α-晶状体蛋白对维持晶状体透明性的功能研究进展

α-晶状体蛋白是眼晶状体细胞质的主要成分,由小热休克蛋白家族中的αA-晶状体蛋白和αB-晶状体蛋白组成.α-晶状体蛋白具有分子伴侣活性,还有调控细胞周期、增强基因组稳定性、防止压力诱导性细胞凋亡的作用.α-晶状体蛋白相关基因突变常引起遗传性白内障,是目前儿童盲的主要原因,而α-晶状体蛋白的多种改变可导致年龄相关性白内障.了解α-晶状体蛋白的功能有助于理解晶状体的发育及其正常功能的维持,为预防及治疗α-晶状体蛋白相关性白内障提供理论依据.就白内障发生机制、遗传定位及晶状体蛋白的功能进行综述.     

Ontogeny of alpha A and alpha B crystallin polypeptides during Rana temporaria lens development

The ontogeny and localization of alpha A and alpha B polypeptide chains of alpha-crystallin were investigated in the developing lens of Rana temporaria, an anuran amphibian, using the indirect immunofluorescence staining method with heterologous antibodies directed against these two polypeptides. alpha A and alpha B crystallins are primary gene products and are translated by different mRNAs in mammals. Although they show about 6000 amino-acid sequence homology (Bloemendal, 1977), the alpha A cDNA of rat and mouse does not hybridize to alpha B mRNA (Dodemont et al., 1981; King and Piatigorsky, 1983). Antigenically too, alpha A and alpha B polypeptides have been shown to be different. These two polypeptides were isolated from mouse lens native alpha-crystallin by SDS-gel electrophoresis and were injected into young rabbits to raise antibodies. These antibodies were tested by immunoblotting against R. temporaria total lens soluble proteins before their use in the present investigation. Results presented here show that in the developing lens of R. temporaria, alpha A appears earlier than alpha B, suggesting a differential gene activation. In addition, these two polypeptides could not be detected either in the developing lens epithelium or in the epithelium of young froglets (2-3 weeks post-metamorphosis).     

Antisera to alpha crystallin as probes to study changes in lens proteins during human cataractogenesis

Antisera have been made to synthetic peptides that correspond to eight different regions of the alpha A molecule. Together with a solid phase radioimmunoassay, these antisera have been used to quantitatively assess binding to enriched alpha crystallin preparations from six different cataractous and six different normal lenses. Seven of the eight antisera show no difference in binding to alpha crystallin from cataractous versus normal lenses, whereas the antiserum directed against the alpha A sequence 120-130 shows a statistically significant decrease in binding to the alpha crystallin from cataractous lenses. Together, these studies demonstrate the feasibility of using antipeptide sera as probes of polypeptide changes during cataractogenesis and suggest that the region of the alpha A crystallin molecule encompassing residues 120-300 may undergo covalent and/or noncovalent structural modification during the process of opacification in the human senile lens.     

The Nitrite/alpha crystallin reaction: a possible mechanism in lens matrix damage

Recent work has explored the potential deleterious role that nitric oxide (NO) and its derivatives may have in human disease. The many by-products of NO include nitrite ion, which accumulates in the anterior chamber during ocular inflammation and can be derived from cigarette smoking. Cigarette smoking has been strongly linked to nuclear cataract formation, although the mechanism remains unknown. We have previously observed that nitrite reactions with the matrix proteins elastin and collagen produce damaging effects that mimic those observed in age- and smoking-related illnesses. In the present study we report on the reaction of nitrite with alpha crystallin, the major lens matrix protein. Incubations at neutral pH and body temperature of nitrite with alpha crystallin resulted in protein modifications indicative of oxidative damage and similar to changes seen in the aging lens as well as cataracts. These include increased fluorescence, yellowing and protein cross-linking. L-kynurenine, a tryptophan derivative, was identified as a reaction product. L-kynurenine was also formed from the reaction of nitrite with free tryptophan. Thus, this non-enzymatic nitration of alpha crystallin provides a novel mechanism by which lens proteins may be damaged in vivo. Since human exposure to nitrite is increased by cigarette smoking, this reaction could provide an explanation for the association between nuclear cataracts and smoking.     

