectomy, elevated mulberry-like gelatinous lesions companied with brown-yellow droplets in the superficial cornea in both eyes were found. Histological analysis of corneal tissue revealed subepithelial amorphous deposits stained positively with Congo red, typical of GDLD. Meanwhile, eosinophilic globular deposits with irregular peripheral margins and various sizes, which were characteristics of spheroidal degeneration, were found. Sequencing of TACSTD2 from the patient revealed a novel homozygous missense mutation c.354G>C, leading to amino acid substitution Q118H in the patient.
Conclusions
This is the first report indicating a new type of gelatinous drop-like corneal dystrophy (GDLD) combined with spheroidal degeneration. Molecular analysis demonstrated a novel mutation in TACSTD2, which may expand the spectrum of mutations in TACSTD2.
PMCID: PMC2927379  PMID: 20806038
5.  Two novel mutations of TACSTD2 found in three Japanese gelatinous drop-like corneal dystrophy families with their aberrant subcellular localization 
Molecular Vision  2011;17:965-970.
Purpose
To report two novel mutation of the tumor-associated calcium signal transducer 2 (TACSTD2) gene in 3 Japanese patients with gelatinous drop-like corneal dystrophy (GDLD).
Methods
Genomic DNAs were extracted from the peripheral blood of 3 Japanese families. The coding region of TACSTD2 was amplified by polymerase chain reaction (PCR) and subjected to direct sequencing analysis. Plasmid vectors harboring normal and mutated TACSTD2 were transfected to the immortalized human corneal epithelial cells to identify the subcellular localization of the normal and mutated TACSTD2 gene products.
Results
Sequencing analysis of TACSTD2 revealed two novel homozygous mutations (c.840_841insTCATCATCGCCGGCCTCATC and c.675C>A which may result in frameshift (p.Ile281SerfsX23) and nonsense (p.Tyr225X) mutations, respectively) in the 3 GDLD patients. Protein expression analysis showed that the mutated gene product was distributed diffusely in the cytoplasm, whereas the normal gene product accumulated at the cell-to-cell borders.
Conclusions
This study reports two novel mutations in 3 GDLD families and expands the spectrum of mutations in TACSTD2 that may cause pathological corneal amyloidosis.
PMCID: PMC3084224  PMID: 21541270
6.  Advanced glycation end product deposits in climatic droplet keratopathy 
Background
Climatic droplet keratopathy (CDK), known as spheroid degeneration of the cornea, is one of the most frequent degenerative corneal disorders affecting visual function. However, the histochemical nature of the deposits seen in CDK is still unclear.
Aim
To investigate the pathogenesis of CDK, we investigated the immunohistochemical localisation of advanced glycation end products (AGEs) in surgical specimens of CDK.
Methods
Immunohistochemical localisation of Nε‐(carboxymethyl)‐L‐lysine (CML), Nε‐(carboxyethyl)‐L‐lysine (CEL), pyrraline, pentosidine and imidazolone was examined in three corneas with CDK, six corneas with bullous keratopathy and three corneas without any corneal diseases.
Results
In all the specimens with CDK, immunoreactivity was strong in CML, moderate in pyrraline and pentosidine, and weak in imidazolone. Immunoreactivity was absent in CEL. In contrast, no immunoreactivity to CML, pyrraline, pentosidine, imidazolone or CEL was detected in corneas with bullous keratopathy, or in corneas without any corneal diseases.
Conclusions
CDK is caused by an aggregation of AGE‐modified proteins. The result is consistent with etiological findings that ultraviolet irradiation and ageing, both of which are accelerators of AGE formation, are closely related to the development of CDK.
doi:10.1136/bjo.2006.099812
PMCID: PMC1857579  PMID: 16973666
7.  Limbal Stem Cell Transplantation for Gelatinous Drop-like Corneal Dystrophy 
Purpose
To report the outcomes of allograft limbal stem cell transplantation for recurrent gelatinous drop-like corneal dystrophy (GDLD).
Methods
In this non-comparative interventional case series, 4 eyes of 3 consecutive patients with recurrent GDLD underwent allograft limbal stem cell transplantation. Two eyes underwent concomitant penetrating keratoplasty while the other two underwent simultaneous superficial keratectomy. Main outcome measures were best spectacle corrected visual acuity, intraocular pressure and corneal clarity.
