Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.     

Transglutaminase-mediated cross-linking of alpha-crystallin: structural and functional consequences.

Aggregation and covalent cross-linking of the crystallins, the major structural proteins of the eye lens, increase light scattering by the lens leading to opacification and cataract. Disturbance of calcium homeostasis in the tissue is one of the factors implicated in cataractogenesis. Calcium-activated transglutaminase (TG)-catalyzed cross-linking of some lens proteins has been reported earlier. We show here that alpha-crystallin, a major structural protein in the lens and a member of the small heat shock protein family, is also a substrate for TG-mediated cross-linking, indicating the presence of donor Lys and acceptor Gln residues in the protein. Upon TG-catalyzed dimerization, the secondary and tertiary structures of the protein are altered, and its surface hydrophobicity reduced. The chaperone-like property of the protein, suspected to be one of its functions in situ, is substantially reduced upon such cross-linking. These results, taken together with earlier ones on lens beta-crystallins and vimentin, suggest that TG-mediated events might compromise lens function. Also, since alpha-crystallin occurs not only in the lens but in other tissues as well, such TG-catalyzed cross-linking and the associated alterations in its structure and activity would be of general pathological interest.     

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.     

Changes in Casein and Egg Albumin due to Reactions with Oxidizing Methyl Linoleate in Dehydrated Systems

Casein and egg albumin were allowed to react with methyl linoleate (ML) at a relative humidity (RH) of 0% or 80% at 50°C for 10 days (protein: ML= 1:0.2 or 1:1, w/w). Changes in the molecular sizes of the reacting proteins were examined by gel filtration and gel electrophoresis. Both proteins showed similar changes, whereas the reaction at RH 80% (protein: ML= 1:1) resulted in insolubilization because of polymerization. Changes in the amino acid residues of the reacting proteins were investigated after acid (6 n HC1) and enzymatic (pepsin-pancreatin, followed by aminopeptidase-prolidase) hydrolyses. Insignificant changes were observed in the amino acid composition of proteins reacted at RH 0%. After reaction at RH 80% (protein: ML =1:1), Lys, His and Met were the only amino acids affected. The percentage loss of these amino acids after acid hydrolysis was Lys (22%), His (41%), Met (9%) for casein and Lys (22%), His (31%), Met (1%) for egg albumin. This percentage loss after enzymatic hydrolysis was Lys (41%), His (49%), Met (94%) for casein and Lys (37%), His (42%), Met (88%) for egg albumin. Some differences between our results and other researchers were also discussed.     

Mass spectrometric evidence for long-lived protein adducts of 4-oxo-2-nonenal

Substantial work has been carried out to elucidate the nature of protein modification by 4-hydroxy-2-nonenal (HNE) and its relatives. Its keto cousin, 4-oxo-2-nonenal (ONE), which arises from linoleic acid oxidation independently of HNE, was previously reported to form Michael adducts with His and Cys that can subsequently, in part, condense with Lys residues to give imidazolylpyrrole cross-links. Despite mass spectrometric evidence also for ONE-Lys Michael adducts, the latter do not accumulate in solution. A long-lived adduct that has the same mass as the ONE Lys Michael adduct is suggested instead to be the isomeric 4-ketoamide that arises, along with other adducts, from the reversibly-formed ONE Lys Schiff base. The Lys-ketoamide and His-Lys imidazolylpyrrole cross-links appear to be unusually prominent markers of stable protein modification by ONE.     

Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens

Over 95% of the dry mass of the eye lens consists of specialized proteins called crystallins. Aged lenses are subject to cataract formation, in which damage, cross-linking, and precipitation of crystallins contribute to a loss of lens clarity. Cataract is one of the major causes of blindness, and it is estimated that over 50,000,000 people suffer from this disability. Damage to lens crystallins appears to be largely attributable to the effects of UV radiation and/or various active oxygen species (oxygen radicals, ¹O₂, H₂O₂, etc.). Photooxidative damage to lens crystallins is normally retarded by a series of antioxidant enzymes and compounds. Crystallins which experience mild oxidative damage are rapidly degraded by a system of lenticular proteases. However, extensive oxidation and cross-linking severely decrease proteolytic susceptibility of lens crystallins. Thus, in the young lens the combination of antioxidants and proteases serves to prevent crystallin damage and precipitation in cataract formation. The aged lens, however, exhibits diminished antioxidant capacity and decreased proteolytic capabilities. The loss of proteolytic activity may actually be partially attributable to oxidative damage which proteases (like any other protein)_can sustain. We propose that the rate of crystallin damage increases as antioxidant capacity declines with age. The lower protease activity of aged lens cells may be insufficient to cope with such rates of crystallin damage, and denatured crystallins may begin to accumulate. As the concentration of oxidatively denatured crystallins rises, cross-linking reactions may produce insoluble aggregates which are refractive to protease digestion. Such a scheme could explain many events which are known to contribute to cataract formation, as well as several which have appeared to be unrelated. This hypothesis is also open to experimental verification and intervention.     