Immunochemical studies on lens protein-protein complexes III. Age-related changes in the stability of lens alpha crystallin containing compleces

The age dependence of the stability of lens crystallin complexes was investigated. The lens protein fractions retained on Diaflo XM-300 membranes were subsequently separated on Bio-gel A 15 m columns, yielding one calf cortical α-β crystallin complex and two different bovine nuclear α-β-γ crystallin complexes. Separate samples of these different lens crystallin complexes were exposed for various lengths of time to acidic or alkaline pH, or to high concentrations of salts at neutral pH. Acidic pH, three units below the neutral, had a more pronounced dissociating effect on the crystallin complexes than alkaline pH, three units above the neutral, or a high salt concentration. The dissociation of the various complexes was tested by ultrafiltration on Diaflo XM-300 membranes and by immunochemical analysis of the fractions retained and filtered.Comparison of the calf cortical and bovine nuclear complex preparations indicated that the stability of the crystallin complexes increases with progressive aging of the lens. The crystallin complexes investigated did dissociate in a constant electrical field, yielding free α-, β- and γ-crystallin molecules.     

Inhibition of unfolding and aggregation of lens protein human gamma D crystallin by sodium citrate

Cataract affects 1 in 6 Americans over the age of 40, and represents a global health problem. Mature onset cataract is associated with the aggregation of partially unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the primary effective treatment for cataract. As an alternative preventive approach, small molecules have been suggested as potential therapeutic agents. In this work, we study the effect of sodium citrate on the stability of Human γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding–refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-terminal domain (N-td) of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-terminal domain (C-td) by 0.6 M GuHCl, and the single transition of I90F HγD-Crys by 0.4 M GuHCl. In kinetic unfolding reactions, sodium citrate stabilization effect was observed only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-Crys was observed, which was not populated in the absence of citrate. The rates of aggregation were measured using solution turbidity. Sodium citrate demonstrated negligible effect on rate of aggregation of WT HγD-Crys, but considerably slowed the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slowed refolding of WT HγD-Crys and I90F HγD-Crys, but had a significantly smaller effect on the refolding of L5S HγD-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results indicate that assessment of potential anti-cataract agents needs to include effects on the unfolding and aggregation pathways, as well as the native state.     

Oxidation of the N-terminal methionine of lens alpha-A crystallin.

Antiserum against the N-terminal peptide of bovine alpha-A crystallin has been used to monitor purification of two different seropositive peptides (i.e. T1a and T1b) from a tryptic digest of bovine lens proteins. Both these peptides have similar amino acid compositions, but peptide T1b has a molecular weight 16 atomic mass units larger than T1a, suggesting posttranslational modification. Analysis of ionization fragments of the T1b peptide by mass spectrometry demonstrates that this difference in molecular weight is due to the in vivo oxidation of the N-terminal met residue of the alpha-A crystallin molecule.     

An assay for intermolecular exchange of alpha crystallin.

An affinity column of alpha crystallin linked to cyanogen bromide-activated Sepharose was developed to study the exchange of alpha subunits. Alpha crystallin bound to the Sepharose-alpha complex was dissociated with 8 mol/l urea, followed by quantitation using high-performance reverse-phase liquid chromatography. The time course of binding at 37 degrees C showed a hyperbolic binding pattern reaching equilibrium between 6-18 hr. Under these conditions, binding of beta and gamma crystallins to the same matrix was less than 10% of the alpha values, as was binding of alpha to glycine-coupled Sepharose. This assay was used to demonstrate changes in the subunit exchange of alpha crystallins present in high molecular weight versus lower molecular weight aggregates of the human lens. These results show that this binding procedure was a specific reproducible assay that might be used to study intermolecular interactions of the alpha crystallins.     

Binding of actin to lens alpha crystallins.

Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.     

Identification of a major continuous epitope of human alpha crystallin.

Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.     

Age-dependent loss of the C-terminal amino acid from alpha crystallin.

Antiserum made against the C-terminal region of alpha-A crystallin was used to monitor the purification of a tryptic peptide containing the C-terminus of the molecule from fetal versus adult bovine lenses. Mass spectral analysis of the peptide preparations obtained from these lenses demonstrated the presence of a peptide (T20) containing an intact C-terminus from fetal lenses and the presence of an additional peptide (T20') from older lenses that contained a cleaved C-terminal serine. These results demonstrate an age-dependent processing of alpha-A crystallin in the bovine lens, resulting in removal of the C-terminal amino acid residue.     

Mass spectrometric analysis of the structure of gamma II bovine lens crystallin.