Results
Patient age ranged from 28 to 63 years. Mean follow-up after surgery was 23±10 (median, 22; range, 12-36) months. Mean visual acuity improved from 2.70±0.61 logMAR preoperatively to 1.05±0.06 logMAR at final postoperative visit (P=0.066). Intraocular pressure was normal in all eyes at baseline and remained within normal limits at all postoperative visits. All corneas remained smooth and clear during the follow-up period with no episode of rejection or recurrence. All patients maintained ambulatory vision until final follow-up.
Conclusion
The present study provides further evidence that limbal stem cell transplantation may be an effective therapeutic alternative in patients with GDLD.
PMCID: PMC3740461  PMID: 23943684
Corneal Dystrophy; Gelatinous Drop-Like Dystrophy; Limbal Stem Cell Transplantation
8.  Two novel mutations identified in two Chinese gelatinous drop-like corneal dystrophy families 
Molecular Vision  2007;13:988-992.
Purpose
To identify the genetic defect in the TACSTD2 gene that causes gelatinous drop-like corneal dystrophy (GDLD) in two unrelated consanguineous Chinese families.
Methods
Genomic DNA was prepared from leukocytes of peripheral venous blood. The coding region of the TACSTD2 gene was evaluated by means of polymerase chain reaction and direct sequencing.
Results
Sequencing of the TACSTD2 gene of the two probands revealed two novel homozygous frameshift mutations: c.84insG and c.480delC. The identified molecular defect cosegregates with the disease among affected members of the families and is not found in 50 unaffected controls.
Conclusions
This study reports two novel mutations in two GDLD families and expands the spectrum of mutations in TACSTD2 gene that may cause pathological corneal amyloidosis.
PMCID: PMC2774463  PMID: 17653040
9.  Apolipoproteins J and E co-localise with amyloid in gelatinous drop-like and lattice type I corneal dystrophies 
The British Journal of Ophthalmology  1999;83(10):1178-1182.
AIMS—Apolipoprotein J (apoJ) and apolipoprotein E (apoE) are thought to contribute to amyloid formation in patients with Alzheimer's disease. The aim of this investigation was to discover whether or not these apolipoproteins associate with corneal amyloid in gelatinous drop-like corneal dystrophy (GDCD) and lattice corneal dystrophy type I (LCD-I).
METHODS—Corneas from three eyes of three patients with GDCD and one eye of one patient with LCD-I were examined immunohistochemically using antibodies against apoJ and apoE. Two normal corneas were similarly examined. Tissue sections of brain from a patient with Alzheimer's disease were used as positive controls for the antibodies. For all negative controls, mouse IgG was used instead of the primary antibody.
RESULTS—Intense apoJ and apoE immunoreactivities were found in congophilic amyloid deposits in GDCD and LCD-I. These deposits were located subepithelially in GDCD, and subepithelially and intrastromally in LCD-I. In GDCD, immunostaining of subepithelial amyloid with anti-apoJ was noticeably stronger than with anti-apoE.
CONCLUSIONS—As in senile plaques in brain from a patient with Alzheimer's disease, apoJ and apoE co-localise with amyloid in corneas with GDCD and LCD-I.


PMCID: PMC1722813  PMID: 10502582
10.  Gelsolin-related amyloidosis. Identification of the amyloid protein in Finnish hereditary amyloidosis as a fragment of variant gelsolin. 
Journal of Clinical Investigation  1991;87(4):1195-1199.
The Finnish type of familial amyloidosis is a systemic disease characterized by progressive cranial neuropathy, corneal lattice dystrophy, and distal sensimotor neuropathy. Amyloid fibrils were isolated from the kidney and heart of a patient with Finnish amyloidosis. After solubilization, the amyloid proteins were fractionated by gel filtration and purified by reverse-phase HPLC. Complete amino acid sequence analyses show that the two amyloid components obtained are fragments of gelsolin, an actin-modulating protein occurring in plasma and the cytoskeleton. The larger component represents residues 173-243 and the minor component residues 173-225, respectively, of mature gelsolin. When compared with the predicted primary structure of human gelsolin a single amino acid substitution is present in amyloid: at position 15 of the amyloid proteins an asparagine is found instead of an aspartic acid residue at the corresponding position (187) in gelsolin. Antibodies to a dodecapeptide of the amyloidogenic region of gelsolin specifically stain the tissue amyloid deposits in Finnish hereditary amyloidosis. The results show that the amyloid subunit protein in Finnish hereditary amyloidosis represents a new type of amyloid that is derived from an actin filament-binding region of a variant gelsolin molecule by limited proteolysis.