In vivo carbamylation and acetylation of water-soluble human lens alphaB-crystallin lysine 92

Several post-translational modifications of lysine residues of lens proteins have been implicated in cataractogenesis. In the present study, the molecular weight of an alpha-crystallin isolated from the water-soluble portion of a cataractous human eye lens indicated that it was a modified alphaB-crystallin. Further analysis by mass spectrometry of tryptic digests of this modified protein showed that Lys 92 was modified and that the sample was structurally heterogeneous. Lys 92 was acetylated in one population and carbamylated in another. Although carbamylation of lens crystallins has been predicted, this is the first documentation of in vivo carbamylation of a specific site. These results are also the first documentation of in vivo lysine acetylation of alphaB-crystallin. Both modifications alter the net charge on alphaB-crystallin, a feature that may have significance to cataractogenesis.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Inhibitors of advanced glycation end product-associated protein cross-linking

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.     

Acceptor-donor relationships in the transglutaminase-mediated cross-linking of lens beta-crystallin subunits

Following the isolation of the N epsilon-(gamma-glutamyl)lysine-containing polymers from human cataracts, our efforts were directed to induce such cross-links experimentally in rabbit lens, and evidence was obtained for the selective reactivities of certain beta-crystallin subunits in this transglutaminase-catalyzed event. In the present work, we examined the enzymatic cross-linking of purified crystallins individually (alpha, beta H, beta L, and gamma) and in combinations, with particular emphasis on forming the approximately 55K dimer. This species was the primary product in the cross-linking of beta H-crystallins; beta L also reacted with transglutaminase. Neither alpha- nor gamma-crystallins formed appreciable amounts of cross-linked structures with transglutaminase. Dansylcadaverine, known to compete against the reactive lysines of proteins in forming N epsilon-(gamma-glutamyl)lysine cross-bridges, was shown to inhibit the generation of dimeric and higher ordered oligomers from beta H and beta L. The fluorescent amine specifically labeled only two subunits in beta H (approximately 29-30K and approximately 26K) and one in beta L (approximately 26K), identifying these substrates as possessing transglutaminase-reactive endo-gamma-glutaminyl residues. An antiserum to bovine beta Bp recognized the approximately 23K subunit of rabbit beta-crystallins and also the approximately 55K dimer, suggesting that the approximately 23K protein participates as a lysine donor in generating the cross-linked dimer with transglutaminase. Inasmuch as the same antiserum reacts with an approximately 50K material reported to appear in increasing amounts with age in human lens, the results lend added support to the physiological significance of transglutaminase in the aging of lens.     

Helices VII and X in the lactose permease of Escherichia coli: proximity and ligand-induced distance changes.

By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N(7)/C(5) split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-specific cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A), N,N'-o- phenylenedimaleimide (6 A) and N,N'-p-phenylenedimaleimide (10 A). Mutant Asp240-->Cys (helix VII)/Lys319-->Cys (helix X) cross-links most efficiently with the 3 A reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H(+) symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H(+) from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).     

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Solution properties of γ‐crystallins: Compact structure and low frictional ratio are conserved properties of diverse γ‐crystallins

γ‐crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ‐crystallins have high contents of sulfur‐containing methionine and cysteine, but this reaches extremes in fish γM‐crystallins with up to 15% Met. In addition, fish γM‐crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ‐crystallins and in the related β‐crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ‐crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β‐sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full‐length and truncated versions of human αB‐crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ‐crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ‐crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.     

Conjugation of glutathione to oxidized tyrosine residues in peptides and proteins

Tyrosine residues are sensitive to oxidation and can be converted to hydroperoxides either by superoxide reacting with the Tyr radical or by singlet oxygen. These hydroperoxides rearrange to bicyclic derivatives that are readily reduced to more stable hydroxides. The aromatic character of tyrosine is lost, but the product contains an α-β unsaturated carbonyl group and is, therefore, an electrophile. We have generated hydroxide derivatives of several Tyr-containing peptides and shown using liquid chromatography/mass spectrometry that they undergo Michael addition with GSH. For Tyr-Gly, rate constants of 9.2 and 11.8 m(-1)min(-1) were measured for the two chromatographically distinct isomers. Unusual for GSH addition to an electrophile, the reaction is reversible, with a half-life of many hours for the reverse reaction. These kinetics indicate that with a typical cellular concentration of 5 mm GSH, >95% Tyr-Gly hydroxide would become conjugated with a half-life of ～15 min. Sperm whale myoglobin forms a hydroperoxide on Tyr-151 in a hydrogen peroxide/superoxide-dependent reaction. We show that its hydroxide derivative reacts with GSH to form a conjugate. Detection of the conjugate required stabilization by reduction; otherwise, the reverse reaction occurred during tryptic digestion and analysis. Our findings represent a novel mechanism for peptide or protein glutathionylation involving a carbon-sulfur cross-link between oxidized Tyr and Cys. As with other electrophiles, the oxidized Tyr should undergo a similar reaction with Cys residues in proteins to give intramolecular or intermolecular protein cross-links. This mechanism could give rise to protein cross-linking in conditions of oxidative stress.     

