High-molecular-weight protein aggregates of calf and cow lens: Spectroscopic evaluation

doi:10.1016/0014-4835(88)90001-2

Experimental Eye Research

Volume 47, Issue 2, August 1988, Pages 173-183

https://doi.org/10.1016/0014-4835(88)90001-2 Get rights and content

Abstract

To gain insight into the molecular features of the high-molecular-weight (HMW) fraction of soluble lens proteins and their changes in aging, we isolated this fraction from the nucleus of calf and cow lenses and measured fluorescence and circular dichroism (CD) properties of the samples. Not only was there an increase in the HMW fraction in the older lens, but there was also an agerelated difference in tertiary structure that was clearly manifested in the fluorescence and CD parameters. The far-u.v. CD of low- and high-molecular-weight proteins do not differ significantly in band position and magnitude, but the near-u.v. CD of HMW protein does differ distinctly from that of all other crystallins (α, β and γ); the entire CD spectrum of this protein is displayed in the negative region. Millipore filtration further revealed that HMW aggregates are essentially a polydisperse population of different conformation (tertiary structure) and that these aggregates are associated by non-convalent interactions. This association is caused mainly by the apolar (hydrophobic) nature of the constituent protein, α-Crystallin has more hydrophobic domain along the peptide chain than do other crystallins and thus is likely to be the predominant protein in HMW aggregates.