Images
PMCID: PMC295133  PMID: 1849145
11.  Denaturation and solvent effect on the conformation and fibril formation of TGFBIp 
Molecular Vision  2009;15:2617-2626.
Purpose
Transforming growth factor beta-induced protein (TGFBIp) aggregates into the phenotypic amyloid fibrils and/or non-amyloid deposits in corneal dystrophies and other disorders. While significant progress has been made in molecular genetics to successfully establish the link between the missense mutations of TGFBI and TGFBIp-related corneal dystrophies, the underlying mechanism for the abnormal aggregation remains elusive due to the lack of insights into the conformational perturbations induced by mutations. In the present study, we examined the effects of denaturants and a co-solvent on recombinant TGFBIp, with a focus on protein conformational changes and amyloid fibril formation.
Methods
Recombinant TGFBIp was subjected to various spectroscopic studies, such as far-ultraviolet circular dichroism (far-UV CD), intrinsic tryptophan fluorescence and quenching, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, under various denaturing conditions (urea and guanidine hydrochloride [GndHCl], acidic pH, and trifluoroethanol [TFE, co-solvent]). A thioflavin T (ThT) fluorescence assay was used to determine the fibril formation of TGFBIp. In addition, a rabbit polyclonal antibody against the oligomer precursors that initiate the formation of amyloid fibrils was also used in dot blot experiments to detect the formation of prefibrillar precursors.
Results
The purified recombinant TGFBIp is in the folded state according to its intrinsic tryptophan fluorescence analyses. A single-step unfolding process was observed in the GndHCl denaturation experiment. Results from far-UV CD, intrinsic tryptophan fluorescence, and ANS fluorescence experiments showed that TFE exerted its solvent effects by initially unfolding and transforming TGFBIp to a β-sheet-enriched conformer at 20%. When increased to 40%, TFE changed TGFBIp into a non-native α-helix conformer. Although GndHCl and TFE led to protein unfolding, enhanced fibril formation could only be observed in the presence of TFE and at acidic pH, according to the ThT fluorescence assays. The paradigmatic protofibrillar TGFBIp oligomers were also detected during the fibril formation by the dot blot experiment.
Conclusions
Our results suggest that protein unfolding may serve as the prerequisite but is not sufficient for the fibrillogenesis. Other factors, such as the solvent used, fragmentation, or pH, may also be crucial for the formation of TGFBIp fibrils.
PMCID: PMC2790481  PMID: 20011632
12.  Ultrastructural morphology and expression of proteoglycans, βig-h3, tenascin-C, fibrillin-1, and fibronectin in bullous keratopathy 
AIMS—To investigate the ultrastructural localisation of proteoglycans (PG), βig-h3 (keratoepithelin), tenascin-C (TN-C)), fibrillin, and fibronectin in bullous keratopathy (BK) corneas.
METHODS—Five corneas from cases of pseudophakic bullous keratopathy (BK) were examined by electron microscopy. PG were demonstrated using cuprolinic blue, and the proteins βig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-βig-h3, mouse anti-TN-C (BC10 and TN2), mouse anti-fibrillin-1 (MAB2502), mouse anti-fibrillin (MAB1919), and rabbit anti-fibronectin by using a standard immunogold technique.
RESULTS—Epithelial cells contained numerous vacuoles. Epithelial folds and large, electron lucent subepithelial bullae were present. Basal lamina was thickened and traversed by disrupted anchoring filaments. In the stroma, interfibrillar collagen spacing was increased and abnormally large PG were present. Descemet's membrane (DM) contained lucent spaces in which there were small filaments. Keratocyte and endothelial cells contained melanin granules. A posterior collagenous layer (PCL) contained numerous microfilaments and wide spacing collagen fibres with a periodicity of 100 nm. Large quantities of abnormal PG were observed at the endothelial face of the PCL. Very strong labelling with βig-h3 antibody was observed in the basement membrane, Bowman's layer, stroma, DM, and PCL, but not in keratocytes and endothelial cells. Strong labelling with BC10 and TN2 was seen below the epithelium, in electron lucent spaces where the hemidesmosomes were absent, in the fibrotic pannus, in parts of Bowman's layer, the stroma, and Descemet's membrane. Labelling with BC10 was stronger and more evenly distributed than with TN2. Fibrillin-1 (MAB2502) and fibrillin (MAB1919) labelling was similar to TN-C labelling. Fibrillin (MAB1919) labelling was stronger than fibrillin-1 (MAB2502) labelling.
