αA-Crystallin Peptide 66 SDRDKFVIFLDVKHF 80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

Background

The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood.

Methodology/Principal Findings

We found that αA-crystallin-derived peptide, ⁶⁶ SDRDKFVIFLDVKHF ⁸⁰, which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation.

Conclusions/Significance

These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

The site at which 4-iodoacetamidosalicylate reacts with glutamate dehydrogenases

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded N^ε-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119–130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of β-casein (β-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of β-CN f7-14, f47-52, and f169-175 and κ-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the β-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC₅₀s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC₅₀s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.     

Studies on the biosynthesis of protein by lactating guinea-pig mammary gland: Characteristics of the synthesis of α-lactalbumin and total protein by slices and cell-free systems

1. Slices of lactating guinea-pig mammary gland were incubated with radioactive amino acids and the various subcellular fractions separated by centrifugation after disruption of the cells by mincing and homogenization. The most active fraction for protein synthesis appeared to be the `mitochondrial'. 2. When the subcellular fractions were prepared without previous incubation of the cells and were then incubated with radioactive amino acid and an energy-generating system, the `mitochondrial fraction' was at least as active for protein synthesis as the `microsomal fraction'. 3. The ribosomes in the microsomal fraction are mainly unattached to membrane whereas those in the mitochondrial fraction are probably attached to fragments of the rough-surfaced endoplasmic reticulum. This latter fraction contains few mitochondria. 4. The combined mitochondrial and microsomal fractions incorporated radioactive amino acids into α-lactalbumin. 5. The radioactive leucine isolated from tryptic and chymotryptic peptides of α-lactalbumin synthesized in the cell-free system was not of uniform specific radioactivity. This was consistent with the polypeptide being assembled by the sequential addition of amino acids. 6. Evidence is presented for the polypeptide chain of α-lactalbumin being assembled from the N-terminus and for chain initiation in the cell-free system. 7. It is concluded that cell-free extracts of lactating mammary gland synthesize α-lactalbumin.     

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC₅₀) of 0.8 mg protein/ml compared to captopril, which had an IC₅₀ value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to α_s1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC₅₀ values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.     

Cross-Reactivity of Antibodies against Leptospiral Recurrent Uveitis-Associated Proteins A and B (LruA and LruB) with Eye Proteins

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be α-crystallin B and vimentin. Similarly, mass spectrometric analyses identified β-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human α-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant β-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized α-crystallin B, β-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.     

Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d₀ and d₄) cross-linker–BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.     

Hydrolysis of Casein-Derived Peptides αS1-Casein(f1-9) and β-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase

Peptides derived from hydrolysis of α_S1-casein(f1-9) [α_S1-CN(f1-9)] and β-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (ΔpepC, ΔpepE, ΔpepN, ΔpepO, and ΔpepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of β-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.     

beta-1, 3-Glucan Synthase from Lilium longiflorum Pollen

A particulate fraction from pollen tubes and ungerminated pollen of Lilium longiflorum incorporated ¹⁴C-glucose from UDP-glucose-¹⁴C into a lipid fraction and into β-1, 3-glucan. Partial hydrolysis of the glucan yielded laminaribiose as the only radioactive disaccharide. The preferred substrate was UDP-glucose, and enzyme activity was stimulated by glucose and by β-linked di- and trisaccharides. Enzyme from growing pollen tubes synthesized β-1, 3-glucan more rapidly and produced a higher proportion of alkali-insoluble glucan than did enzyme from ungerminated pollen. The onset of pollen tube growth may be dependent on altered activity of β-1, 3-glucan synthase.     

