Jack N. Liang

CHANGES IN TERTIARY STRUCTURE OF CALF-LENS α-CRYSTALLIN BY NEAR-UV IRRADIATION: ROLE OF HYDROGEN PEROXIDE

The effect of 300 nm irradiation on the three lens crystallins, α‐, β‐, and γ‐, was studied by using fluorescence and circular dichroism techniques. α‐Crystallin showed a pronounced change in tertiary structure as manifested in fluorescence and circular dichroism measurements. This finding is in agreement with our earlier findings that the tryptophan residues of α‐crystallin are more exposed than those of the other two crystallins. The results of studies using inhibitors specific for the different active species of oxygen suggest that H2O2‐mediated damage is involved in the change of tertiary structure of the proteins. Analyses of circular dichroism spectra indicate that, upon irradiation, the secondary structure of α‐crystallin remains virtually unaltered, and that the change in tertiary structure results primarily from photoinduced damage to the tryptophan residues.

In vitro non-enzymatic glycation and formation of browning products in the bovine lens α-crystallin

Calf lens LMW alpha-crystallin was glycated by incubating with various sugars (glucose, glucose-6-P and ribose) for 21 days. All sugars induced disulfide formation, but ribose also produced higher molecular weight cross-linked species. The ribocated protein turned yellow in color and had a strong blue fluorescence (Ex/Em = 370/450 nm) typical for a browning product. The chromophore of the browning product showed a new circular dichroism (CD) band at 320-330 nm. Conformational study indicated that the browning reaction destablized protein and may play a significant role in protein aggregation and insolubilization.

Age-related changes in protein conformation in bovine lens crystallins

In order to investigate the conformational changes associated with the aging process, circular dichroism (CD), absorption and fluorescence measurements of bovine lens crystallins isolated from the nucleus of old (cow) and young (calf) animals are reported. Results show considerable differences in spectroscopic parameters between the young and old alpha-crystallin; however, no such changes were observed for beta- and gamma-crystallins. Age-related changes include an increased absorption in near-u.v. and decreased intensity in the far-u.v. region; near-u.v. circular dichroism shows a considerable difference, whereas the dichroism in far-u.v. remains the same. The decrease in tryptophan fluorescence of old alpha-crystallin is of the same magnitude as is the increase in non-tryptophan fluorescence. The fluorescence of the sulfhydryl (SH) specific probe, 2-(4-maleimidylanilino) naphthalene-6-sulfonate, indicates that accessible (to the probe) SH groups of cow alpha-crystallin are fewer than those of calf, and they are also in a more polar environment. This study demonstrates that, with aging, alpha-crystallin undergoes a change in the tertiary structure involving tryptophan, tyrosine and cysteine residues. This conformational change has been explained by the suggestion that a large portion of the protein unfolds during the aging process, resulting in a change in interaction properties between the aromatic amino acid residues and between the residues and the peptide backbone. The unfolding is also associated with the accessibility, reactivity and spatial arrangement of these residues, including the cysteine by which aggregation or cross-linking of the protein is likely to occur.

Sugar-induced change in near ultraviolet circular dichroism of α-crystallin

Abstract In order to study the effect of sugar molecules on the structure of α-crystallin, the protein from bovine lenses was isolated and treated with D-glucose and D-glucose-6-phosphate. Structural studies of the products were carried out using circular dichroism (CD) measurements. In the near-ultraviolet (UV) region, three positive and two negative CD bands are observed for α-crystallin. Upon incubation with the saccharides, the positive ellipticities of these near-UV CD bands increase greatly. The CD spectrum of α-crystallin in the far-UV region, which is typical of the β-conformation of a protein, does not change. The increase does not appear to be caused by glycosylation, or by sugar metabolites such as sorbitol or glycolytic products. When the concentration of sugar is higher than that has been used in this study, the protein tends to aggregate. The results strongly suggest that sugar, upon interaction with α-crystallin, induces a change in the tertiary structure of the protein while its secondary structure remains intact. The interaction of sugar molecules causing such change is weak, noncovalent, and temperature-dependent. The near-UV CD of α-crystallin appears to be a useful probe for the study of such structural changes, which may be significant in the pathogenesis of cataract.