CONCLUSIONS—Immunoelectron microscopy showed precise labelling of proteins at both the cellular and the subcellular level. Expression of proteins βig-h3, TN-C, fibrillin, and fibronectin was highly increased compared with normal cornea. In the oedematous stroma, increased collagen fibril separation may facilitate a wider distribution of some soluble proteins, such as βig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response.


doi:10.1136/bjo.85.6.720
PMCID: PMC1724008  PMID: 11371495
13.  Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene 
Experimental Eye Research  2011;96(1):163-170.
Different types of granular corneal dystrophy (GCD)1 and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo.
doi:10.1016/j.exer.2011.11.014
PMCID: PMC3311163  PMID: 22155582
corneal dystrophy; TGFBI; mutation; laser capture microdissection; proteomics; emPAI; amyloidosis; serine protease HtrA1
14.  Distinct position-specific sequence features of hexa-peptides that form amyloid-fibrils: application to discriminate between amyloid fibril and amorphous β-aggregate forming peptide sequences 
BMC Bioinformatics  2013;14(Suppl 8):S6.
Background
Comparison of short peptides which form amyloid-fibrils with their homologues that may form amorphous β-aggregates but not fibrils, can aid development of novel amyloid-containing nanomaterials with well defined morphologies and characteristics. The knowledge gained from the comparative analysis could also be applied towards identifying potential aggregation prone regions in proteins, which are important for biotechnology applications or have been implicated in neurodegenerative diseases. In this work we have systematically analyzed a set of 139 amyloid-fibril hexa-peptides along with a highly homologous set of 168 hexa-peptides that do not form amyloid fibrils for their position-wise as well as overall amino acid compositions and averages of 49 selected amino acid properties.
Results
Amyloid-fibril forming peptides show distinct preferences and avoidances for amino acid residues to occur at each of the six positions. As expected, the amyloid fibril peptides are also more hydrophobic than non-amyloid peptides. We have used the results of this analysis to develop statistical potential energy values for the 20 amino acid residues to occur at each of the six different positions in the hexa-peptides. The distribution of the potential energy values in 139 amyloid and 168 non-amyloid fibrils are distinct and the amyloid-fibril peptides tend to be more stable (lower total potential energy values) than non-amyloid peptides. The average frequency of occurrence of these peptides with lower than specific cutoff energies at different positions is 72% and 50%, respectively. The potential energy values were used to devise a statistical discriminator to distinguish between amyloid-fibril and non-amyloid peptides. Our method could identify the amyloid-fibril forming hexa-peptides to an accuracy of 89%. On the other hand, the accuracy of identifying non-amyloid peptides was only 54%. Further attempts were made to improve the prediction accuracy via machine learning. This resulted in an overall accuracy of 82.7% with the sensitivity and specificity of 81.3% and 83.9%, respectively, in 10-fold cross-validation method.
Conclusions
Amyloid-fibril forming hexa-peptides show position specific sequence features that are different from those which may form amorphous β-aggregates. These positional preferences are found to be important features for discriminating amyloid-fibril forming peptides from their homologues that don't form amyloid-fibrils.
doi:10.1186/1471-2105-14-S8-S6
PMCID: PMC3654898  PMID: 23815227
15.  Molecular Structures of Amyloid and Prion Fibrils: Consensus vs. Controversy 
Accounts of chemical research  2013;46(7):1487-1496.
Conspectus
Many peptides and proteins self-assemble into amyloid fibrils, including polypeptides that are associated with human amyloid diseases, mammalian and fungal prion proteins, and proteins that are believed to have biologically functional amyloid states. Proper understanding of the common propensity for polypeptides to form amyloid fibrils depends on elucidation of the molecular structures of these fibrils, as does rational design of amyloid inhibitors and imaging agents. Whereas amyloid fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information now exists, with important contributions from solid state nuclear magnetic resonance (NMR) measurements. This article reviews results from our laboratories and discusses several structural issues that have been sources of controversy.