Association Properties and Unfolding of a βγ-Crystallin Domain of a Vibrio-Specific Protein

The βγ-crystallin superfamily possesses a large number of versatile members, of which only a few members other than lens βγ-crystallins have been studied. Understanding the non-crystallin functions as well as origin of crystallin-like properties of such proteins is possible by exploring novel members from diverse sources. We describe a novel βγ-crystallin domain with S-type (Spherulin 3a type) Greek key motifs in protein vibrillin from a pathogenic bacterium Vibrio cholerae. This domain is a part of a large Vibrio-specific protein prevalent in Vibrio species (found in at least fourteen different strains sequenced so far). The domain contains two canonical N/D-N/D-X-X-S/T-S Ca²⁺-binding motifs, and bind Ca²⁺. Unlike spherulin 3a and other microbial homologues studied so far, βγ-crystallin domain of vibrillin self-associates forming oligomers of various sizes including dimers. The fractionated dimers readily form octamers in concentration-dependent manner, suggesting an association between these two major forms. The domain associates/dissociates forming dimers at the cost of monomeric populations in the presence of Ca²⁺. No such effect of Ca²⁺ has been observed in oligomeric species. The equilibrium unfolding of both forms follows a similar pattern, with the formation of an unfolding intermediate at sub-molar concentrations of denaturant. These properties exhibited by this βγ-crystallin domain are not shown by any other domain studied so far, demonstrating the diversity in domain properties.     

The Congenital Cataract-Linked A2V Mutation Impairs Tetramer Formation and Promotes Aggregation of βB2-Crystallin

β/γ-Crystallins, the major structural proteins in human lens, are highly conserved in their tertiary structures but distinct in the quaternary structures. The N- and C-terminal extensions have been proposed to play a crucial role in mediating the size of β-crystallin assembly. In this research, we investigated the molecular mechanism underlying the congenital hereditary cataract caused by the recently characterized A2V mutation in βB2-crystallin. Spectroscopic experiments indicated that the mutation did not affect the secondary and tertiary structures of βB2-crystallin. The mutation did not affect the formation of βB2/βA3-crystallin heteromer as well as the stability and folding of the heteromer, suggesting that the mutation might not interfere with the protein interacting network in the lens. However, the tetramerization of βB2-crystallin at high protein concentrations was retarded by the A2V mutation. The mutation slightly decreased the thermal stability and promoted the thermal aggregation of βB2-crystallin. Although it did not influence the stability of βB2-crystallin against denaturation induced by chemical denaturants and UV irradiation, the A2V mutant was more prone to be trapped in the off-pathway aggregation process during kinetic refolding. Our results suggested that the A2V mutation might lead to injury of lens optical properties by decreasing βB2-crystallin stability against heat treatment and by impairing βB2-crystallin assembly into high-order homo-oligomers.     

UPR Activation and the Down–Regulation of α-Crystallin in Human High Myopia-Related Cataract Lens Epithelium

Purpose

To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts.

Methods and Materials

The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6.

Results

In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control.

Conclusion

These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract.     

Antioxidant activity of the simulated gastro-intestinal digestion hydrolysate of two edible Nigerian marine molluscs: Tympanatonus fuscatus var radula (L.) and Pachymelania aurita (M.)

The multifunctional nature of antioxidant peptides makes them more attractive candidates as dietary ingredients in health maintenance. Therefore, food protein-derived antioxidant peptides are continuously investigated. This study investigated the in vitro antioxidant properties of hydrolysate and ultrafiltered peptide fractions of Pachymelania aurita and Tympanatonus fuscatus var radula-two commonly consumed marine molluscs known as periwinkles in southern Nigeria. Simulated gastrointestinal digestion (SGID) of soluble proteins of T. fuscatus and P. aurita was carried out using pepsin, trypsin and chymotrypsin, and the SGID hydrolysates were fractionated using a 3 kDa membrane filter. The hydrolysates and their fractions were investigated for anti-lipid peroxidation, hydroxyl radical scavenging activity (HRSA), ferric reducing antioxidant property (FRAP) and metal chelation activity, and they demonstrated clear antioxidant properties in all the assay models used. Low molecular weight fractions of the hydrolysates demonstrated more potent antioxidant activity than higher molecular weight fractions. This is profound in the metal chelation assay, where low molecular weight peptide fractions, T ≤ 3 kDa and P ≤ 3 kDa (IC₅₀ values of 8.10 ± 0.011 and 5.56 ± 0.50 µg/ml respectively) had activity that is similar to that of EDTA (11.84 ± 0.89 µg/ml). Similar activity effects were observed in other assays where there was about 3-fold higher activity in low molecular weight fractions. These results demonstrate the presence of antioxidant peptide(s) in the protein hydrolysates of the periwinkles.     

