Association of Sugar with Human Lens Protein

Cerium oxide nanoparticles (nanoceria) are generally known for their recyclable antioxidative properties making them an appealing biomaterial for protecting against physiological and pathological age-related changes that are caused by reactive oxygen species (ROS). Cataract is one such pathology that has been associated with oxidation and glycation of the lens proteins (crystallins) leading to aggregation and opacification. A novel coated nanoceria formulation has been previously shown to enter the human lens epithelial cells (HLECs) and protect them from oxidative stress induced by hydrogen peroxide (H2O2). In this work, the mechanism of nanoceria uptake in HLECs is studied and multiple anti-cataractogenic properties are assessed in vitro. Our results show that the nanoceria provide multiple beneficial actions to delay cataract progression by (1) acting as a catalase mimetic in cells with inhibited catalase, (2) improving reduced to oxidised glutathione ratio (GSH/GSSG) in HLECs, and (3) inhibiting the non-enzymatic glucose-induced glycation of the chaperone lens protein α-crystallin. Given the multifactorial nature of cataract progression, the varied actions of nanoceria render them promising candidates for potential non-surgical therapeutic treatment.

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Surface plasmon resonance study of the interaction of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (bis-ANS) and adenosine triphosphate (ATP) with oligomeric recombinant human lens αA-crystallin

Canadian Journal of Chemistry ◽

10.1139/cjc-2018-0412 ◽

2019 ◽

Vol 97 (6) ◽

pp. 504-511

Author(s):

Srabani Karmakar ◽

Shrutidhara Biswas ◽

Kali P. Das ◽

Umakanta Tripathy

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Age Related Macular Degeneration ◽

Disulfonic Acid ◽

Human Lens ◽

Lens Protein ◽

Retinal Diseases ◽

Dipotassium Salt ◽

Αb Crystallin

α-Crystallin, an abundant mammalian lens protein made up of two subunits (αA- and αB-crystallin), is involved in the maintenance of the optimal refractive index in the lens. The protein is implicated in the pathophysiology of a large number of retinal diseases including cataract, age-related macular degeneration, diabetic retinopathy, and uveitis. α-Crystallin belongs to the small heat shock protein (sHSP) family, forms large oligomeric structures, and functions as a molecular chaperone appearing very early during embryonic development. To gain mechanistic insight into the structural and functional role of α-crystallin and its alterations in various retinal diseases, it is important to study the interaction chemistry with its known partners. The hydrophobic sites in α-crystallin have been studied extensively using environmentally sensitive fluorescent probes such as 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (bis-ANS) that interacts with both subunits of α-cystallin in 1:1 stoichiometry at 37 °C and diminishes the chaperone-like activity of the protein. Furthermore, it has been shown that ATP plays a crucial role in the association of α-crystallin with substrate proteins. We use surface plasmon resonance (SPR) to monitor the interactions of immobilized oligomeric recombinant αA subunit of human α-crystallin protein with bis-ANS and ATP. We assess the thermodynamic parameters and kinetics of such interactions at various temperatures. Our results indicate that bis-ANS binds to αA-crystallin with higher affinity when compared with ATP, although both αA-crystallin and αB-crystallin display fast interaction kinetics.

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Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2)

Cells ◽

10.3390/cells9122670 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2670

Author(s):

Naoki Yamamoto ◽

Shun Takeda ◽

Natsuko Hatsusaka ◽

Noriko Hiramatsu ◽

Noriaki Nagai ◽

...

Keyword(s):

Amyloid Β ◽

Lens Epithelial Cell ◽

Human Lens ◽

Epithelial Cell Line ◽

Lens Protein ◽

Medium Temperature ◽

Tropical Regions ◽

Human Lens Epithelial Cell ◽

In Silico Simulation ◽

Temperature Ranges

The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid β (Aβ)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aβ1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.

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Role of Glycation in Human Lens Protein Structure Change

European Journal of Ophthalmology ◽

10.1177/112067219600600211 ◽

1996 ◽

Vol 6 (2) ◽

pp. 155-161 ◽

Cited By ~ 12

Author(s):

J.S. Ramalho ◽

C. Marques ◽

P.C. Pereira ◽

M.C. Mota

Keyword(s):

Protein Structure ◽

Structure Change ◽

Human Lens ◽

Lens Protein

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Changes in the nanostructure of non-renewable human lens protein as determined by optical measurements

Frontiers in Optics 2004/Laser Science XXII/Diffractive Optics and Micro-Optics/Optical Fabrication and Testing ◽

10.1364/fio.2004.ftue6 ◽

2004 ◽

Author(s):