An essential role for the 2-sulfamino group in the interaction of calcium ion with heparin

Abstract Differences in the circular dichroism and n.m.r. spectra of the sodium and calcium salts of heparin concur in suggesting that a chelate complex is formed between 1 mol of calcium ion and a disaccharide repeating segment of the polymer. Upon selective hydrolysis of the N -sulfate group of heparin, this specific binding of calcium is no longer observable. It is proposed, among other possibilities, that the carboxylic group strongly binds the cation, and that the sulfamino group of an adjacent sugar residue simultaneously engages in a weaker electrostatic interaction, so as to stabilize the complex. Replacement of the N -sulfate by an N -acetyl group also eliminates the specific calcium-binding capability of heparin.

Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione

Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH groups after the exchange reaction in alpha-crystallin and gamma-crystallin, respectively. The exchange reaction was further shown by an acidic shifting in IEF pattern. The near ultraviolet CD shows a slight decrease in the GSSG-treated crystallins. The fluorescence measurements of the SH specific probe IANBD, 4-(N-iodoacetoxy)ethyl-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole, indicate that the surface SH groups were oxidized in the GSSG-treated samples. The labeling with amine selectively reactive probe FITC, fluorescein-5-isothiocyanate, indicates an increase of amine reactivity with mixed disulfide formation. Polarization measurements show that bound FITC probes are in a less rigid structure in the mixed disulfide rich crystallin. All these results point out that the formation of mixed disulfide partially unfolds protein.

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins.

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components—ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)—suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, βB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Emu2009=u2009400/400xa0nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, βB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.

Spectroscopic studies on pepsin-solubilized vitreous and cartilage collagens.

Circular dichroism and the fluorescent probe, 2-p-toluidinyl-naphthalene-6-sulfonate, were used to compare the molecular properties of pepsin-solubilized vitreous collagen with cartilage and calfskin collagens. Type II vitreous and cartilage collagens have more hydrophobic regions along their molecular domain than does type I calfskin collagen. The rate of fibril growth is faster in type II collagens than in type I. The increased hydrophobicity of type II collagens is attributed to high carbohydrate content and compositional variations. Although the secondary structures of the three collagens do not differ significantly, differences in carbohydrate content, composition, and hydrophobic character may cause some variations in the tertiary structures. It is suggested that the tertiary structure plays an important role in the nature and rate of fibril growth. Differences between cartilage and vitreous collagen in fluorescence behavior, fibril growth, and melting temperature indicate that vitreous collagen may be a special type II collagen.

Protein–protein interactions involving congenital cataract T5P γC-crystallin mutant: A confocal fluorescence microscopy study

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects protein-protein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP-RFP (donor-acceptor) plasmid pairs was cotransfected into HeLa cells: gammaC-gammaC, gammaC-gammaCT5P, gammaCT5P-gammaCT5P, alphaA-gammaC, and alphaA-gammaCT5P. After culture, confocal fluorescence cell images were taken. Protein-protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P gammaC-crystallin contain many protein aggregates, but cells co-expressing with either gammaC- or alphaA-crystallin reduce the aggregation considerably. FRET determination indicates that gammaCT5P-gammaCT5P shows less protein-protein interaction than either gammaC-gammaC or gammaC-gammaCT5P. Cotransfection with alphaA-crystallin (alphaA-gammaC or alphaA-T5PgammaC) increases nFRET compared with gammaC-gammaC or gammaC-T5PgammaC. Our results demonstrate that T5P gammaC-crystallin shows more protein aggregates and less protein-protein interaction than WT gammaC-crystallin. Chaperone alphaA-crystallin can rescue T5P gammaC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein-protein interactions and increased aggregation from an insufficient amount of alpha-crystallin for protection.