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K_m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

CRYβA3/A1-Crystallin Knockout Develops Nuclear Cataract and Causes Impaired Lysosomal Cargo Clearance and Calpain Activation

βA3/A1-crystallin is an abundant structural protein of the lens that is very critical for lens function. Many different genetic mutations have been shown to associate with different types of cataracts in humans and in animal models. βA3/A1-crystallin has four Greek key-motifs that organize into two crystallin domains. It shown to bind calcium with moderate affinity and has putative calcium-binding site. Other than in the lens, βA3/A1 is also expressed in retinal astrocytes, retinal pigment epithelial (RPE) cells, and retinal ganglion cells. The function of βA3/A1-crystallin in the retinal cell types is well studied; however, a clear understanding of the function of this protein in the lens has not yet been established. In the current study, we generated the βA3/A1-crystallin knockout (KO) mouse and explored the function of βA3/A1-crystallin in lens development. Our results showed that βA3-KO mice develop congenital nuclear cataract and exhibit persistent fetal vasculature condition. At the cellular level KO lenses show defective lysosomal clearance and accumulation of nuclei, mitochondria, and autophagic cargo in the outer cortical region of the lens. In addition, the calcium level and the expression and activity of calpain-3 were increased in KO lenses. Taken together, these results suggest the lack of βA3-crystallin function in lenses, alters calcium homeostasis which in turn causes lysosomal defects and calpain activation. These defects are responsible for the development of nuclear cataract in KO lenses.     

Thermal Stress Induced Aggregation of Aquaporin 0 (AQP0) and Protection by α-Crystallin via Its Chaperone Function

Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.     

Effects of temperature and concentration on bovine lens α-crystallin secondary structure: a circular dichroism spectroscopic study

Elucidation of the structure of α-crystallin, the major protein in all vertebrate lenses, is important for understanding its role in maintaining transparency and its function in other tissues under both normal and pathological conditions. Progress toward a unified consensus concerning the tertiary and quaternary structures of α-crystallin depends, in part, on an accurate estimation of its secondary structure. For the first time, three algorithms, SELCON, K2D and CONTIN were used to analyze far ultra-violet circular dichroism (UV–CD) spectra of bovine lens α-crystallin to estimate the secondary structure and to determine the effects of temperature and concentration. Under all experimental conditions tested, the analyses show that α-crystallin contains 14% α-helix, 35% β-sheet and the remainder, random coil and turns. The results suggest that α-crystallin is best classified as a mixed protein. In addition, increased temperature and concentration of α-crystallin result in increased α-helices with a compensatory decrease in β-sheets. Such structural alterations in α-crystallin may be functionally important during terminal differentiation of the lens fiber cells that is accompanied by increased protein concentrations and its role as a chaperone-like protein.     

The covalent structure of cartilage collagen. Amino acid sequence of residues 552–661 of bovine α1(II) chains

The covalent structure of the first 111 residues from the N-terminus of peptide α1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552–661 of the α1(II) chain. The sequence was determined by automated Edman degradation of peptide α1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the α1(II) chain with the homologous segment of the α1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the α1(II) chain (Butler, Miller & Finch (1976) Biochemistry 15, 3000–3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the α1(II) chain were compared with the homologous regions of the α1(I) and α2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the α1(I) chain, it appears that the α1(II)-chain sequence presented here contains three more amino acids than that reported for the α1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide α1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.