Inhibition of alpha-crystallin aggregation by gamma-crystallin

The transparency of the mammalian lens is primarily maintained by short range order among the major proteins of the lens fiber cells, the crystallins. Although these proteins are highly conserved at the amino acid sequence level, it has proven difficult to establish that they possess other than structural functions. We find that when non-lens proteins are added to concentrated solutions of alpha-crystallin, aggregation is induced, presumably through excluded volume effects. In contrast, the monomeric gamma-crystallins and the low molecular weight form of beta-crystallin (beta L) cause a decrease in the size of alpha-crystallin. When the naturally aggregated form of alpha-crystallin is examined, gamma- and beta L-crystallin, as well as a reducing agent, also cause partial dissociation as detected by dynamic light scattering and size exclusion chromatography, while no effect is seen with non-crystallin proteins. Furthermore, the chemical cross-linking of alpha-crystallin is inhibited by gamma- and beta L-crystallin but not by other proteins. The ability of gamma-crystallin to inhibit the association of alpha-crystallin is primarily localized to the gamma-II form which contains a high degree of exposed thiols. Only small amounts of gamma- and beta L-crystallin, however, can be cross-linked to alpha-crystallin in mixtures of the three proteins even at very high protein concentrations. These results suggest that one possible role for the lower molecular weight crystallins may be to minimize through a reductive effect the intrinsic tendency of alpha-crystallin to aggregate, an association reaction implicated in the loss of lens transparency.     

A systematic approach to evaluate the modification of lens proteins by glycation-induced crosslinking

To systematically evaluate the modification of lens proteins by aldose and dicarbonyl sugars during the glycation process, the sugar-dependent incorporation of Lys and Arg, SDS–PAGE profile, amino acid analysis, and fluorophore formation (excitation 370 nm/emission 440 nm) were determined. Reaction mixtures with glycolaldehyde, glyceraldehyde, threose and 3-deoxythreosone showed the greatest extent of Lys crosslinking and fluorescence formation. An increase in fluorescence intensity, but a decrease in Lys and Arg crosslinking, was found with glyoxal, methylglyoxal, hydroxypyruvaldehyde and threosone. In addition glyoxal, methylglyoxal and hydroxypyruvaldehyde caused the specific loss of Arg residues in lens proteins. Reaction mixtures with xylose, xylosone, glucose, glucosone and 3-deoxyglucosone exhibited the least protein modifications; however, incubation with 3-deoxyxylosone resulted in extensive loss of Lys and Arg residues, a higher extent of Lys or Arg crosslinking and significant fluorophore formation. Each sugar exhibited unique characteristics in the modification of lens proteins by glycation. To validly compare the protein modifications occurring during glycation reactions, a systematic approach was employed to evaluate the potential role of aldose and dicarbonyl sugars in protein modification.     

Novel intra- and inter-molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation.

Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, approximately 85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 +/- 1, and 20707 +/- 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met(36/73) were converted to Met(36/73) sulfoxides. An additional product with an unusual mass addition of m/z 14 (+/-0.2) was identified and corresponded to the addition of oxygen to Cys(41), conjugation to various epsilon-amines of Lys(6), Lys(34/35), or Lys(87) with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys(34/35) to Cys(41) sulfinamide bonds. HOCl oxidation of mutants lacking Cys(41) (Ala(41)S100A8) or specific Lys residues (e.g. Lys(34/35), Ala(34/35)S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H(2)O(2) and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products(.) In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.     

Age‐related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old‐age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C‐terminus with significantly higher frequency compared to other residues, suggesting that trypsin‐like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA‐crystallin peptide (αA_57‐65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA_57‐65 formation escalated significantly. Using 2D gel electrophoresis/nanoLC‐ESI‐MS/MS, 12 protein complexes of 40–150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross‐linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes. Proteins 2015; 83:1878–1886. © 2015 Wiley Periodicals, Inc.     

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

Sauvagine cross-links to the second extracellular loop of the corticotropin-releasing factor type 1 receptor

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr(0),Gln(1)]SVG ((125)I-YQS) and [Tyr(0),Gln(1), Leu(17)]SVG ((125)I-YQLS) were examined. (125)I-YQLS or (125)I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the epsilon amino groups of lysine residues that have a molecular distance of 11.4 A. DSS specifically and efficiently cross-linked (125)I-YQLS and (125)I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys(110), Lys(111), Lys(113), Lys(257), and Lys(262)) that can cross-link to the 4 lysine residues (Lys(16), Lys(22), Lys(25), and Lys(27)) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to (125)I-YQLS or (125)I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys(16) of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys(257) in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys(257) of CRFR1 lies in close proximity (11.4 A) to Lys(16) of SVG.