In many cases, the molecular structures of amyloid fibrils are not determined uniquely by their amino acid sequences. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, when in fact different laboratories are simply studying different polymorphs. For 40-residue β-amyloid (Aβ1–40) fibrils associated with Alzheimer’s disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs, which we found to have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR, and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR.
The in-register parallel β-sheet organization found in β-amyloid fibrils has also been found in many other fibril-forming systems by solid state NMR and electron paramagnetic resonance (EPR), and is attributable to stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, antiparallel β-sheets have been identified and characterized by solid state NMR in certain fibrils formed by the D23N mutant of Aβ1–40, which is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ1–40 fibrils are metastable with respect to conversion to parallel structures, and therefore represent an off-pathway intermediate in the amyloid fibril formation process. Evidence for antiparallel β-sheets in other amyloid-formation intermediates has been obtained recently by other methods.
As an alternative to simple parallel and antiparallel β-sheet structures, β-helical structural models have been proposed for various fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures, but the structure of infectious PrP aggregates is not yet known definitively. The fungal HET-s prion protein has been shown by solid state NMR to have a β-helical structure, but all yeast prions studied by solid state NMR (i.e., Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with the fibril core being formed by their Gln- and Asn-rich N-terminal segments.
doi:10.1021/ar300282r
PMCID: PMC3632659  PMID: 23294335
16.  Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain κIII subtype in a small formalin fixed, paraffin wax embedded tissue sample 
Journal of Clinical Pathology  2005;58(5):539-542.
Background: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins.
Aims: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type.
Methods: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing.
Results: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain κIII subtype (AL-κIII) (16, 8, and 3 kDa).
Conclusions: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.
doi:10.1136/jcp.2004.022517
PMCID: PMC1770653  PMID: 15858128
immunoglobulin light chains; microtechnique; orbital amyloidosis
17.  Bacterial Inclusion Bodies Contain Amyloid-Like Structure  
PLoS Biology  2008;6(8):e195.
Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized β-sheet–rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-β structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation.
Author Summary
Protein aggregation is a process by which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are associated with several pathological conditions in humans, including Alzheimer disease, Parkinson disease, and diabetes type II. Furthermore, protein aggregation is a major concern in the biotechnological production of recombinant proteins and the storage of proteins, and is a central mechanism of protein folding. In general, two classes of protein aggregates are classified: first, highly ordered aggregates can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or reorganized into β-sheet–rich aggregates, termed amyloid-like fibrils; and second, amorphous aggregates that lack any long-range order. Here, we demonstrate that bacterial inclusion bodies, which have been believed to be made up of amorphous aggregates, are in fact amyloid-like, comprising cross-β structure that is dependent on amino-acid sequence. These findings suggest that inclusion bodies are structured, that amyloid formation is a process present in both eukaryotes and prokaryotes, that amino acid sequences can evolve to avoid the amyloid conformation, and that there might be no amorphous state of a protein aggregate.
Bacterial inclusion bodies that have been classified as amorphous protein aggregates are in fact structured, comprising a cross-β-sheet structure reminiscent of amyloid fibrils.
doi:10.1371/journal.pbio.0060195
PMCID: PMC2494559  PMID: 18684013
18.  Pathogenesis, diagnosis and treatment of systemic amyloidosis. 
Amyloidosis is a disorder of protein folding in which normally soluble proteins are deposited as abnormal, insoluble fibrils that disrupt tissue structure and cause disease. Although about 20 different unrelated proteins can form amyloid fibrils in vivo, all such fibrils share a common cross-beta core structure. Some natural wild-type proteins are inherently amyloidogenic, form fibrils and cause amyloidosis in old age or if present for long periods at abnormally high concentration. Other amyloidogenic proteins are acquired or inherited variants, containing amino-acid substitutions that render them unstable so that they populate partly unfolded states under physiological conditions, and these intermediates then aggregate in the stable amyloid fold. In addition to the fibrils, amyloid deposits always contain the non-fibrillar pentraxin plasma protein, serum amyloid P component (SAP), because it undergoes specific calcium-dependent binding to amyloid fibrils. SAP contributes to amyloidogenesis, probably by stabilizing amyloid fibrils and retarding their clearance. Radiolabelled SAP is an extremely useful, safe, specific, non-invasive, quantitative tracer for scintigraphic imaging of systemic amyloid deposits. Its use has demonstrated that elimination of the supply of amyloid fibril precursor proteins leads to regression of amyloid deposits with clinical benefit. Current treatment of amyloidosis comprises careful maintenance of impaired organ function, replacement of end-stage organ failure by dialysis or transplantation, and vigorous efforts to control underlying conditions responsible for production of fibril precursors. New approaches under development include drugs for stabilization of the native fold of precursor proteins, inhibition of fibrillogenesis, reversion of the amyloid to the native fold, and dissociation of SAP to accelerate amyloid fibril clearance in vivo.
