Post-translationally modified human lens crystallin fragments show aggregation in vitro

BackgroundCrystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties.MethodsFour crystallin fragments-containing fractions (Fraction I [～3.5 kDa species], Fraction II [～3.5–7 kDa species], Fraction III [～7–10 kDa species] and Fraction IV [>10–18 kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50–70 year old-donors. The crystallin fragments of the Fractions I–IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I–IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins.ResultsCrystallin fragments in Fractions I–IV were derived from α-, β- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in M_r from 5.5×10⁵ to 1.0×10⁸ Da, with diameters of 10–28 nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins.ConclusionThe crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development.General significanceCrystallin fragments play a major role in human cataract development.     

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).     

Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, alphaB-(1-18) (MDIAIHHPWIRRPFFPFH) and betaA3/A1-(59-74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, gammaS-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact alpha-, beta-, and gamma-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of alphaB-(1-18) and betaA3/A1-(59-74) peptides increased the scattering of light by beta- and gamma-crystallin and alcohol dehydrogenase. The ability of alpha-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with alphaB-(1-18) and betaA3/A1-(59-74) peptides, whereas gammaS peptide had no effect on chaperone-like activity of alpha-crystallin. The betaA3/A1-(59-74 peptide caused a 5.64-fold increase in alphaB-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in alphaB-(1-18) and betaA3/A1-(59-74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

On the stability of bovine gamma II crystallin.

Bovine gamma II crystallin, undergoes structural changes when lyophilized. The lyophylized powder does not readily dissolve in buffer, although it can be taken up in distilled water. The lyophylized sample, as opposed to a sample concentrated by vacuum distillation at 30 degrees C, does exhibit different migration on isoelectric focusing gels. The hydration and denaturation properties of the two preparations are different. The lyophylized sample possesses a higher non-freezable water content as a function of concentration than its counterpart. The lyophylized sample also shows less denaturation in differential scanning calorimetry scans, up to 110 degrees C, than its counterpart. This indicates that lyophylization may induce a slight denaturation, due to structural-conformational change. On the other hand, the CD spectra of lyophylized and non-lyophylized gamma II crystallins do not differ significantly. This implies that the conformational changes upon lyophylization do not involve the secondary structure of gamma II crystallin.     

Leucine degradation in cell-free extracts of skeletal muscle.

Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.     

Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts

The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water-soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5-phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4-bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1-phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds.     

sHSP in the eye lens: crystallin mutations, cataract and proteostasis

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin "holdase" chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the "α-crystallin domain" of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens β- and γ-crystallins are considered in the context of their possible dependence on the "holdase" chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Low temperature perception in plants: effects of cold on protein phosphorylation in cell-free extracts

Activities of prevalent protein phosphatases decreased by nearly 95% and those of individual protein kinases were differentially reduced at low temperature. Inhibition of phosphatase activity at temperatures below 12°C resulted in marked hyperphosphorylation of a 58-kDa protein (PP58). The temperature threshold for hyperphosphorylation of PP58 coincided with the known threshold for cold-induced calcium influx. Since calcium influx is triggered by several environmental stresses, we propose that the observed direct effects of cold on the phosphorylation of specific proteins enable cells to couple a shared calcium signal to a cold-specific transduction pathway.     

The ageing lens and cataract: a model of normal and pathological ageing

Cataract is a visible opacity in the lens substance, which, when located on the visual axis, leads to visual loss. Age-related cataract is a cause of blindness on a global scale involving genetic and environmental influences. With ageing, lens proteins undergo non-enzymatic, post-translational modification and the accumulation of fluorescent chromophores, increasing susceptibility to oxidation and cross-linking and increased light-scatter. Because the human lens grows throughout life, the lens core is exposed for a longer period to such influences and the risk of oxidative damage increases in the fourth decade when a barrier to the transport of glutathione forms around the lens nucleus. Consequently, as the lens ages, its transparency falls and the nucleus becomes more rigid, resisting the change in shape necessary for accommodation. This is the basis of presbyopia. In some individuals, the steady accumulation of chromophores and complex, insoluble crystallin aggregates in the lens nucleus leads to the formation of a brown nuclear cataract. The process is homogeneous and the affected lens fibres retain their gross morphology. Cortical opacities are due to changes in membrane permeability and enzyme function and shear-stress damage to lens fibres with continued accommodative effort. Unlike nuclear cataract, progression is intermittent, stepwise and non-uniform.