John Schroeder ◽

Gillray L. Kandel

Keyword(s):

Optical Measurements ◽

Human Lens ◽

Lens Protein

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LP2, a differentiation-associated lipid-binding protein expressed in bovine lens

Biochemical Journal ◽

10.1042/bj3200049 ◽

1996 ◽

Vol 320 (1) ◽

pp. 49-54 ◽

Cited By ~ 18

Author(s):

Cynthia JAWORSKI ◽

Graeme WISTOW

Keyword(s):

Cellular Differentiation ◽

Expression Patterns ◽

Complete Sequence ◽

Lipid Binding ◽

Human Lens ◽

Fatty Acid Binding Proteins ◽

Lens Protein ◽

Differentiation Markers ◽

Fatty Acid Binding ◽

Bovine Lens

A 13 kDa protein from bovine lens was identified and characterized by protein microsequencing and by rapid amplification of cDNA ends (RACE) PCR. Its complete sequence shows that this protein belongs to a family of fatty acid-binding proteins (FABPs), including myelin and adipocyte P2, that are associated with cellular differentiation. The bovine lens protein, designated LP2, shows very close similarity to human epidermal FABP (eFABP) and human eFABP was detected in human lens, suggesting that the two proteins might be orthologous. Reverse transcriptase–PCR (RT–PCR) was used to compare expression patterns of LP2 with those for actin and for the differentiation markers γB-crystallin and γs-crystallin in lens. Actin was most abundant in the relatively undifferentiated epithelial cells and decreased with lens cell differentiation. In contrast γB-crystallin and γs-crystallin were detected only in fibres (nuclear and cortical respectively). LP2 transcripts were detected most abundantly in fibre cells and apparently increased with cellular differentiation. Molecular modelling confirms that the sequence of LP2 fits the tertiary template of adipocyte P2 but reveals the presence of two close pairs of cysteine residues that might be susceptible to intramolecular disulphide bond formation under appropriate oxidizing conditions. LP2 is thus another potential target for oxidative stress during cataract formation in lens.

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Structure Elucidation of a Novel Yellow Chromophore from Human Lens Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m405664200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45441-45449 ◽

Cited By ~ 37

Author(s):

Rongzhu Cheng ◽

Qi Feng ◽

Ognyan K. Argirov ◽

Beryl J. Ortwerth

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Water Soluble ◽

Human Lens ◽

Lens Protein ◽

Two Dimensional ◽

Cross Link ◽

Lens Proteins ◽

Lysine Residues ◽

Normal Human

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and1H,13C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the ϵ-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 ± 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract developmentin vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

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Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

10.1101/2020.02.21.960237 ◽

2020 ◽

Author(s):

Calvin J. Vetter ◽

David C. Thorn ◽

Samuel G. Wheeler ◽

Charlie Mundorff ◽

Kate Halverson ◽

...

Keyword(s):

Light Scattering ◽

Molecular Mechanisms ◽

Small Population ◽

Site Directed Mutagenesis ◽

Human Lens ◽

Lens Protein ◽

Triple Mutant ◽

Age Related ◽

Human Lenses ◽

Partially Unfolded

AbstractAge-related cataract is a major cause of blindness worldwide. Yet, the molecular mechanisms whereby large, light scattering aggregates form is poorly understood, because of the complexity of the aggregates isolated from human lenses. The predominant proteins in the lens are structural proteins called crystallins. The γS-crystallin is heavily modified in cataractous lenses by deamidation, which introduces a negative charge at labile asparagine residues. The effects of deamidation at asparagines, N14, N76, and N143, were mimicked by replacing the asparagine with aspartate using site-directed mutagenesis. The effects of these surface deamidations on the stability, unfolding, and aggregation properties of γS were determined using dynamic light scattering, chemical and thermal-denaturation, and hydrogen-deuterium exchange with mass spectrometry. We found that a small population of all the deamidation mimics aggregated directly into large light scattering bodies with a radius greater than 10 nm that contributed 14-60% of the total scattering intensity compared to 7% for WT under the same conditions. A possible mechanism was identified under partially denaturing conditions, where deamidation led to significantly more rapid unfolding and aggregation particularly for N76D compared to WT. The triple mutant was further destabilized, reflecting the enhanced aggregation properties of N14D and N143D. Thus, the effects of deamidation were both site-specific and cumulative. αA-crystallin was ineffective at acting as a chaperone to prevent the aggregation of destabilized, deamidated γS. It is concluded that surface deamidations, while causing minimal structural disruption individually, progressively destabilize crystallin proteins, leading to their unfolding and precipitation in aged and cataractous lenses.

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