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Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses
2008, Experimental Eye Research
Citation Excerpt :
In addition, the same report also showed that human lens WS-HMW protein-associated crystallin fragments were a heterogeneous mixture derived from α-, β-, and γ-crystallins, and were rich in hydrophobic amino acids [i.e., 40.3% and 45.3% of total amino acids were hydrophobic amino acids in 16- to 18-year-old and 60- to 80-year-old lenses, respectively (Srivastava et al., 1996)]. Indeed, previous reports have suggested that WS-HMW proteins contained no covalently bonded multimers but were held primarily via hydrophobic interactions (Meissier and Chakravarti, 1988; Spector, 1982). We recently compared the crystallin species present in the WS-HMW and WI proteins of human cataractous and age-matched normal lenses (Harrington et al., 2004).
The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate–PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M_r between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of α-, β-, and γ-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of α-, β-, and γ-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.
Human lens high-molecular-weight α-crystallin aggregates
2000, Biochemical and Biophysical Research Communications
α-Crystallin high-molecular-weight (HMW) aggregates can be formed in vitro by many mechanisms, but the mechanism of in vivo aggregation has not been clearly established. HMW and LMW (low-molecular-weight) α-crystallins were isolated from human lenses 50–60 years of age and some spectroscopic measurements were performed. Conformational differences were suggested based on data of increased bis-ANS (4,4′-dianilino-1,1′-binaphthalene-5,5′-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW α-crystallin was more hydrophobic than LMW α-crystallin, possibly resulting from partial unfolding of α-crystallin. On the other hand, the increased ThT fluorescence and far-UV CD intensities indicate that an increased amount of β-sheet conformation was involved in aggregation. These data, along with little difference in chaperone-like activity between the LMW and HMW α-crystallins, strongly suggest that HMW α-crystallin aggregates resulted from partial unfolding and disassembling–reassembling of LMW α-crystallin caused by posttranslational modification rather than chaperone complex formation.
α-Crystallin as a molecular chaperone
1999, Progress in Retinal and Eye Research
The role of α-crystallin as a molecular chaperone may explain how the lens stays transparent for so long. α-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by “trapping” the protein in a high molecular weight complex. It also protects enzyme activities. The substrate protein may interact while in a molten globule state. α-Crystallin predominantly binds to proteins very early in the denaturation pathways. The amphiphilic nature of α-crystallin, a polar C-terminal-region and a hydrophobic N-terminal-region are all essential for chaperone function. The flexible C-terminal extension maintains solubility and can bind to opposing charged residues of unfolding proteins. Hydrophobic regions in the N-terminal region then hold the unfolded protein. Specific areas important for chaperone binding and function have been identified throughout the N-terminal-region, connecting peptide and C-terminal extension. After a substantial amount of chemical data and models, cryo-EM images of α-crystallin have confirmed a variable 3D surface with a hollow interior. α-Crystallin taken from the lens nucleus shows an age-dependent decrease in chaperone function. High molecular weight aggregates and α-crystallin found within the nucleus from clear and cataract lenses have reduced chaperone function. Post-translational modifications, known to occur during ageing, such as glycation, carbamylation, oxidation, phosphorylation and truncation cause a decrease in chaperone function. α-Crystallin is expressed outside the lens. αB-Crystallin can be induced by heat shock in many tissues where it is translocated from cytoplasm to nucleus. Increased expression of αB-crystallin has been seen in many pathological states. Conformational disorders, including cataract may have a common aetiology and potentially a common therapy.
NMR spectroscopy of α-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins
1998, International Journal of Biological Macromolecules
The subunit molecular mass of α-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved ¹H NMR spectrum is observed for α-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, αA- and αB-crystallin. These extensions are not involved in interactions with other proteins (e.g. β- and γ-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of αA-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic α-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the αA-crystallin subunit maintains its flexibility whereas the αB-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the ‘substrate’ protein. The conformation of ‘substrate’ proteins when they interact with α-crystallin has been probed by ¹H NMR spectroscopy and it is concluded that α-crystallin interacts with ‘substrate’ proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the ¹H and ³¹P NMR spectra of α-crystallin in the presence of increasing concentations of urea, it is proposed that α-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of α-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with ‘substrate’ proteins during the chaperone action.
Age-related changes in bovine α-crystallin and high-molecular-weight protein
1996, Experimental Eye Research
The high-molecular-weight (HMW) protein from the lens is composed mostly of α-crystallin in a highly aggregated state. Bovine HMW protein was carefully separated from α-crystallin by size-exclusion chromatography. α-Crystallin has chaperone-like ability whereby it stabilizes other proteins under conditions of stress (e.g. heat). Comparison of bovine HMW protein and α-crystallin shows that the HMW protein has a markedly reduced chaperone ability compared to α-crystallin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of α-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the α-crystallin subunits with age have been quantified. Phosphorylation of α-crystallin occurs early in life but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of α-crystallin has little effect on its chaperone ability. As is found in the artificially prepared HMW complex of α- and γ-crystallin, NMR spectroscopy shows that in the naturally occurring HMW protein, the short C-terminal extension of the α_Bsubunit has lost its flexibility whereas the α_Asubunit extension is still flexible. Post-translational modifications therefore seem to have little effect on the chaperone action of α-crystallin, but alterations in the quaternary structure of α-crystallin via incorporation into the HMW aggregate, lead to major changes in the chaperone ability of the protein. The results are consistent with the notion that one of the contributing factors to cataract formation in the lens is the depletion of α-crystallin with age as it is converted into the HMW protein.
Methylisocyanate and actin polymerization: the in vitro effects of carbamylation
1993, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Uremia has been implicated in cataractogenesis due to protein carbamylation by cyanate derived from urea. The present study was designed to directly identify the effects of carbamylation on actin polymerization and the possible contribution to cataract formation. The susceptibility of actin to carbamylation is expected because of the 19 lysines distributed along its length. The lysines of actin were selectively carbamylated by methylisocyanate (MIC) at pH 8.0 and 4°C and actin polymerization assayed by high-shear viscometry, fluorescence and transmission electron microscopy. Our results provide evidence that non-enzymatic carbamylation of the lysine residues prevents the polymerization of actin. In addition, this carbamylated actin inhibited the polymerization of nascent, unmodified actin. High-shear viscosity measurements demonstrated decreased initial apparent rates and decreased steady-states (final specific viscosities) of polymerization. Fluorescence measurements showed decreased relative intensities of fluorescence versus control and confirmed the inhibitory effects of carbamylation by MIC on the steady state of F-actin. Transmission electron microscopy (TEM) showed the presence of disorganized filaments when carbamylated actin was added to polymerizing unmodified actin. Our results suggest that carbamylation of actin can cause a loss of ordered filament structure and shape of the lens fiber cell, thus predisposing it to cataract development.

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High-molecular-weight protein aggregates of calf and cow lens: Spectroscopic evaluation

Abstract

Exp. Eye Res.

Biochem. Biophys. Res. Commun.

Exp. Eye Res.

Exp. Eye Res.

Exp. Eye Res.

Exp. Eye Res.

Exp. Eye Res.

J. Biol. Chem.

Exp. Eye. Res.

Biochem. Biophys. Res. Commun.

Exp. Eye Res.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

Exp. Eye Res.

Biophys. Chem.

Spectroscopic investigations of bovine lens crystallins. 2. Fluorescent probes for polar-apolar nature and sulfhydryl group accessibility

Biochemistry

Changes in tertiary structure of calf-lens α-crystallin by near-u.v. irradiation: role of hydrogen peroxide

Photochem. Photobiol.

Theory of transparency of the eye

Appl. Optics

Lens proteins

CRC Crit. Res. Biochem.