The amino acid sequence of bovine gamma II-crystallin has been verified by a combination of electrospray and fast atom bombardment mass spectrometry. The molecular weight of gamma II, isolated by gel filtration and ion exchange chromatography, was determined to be 20,967 +/- 3 by electrospray mass spectrometry. Another aliquot of gamma II was completely digested by trypsin in a medium of 20% CH3CN and 0.1 M Tris, pH 8.2. The tryptic peptides were separated by reversed phase HPLC and identified by their molecular weights, as determined by fast atom bombardment mass spectrometry (FABMS). The identification of each peptide was confirmed by digesting the peptide further to give new peptides whose molecular weights were also determined by FABMS and related to the proposed amino acid sequences. The data from both types of mass spectrometric analyses were consistent with the sequence previously proposed by Hay et al. (J. Biol. Chem. 1987, 146, 332-338), including threonine at position 119. The FAB mass spectrum of one HPLC fraction suggested that disulfide bonding between Cys 18 and Cys 22 was present in at least half the protein preparation. Whether the Cys 18/Cys 22 disulfide bond was present in native gamma II or was produced during isolation or enzymic digestion could not be determined from these studies. Samples that had been stored for several weeks showed that several of the cysteines had become disulfide bonded. These studies illustrate the power of mass spectrometric techniques to accurately confirm the primary structure of proteins and to identify post-translational modifications.     

Protein aging: Truncation of aquaporin 0 in human lens regions is a continuous age-dependent process

The human lens is ideal for the study of macromolecular aging because cells in the centre, along with their constituent proteins, are present for our entire lives. We examined the major membrane protein, aquaporin 0 (AQP0), in regions of the lens formed at different times during our lifespan, to determine if similar changes could be detected and if they were progressive. Membrane fractions from three concentric lens regions were examined by SDS-PAGE coupled with densitometry, and Western blotting, to assess the time course of truncation. The overall extent of modification was also examined by MALDI mass spectrometry of the undigested proteins. In all regions, AQP0 became progressively more truncated, specifically by the loss of a 2 kDa intracellular C-terminal peptide. The proteolysis increased steadily in all regions such that half of the AQP0 in the barrier region (that part of the lens formed immediately after birth) had been cleaved by age 40-50. MALDI mass spectrometry revealed that in all regions, AQP0 not only was shortened, it also became progressively more heterogeneous with age. Since the lens interior is devoid of active enzymes, it is very likely that the cleavage of AQP0 is chemically induced. We speculate that the loss of this C-terminal peptide ‘spacer’ may allow occlusion of AQP0 pores on the cytoplasmic face of the fibre cell membranes. Once a significant proportion of AQP0 has been cleaved, this occlusion may contribute to the formation of the lens permeability barrier that develops at middle age.     

Differential synthesis of crystallin and noncrystallin polypeptides during lens fiber cell differentiation in vitro.

Cells of the isolated lens epithelium from 6-day-old chick embryos undergo numerous changes characteristic of lens fiber cell differentiation when cultured in the presence of fetal calf serum. In the present study, protein synthesis in these explants was quantitated by monitoring the incorporation of [³H]valine during the first day of lens fiber cell differentiation in vitro. Forty-six lens epithelial cell polypeptides were identified as α-, β-, δ- or nonerystallin on the basis of their native molecular weight (determined by Sephadex G-200 chromatography) and subunit molecular weight (determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis).During the first 5 hr in vitro, the rate of labeling of 45 of the 46 polypeptides increased approximately twofold, while the labeling of one β-crystallin component appeared transiently stimulated about threefold. Between 5 and 24 hr in vitro, the rate of labeling was maintained for α- and most of the β-crystallins, was increased an additional 2·3-fold for δ-crystallin, and was decreased appreciably for most of the noncrystallin polypeptides. “Pulse-chase” experiments, coupled with previous tests of intracellular specific activity of [³H]valine under the present labeling conditions, indicate that the differences in labeling were due principally to differences in rates of synthesis. Thus, lens fiber cell differentiation in vitro is associated with preferential synthesis of the lens crystallins.The β-crystallins of the embryonic lens epithelial cells appear to exist as two native molecular weight classes. The higher class has a molecular weight near 80 000 and is composed of aggregated polypeptides, while the lower class ranges from approximately 20 500 to 34 000 in molecular weight and consists of monomeric polypeptides smaller than those which form the higher class.