Kirsten J. Lampi

Sequence analysis of βA3, βB3, and βA4 crystallins completes the identification of the major proteins in young human lens

A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as αA, αB, βA1, βA3, βA4, βB1, βB2, βB3, γS, γC, and γD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human βA1, βA3, βA4, and βB3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of βA3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.

A Nonsense Mutation in CRYBB1 Associated with Autosomal Dominant Cataract Linked to Human Chromosome 22q

Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the beta-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [theta] 0) and D22S1154 (Z=1.39 at theta=0), which closely flank the genes for betaB1-crystallin (CRYBB1) and betaA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G-->T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human betaB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans.

Deamidation destabilizes and triggers aggregation of a lens protein, βA3-crystallin

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the βA3‐subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of βA3 and were investigated in this study. Deamidation was mimicked by site‐directed mutagenesis at Q42 and N54 on the N‐terminal domain, N133 and N155 on the C‐terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of βA3. Deamidations in the C‐terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N‐terminal domain. Deamidation at N54 and N155 also disrupted the association with the βB1‐subunit. Sedimentation velocity experiments integrated with high‐resolution analysis detected soluble aggregates at 15%–20% in all deamidated proteins, but not in wild‐type βA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other β‐subunits. Understanding the role of deamidation in the long‐lived crystallins has important implications in other aggregation diseases.

New focus on alpha-crystallins in retinal neurodegenerative diseases

The crystallin proteins were initially identified as structural proteins of the ocular lens and have been recently demonstrated to be expressed in normal retina. They are dramatically upregulated by a large range of retinal diseases including diabetic retinopathy, age-related macular degeneration, uveitis, trauma and ischemia. The crystallin family of proteins is composed of alpha-, beta- and gamma-crystallin. Alpha-crystallins, which are small heat shock proteins, have received substantial attention recently. This review summarizes the current knowledge of alpha-crystallins in retinal diseases, their roles in retinal neuron cell survival and retinal inflammation, and the regulation of their expression and activity. Their potential role in the development of new treatments for neurodegenerative diseases is also discussed.

Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses

Purpose. To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. Methods. Total soluble proteins from bovine, human, and rodent lenses, ßH crystallin, or recombinant ßB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Pro-teolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. Results. The in vitro cleavage sites produced by m-calpain on the N-termini of human ßB1, ßA3, and ßB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of a- and ß-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. Conclusions. Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.

Laser light-scattering evidence for an altered association of βB1-crystallin deamidated in the connecting peptide

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in βB1‐crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble βB1 contained 13%–17% deamidation at Gln146 and Asn157. Static and quasi‐elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild‐type (wt) βB1 and the deamidated βB1 mutants, Q146E and N157D. Dimer formation occurred for wt βB1, Q146E, and N157D in a concentration‐dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of βB1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.

Aggregation of deamidated human βB2-crystallin and incomplete rescue by α-crystallin chaperone

Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens betaB1, betaB2 and betaA3-crystallin. Of particular interest, betaB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine betaLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine alpha-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by alpha-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated beta-crystallin aggregates is discussed.

Ubiquitin Proteasome Pathway–Mediated Degradation of Proteins: Effects Due to Site-Specific Substrate Deamidation

PURPOSE The accumulation, aggregation, and precipitation of proteins is etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome pathway (UPP) is protein surveillance and maintenance of protein quality. The purpose of this study was to determine whether deamidation can alter clearance of crystallins by the UPP. METHODS Wild-type (WT) and deamidated crystallins were expressed and (125)I-radiolabeled. Ubiquitination and degradation were monitored separately. RESULTS For betaB2 crystallins, rates of ubiquitination and adenosine triphosphate-dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E>Q162E> Q70E=WT betaB2 using reticulocyte lysate as the source of degradation machinery. Human lens epithelial cell lysates and lens fiber cell lysates also catalyzed ubiquitination but only limited degradation. Supplementation with proteasome failed to enhance degradation. Rates of ubiquitination and degradation of WT and deamidated betaB1 crystallins were rapid and showed little relationship to the site of deamidation using N157D and Q204E mutants. gammaD-Crystallins were not degraded by the UPP. Deamidation altered amine reactivity, circular dichroism spectra, surface hydrophobicity, and thermal stability. CONCLUSIONS These data demonstrate for the first time that, like mild oxidative stress, deamidation of some proteins makes them preferred substrates for ubiquitination and, in some cells, for UPP-dependent degradation. Failure to properly execute ubiquitination and degrade the ubiquitin-conjugates may explain their accumulation on aging and in cataractogenesis.

Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Tissue transglutaminase catalyzes the deamidation of glutamines in lens βB2- and βB3-crystallins

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including alphaB-crystallin and several beta-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked betaB(2)- and betaA(3)-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of betaB(2)- and betaB(3)-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the beta-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.