doi:10.1098/rstb.2000.0766
PMCID: PMC1088426  PMID: 11260801
19.  Trends in the indications for penetrating keratoplasty in Shandong, 2005-2010 
AIM
To identify the current indications and the trend shifts for penetrating keratoplasty (PKP) in Shandong.
METHODS
The medical charts of all patients who underwent PKP at Shandong Eye Institute from June 1, 2005 to May 31, 2010 were analysed retrospectively.
RESULTS
A total of 875 patients (875 eyes) received PKP in this 5-year period, accounting for 61.6% of all corneal transplantation surgeries. The leading indications for PKP were infectious keratitis (37.1%), HSK (19.1%), keratoconus (11.2%), bullous keratopathy (8.5%), regrafting (6.7%) and corneal scarring (4.8%). The percentage of PKP for keratoconus declined year by year, whereas the percentage of bullous keratopathy had a mild annual increase. Fungal infections accounted for 65.2% of the infectious keratitis cases, remaining the leading cause of corneal infection. In addition, 54.1% of bullous keratopathy cases were associated with cataract surgery. The leading initial diagnoses associated with regrafting were infectious keratitis (38.9%), HSK (18.6%) and corneal burn (16.9%). The major causes of regrafting included graft endothelial dysfunction (39.0%), graft ulcer (28.8%) and primary disease recurrence (15.3%).
CONCLUSION
Infectious keratitis remained the leading indication for PKP in Shandong, and fungal infections were still the major cause of corneal infections. There was an increasing trend in the percentage of PKP cases indicated for bullous keratopathy but a decline in the same for keratoconus. Even with a decline in the overall proportion among all corneal transplantation surgeries, PKP is still the major corneal transplant choice in Shandong.
doi:10.3980/j.issn.2222-3959.2011.05.07
PMCID: PMC3340731  PMID: 22553709
penetrating keratoplasty; indication; infectious keratitis
20.  A Simple Lattice Model That Captures Protein Folding, Aggregation and Amyloid Formation 
PLoS ONE  2014;9(1):e85185.
The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to inter-molecular beta strand formation. Such amyloid formation is associated with neurodegenerative disorders like Alzheimer's and Parkinson's. Molecular modelling can play a key role in providing insight into the factors that make proteins prone to fibril formation. However, fully atomistic models are computationally too expensive to capture the length and time scales associated with fibril formation. As the ability to form fibrils is the rule rather than the exception, much insight can be gained from the study of coarse-grained models that capture the key generic features associated with amyloid formation. Here we present a simple lattice model that can capture both protein folding and beta strand formation. Unlike standard lattice models, this model explicitly incorporates the formation of hydrogen bonds and the directionality of side chains. The simplicity of our model makes it computationally feasible to investigate the interplay between folding, amorphous aggregation and fibril formation, and maintains the capability of classic lattice models to simulate protein folding with high specificity. In our model, the folded proteins contain structures that resemble naturally occurring beta-sheets, with alternating polar and hydrophobic amino acids. Moreover, fibrils with intermolecular cross-beta strand conformations can be formed spontaneously out of multiple short hydrophobic peptide sequences. Both the formation of hydrogen bonds in folded structures and in fibrils is strongly dependent on the amino acid sequence, indicating that hydrogen-bonding interactions alone are not strong enough to initiate the formation of beta sheets. This result agrees with experimental observations that beta sheet and amyloid formation is strongly sequence dependent, with hydrophobic sequences being more prone to form such structures. Our model should open the way to a systematic study of the interplay between the factors that lead to amyloid formation.
doi:10.1371/journal.pone.0085185
PMCID: PMC3893179  PMID: 24454816
21.  Aggregation Propensity of the Human Proteome 
PLoS Computational Biology  2008;4(10):e1000199.
Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with β-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context.
Author Summary
Amyloid-like fibrils are insoluble proteinaceous fibrillar aggregates with a characteristic structure (the cross-β core) that form and deposit in more than 40 pathological conditions in humans. These include Alzheimer's disease, Parkinson's disease, type II diabetes, and the spongiform encephalopathies. A number of proteins not involved in any disease can also form amyloid-like fibrils in vitro, suggesting that amyloid fibril formation is an intrinsic property of proteins in general. Recent efforts in understanding the physico-chemical grounds of amyloid fibril formation has led to the development of several algorithms, capable of predicting a number of aggregation-related parameters of a protein directly from its amino acid sequence. In order to study the predicted aggregation behavior of the human proteome, we have run one of these algorithms on the 34,180 human protein sequences. Our results demonstrate that molecular evolution has acted on protein sequences to finely modulate their aggregation propensities, depending on different parameters related to their in vivo environment. Together with cellular control mechanisms, this natural selection protects proteins from aggregation during their lifetime.
doi:10.1371/journal.pcbi.1000199
PMCID: PMC2557143  PMID: 18927604
22.  Fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 are strongly expressed in the epithelium of human granular and lattice type I corneal dystrophies 
Molecular Vision  2012;18:1927-1936.
Purpose
To determine the extracellular matrix proteins involved in the formation of human granular and lattice type I corneal stromal dystrophies, the expression patterns of fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 were compared in human corneal stromal dystrophy samples.
Methods
Ten cases of granular dystrophy, 7 cases of lattice dystrophy, and 6 normal corneal buttons collected during corneal transplantation were examined for their expression patterns of fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 by immunohistochemistry.
Results
Highly elevated fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 were observed in the epithelial layer of both granular and lattice type I dystrophies. Fibrillin-2, tenascin-C, and matrilin-4 in the granular dystrophy and all antibodies in the lattice dystrophy showed statistically significant staining in the corneal stroma (p<0.05). Interestingly, fibrillin-2, matrilin-2, and matrilin-4 stained significantly in amyloid plaques of lattice type 1 dystrophy.
Conclusions
Fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 may be markers of the pathogenesis of either granular or lattice type I corneal dystrophy, as revealed by immunohistochemical analysis. Each molecule seems to be involved in the regeneration and reorganization of the corneal matrix in granular and lattice type I dystrophies.
PMCID: PMC3413442  PMID: 22876117
23.  Visual outcome in corneal grafts: a preliminary analysis of the Swedish Corneal Transplant Register 
Aim: To assess visual outcome and the incidence of complications at 2 years postoperatively in corneal grafts reported to the Swedish Corneal Transplant Register.
Methods: Preoperative and 2 year follow up data were submitted to the Swedish Corneal Transplant Register by surgeons in eight corneal transplant clinics in Sweden. Preoperative data on 1957 grafts and 520 grafts with 2 year follow up were included in the analysis. Data were analysed by multiple linear and logistic regression methods, as appropriate.
Results: The major diagnostic categories were keratoconus (29%), bullous keratopathy (21%), and “other diagnosis” (32%). Fuchs' endothelial dystrophy and stromal dystrophies accounted for 15% and 3% of grafts, respectively. At 2 years the overall incidence of complications, other than rejection and regrafting, was 26%, with an increasing frequency from keratoconus < Fuchs' dystrophy < bullous keratopathy < “other diagnosis.” Rejection was observed in 15% of grafts and was more likely in the bullous keratopathy (OR 3.1, 95% CI 1.1 to 9.0, p=0.04) and “other diagnosis” (OR 2.6, 95% CI 1.1 to 5.9, p=0.03) groups. Regrafting, which occurred in 10% of cases, was not influenced by diagnosis, but it was related to the incidence of rejection (OR 14.8, 95% CI 6.1 to 35.9, p<0.001) and other complications (OR 4.4, 95% CI 1.9 to 10.4, p=0.001), and to the presence of other sight threatening pathology in the eye (OR 3.6, 95% CI 1.3 to 9.9, p=0.01). Visual acuity was improved in a high proportion of the patients, especially those with keratoconus and Fuchs' dystrophy where, respectively, 86% and 54% of grafts achieved a visual acuity of ≥0.5 at 2 years, compared with only 31% with bullous keratopathy and 35% in the “other diagnosis” group. 60% of grafts for keratoconus and Fuchs' dystrophy achieved a visual acuity equal to or better than the other eye. Postoperative astigmatism was higher in the bullous keratopathy (p=0.01) group. Patients with high astigmatism benefited from refractive surgery, showing a reduction from 7.9 (95%CI 6.9, 8.7) to 3.2 (95% CI 2.6, 3.9) dioptres (p<0.001). A centre effect was evident in visual outcome.
Conclusion: The overall incidence of complications was related to diagnosis. Complications other than rejection and regrafting were most likely in the “other diagnosis” group, and further analysis of this group is therefore planned. The best improvement in visual acuity and the lowest astigmatism were achieved in the keratoconus and Fuchs' dystrophy groups; but the influence of diagnosis on astigmatism was small and, overall, the statistical model accounted for only 8% of the variability in astigmatism. Refractive surgery was, however, effective in reducing astigmatism. It is hoped that a better understanding of the factors that determine the visual outcome of grafts will emerge from future analyses of the Swedish Corneal Transplant Register, helping to refine the criteria for patient selection and to guide clinical practice.
PMCID: PMC1771003  PMID: 11815343
corneal graft; astigmatism; visual acuity; Sweden
24.  Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils 
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.
doi:10.7150/ijbs.5380
PMCID: PMC3584918  PMID: 23459392
lysozyme; amyloid fibril formation; core region; tryptophan; egg white.
25.  Insight into the Structure of Amyloid Fibrils from the Analysis of Globular Proteins 
PLoS Computational Biology  2006;2(12):e170.
The conversion from soluble states into cross-β fibrillar aggregates is a property shared by many different proteins and peptides and was hence conjectured to be a generic feature of polypeptide chains. Increasing evidence is now accumulating that such fibrillar assemblies are generally characterized by a parallel in-register alignment of β-strands contributed by distinct protein molecules. Here we assume a universal mechanism is responsible for β-structure formation and deduce sequence-specific interaction energies between pairs of protein fragments from a statistical analysis of the native folds of globular proteins. The derived fragment–fragment interaction was implemented within a novel algorithm, prediction of amyloid structure aggregation (PASTA), to investigate the role of sequence heterogeneity in driving specific aggregation into ordered self-propagating cross-β structures. The algorithm predicts that the parallel in-register arrangement of sequence portions that participate in the fibril cross-β core is favoured in most cases. However, the antiparallel arrangement is correctly discriminated when present in fibrils formed by short peptides. The predictions of the most aggregation-prone portions of initially unfolded polypeptide chains are also in excellent agreement with available experimental observations. These results corroborate the recent hypothesis that the amyloid structure is stabilised by the same physicochemical determinants as those operating in folded proteins. They also suggest that side chain–side chain interaction across neighbouring β-strands is a key determinant of amyloid fibril formation and of their self-propagating ability.
Synopsis
In many fatal neurodegenerative diseases, including Alzheimer, Parkinson, and spongiform encephalopathies, proteins aggregate into specific fibrous structures to form insoluble plaques known as amyloid. The amyloid structure may also play a nonaberrant role in different organisms. Many globular proteins, folding to their biologically functional native structures in vivo, can be induced to aggregate into amyloid-like fibrils under suitable conditions in vitro. One hallmark of amyloid structure is a specific supramolecular architecture called cross-beta structure, held together by hydrogen bonds extending repeatedly along the fibril axis, but intermolecular interactions are yet unknown at the amino-acid level except for very few cases. In this study, the authors present an algorithm, called prediction of amyloid structure aggregation (PASTA), to computationally predict which portions of a given protein or peptide sequence forming amyloid fibrils are stabilizing the corresponding cross-beta structure and the specific intermolecular pattern of hydrogen-bonded amino acids. PASTA is based on the assumption that the same amino acid–specific interactions stabilizing hydrogen bond patterns in native structures of globular proteins are also employed by nature in amyloid structure. The successful comparison of the authors' prediction with available experimental data supports the existence of a unique framework to describe protein folding and aggregation.
doi:10.1371/journal.pcbi.0020170
PMCID: PMC1698942  PMID: 17173479

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