Modification Of Arginine Residues Research Articles

Virtual Screening and In vitro Evaluation Identify Methotrexate and Testosterone are Inhibitors of Protein Arginine Deiminase 4

Peptidyl arginine deiminases (PAD) are proteins that modify arginine residues to citrulline. In humans there are five isoforms PAD1-4 and PAD6. PAD4 is involved in several human diseases such as Alzheimer, breast cancer, rheumatoid arthritis and therefore has been recognized as a potential pharmacological target. In this work, 3175 molecules retrieved from ZINC12 Food and Drug Administration (FDA) catalog database were assayed through a structure-based virtual screening to identify potential PAD4 binders. Three were confirmed through enzyme inhibition studies. Flow cytometry analysis were used to check for drug cytotoxic effects on blood cells from three healthy donors. Molecular docking in a wild-type PAD4 structure showed the top 20 FDA molecules with the best binding energies. Those molecules were distributed in clusters of mixed binding energies but with similar chemical structures. These patterns along with the FDA information were used to aid in the rational selection of methotrexate, testosterone and leucovorin compounds for in vitro evaluations. Time course fluorescent enzyme kinetics confirmed that methotrexate (0.01–1 mmol) and testosterone enanthate (49.9–12.5 mmol) inhibit PAD4 at similar levels to the known inhibitor BB-Cl-amidine (8.8 mmol), except leucovorin. The chosen concentrations lack cytotoxic effects on blood cells. These findings encourage repositioning campaigns in the quest of PAD4 inhibitors.

Legionella pneumophila temporally regulates the activity of ADP/ATP translocases by reversible ADP‐ribosylation

AbstractThe mitochondrion is an important signaling hub that governs diverse cellular functions, including metabolism, energy production, and immunity. Among the hundreds of effectors translocated into host cells by the Dot/Icm system of Legionella pneumophila, several are targeted to mitochondria but the function of most of them remains elusive. Our recent study found that the effector Ceg3 inhibits the activity of ADP/ATP translocases (ANTs) by ADP‐ribosylation (ADPR). Here, we show that the effect of Ceg3 is antagonized by Larg1, an effector encoded by lpg0081, a gene that is situated next to ceg3. Larg1 functions to reverse Ceg3‐mediated ADPR of ANTs by cleaving the N‐glycosidic bond between the ADPR moiety and the modified arginine residues in ANTs, leading to restoration of their activity in ADP/ATP exchange. Structural analysis of Larg1 and its complex with ADPR reveals that this ADPR glycohydrolase harbors a unique macrodomain that catalyzes the removal of ADPR modification on ANTs. Our results also demonstrate that together with Ceg3, Larg1 imposes temporal regulation of the activity of ANTs by reversible ADPR during L. pneumophila infection.

Antiviral activity of glucose-derived reactive metabolite, methylglyoxal against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Background: The ongoing coronavirus disease 2019 (COVID-19) pandemic has resulted in a global health emergency. With incomplete vaccination and incomplete protection of the vaccinated population, there remains an urgent need to develop drugs to treat COVID-19. Our previou in silico analysis suggested vulnerability of SARS-CoV-2 to inactivation by the endogenous reactive metabolite, methylglyoxal (MG), by modification of arginine residues in the functional domains of viral spike and nucleocapsid proteins1–3. In this study, the antiviral activity of MG against wild-type SARS-CoV-2 using in vitro assays was evaluated. Methods: Wild-type SARS-CoV-2 with titers of multiplicities of infection (MOI) 0.8, 0.2, 0.02, and 0.01 were incubated with 2-fold serial dilutions of MG (7.8 μM to 500 μM) in infection medium for six hour. MG-treated and untreated control SARS-CoV-2 were incubated with confluent cultures of Vero cells in vitro for one hour, cultures were washed and then incubated in a fresh infection medium at 37°C for 4 - 5 days until 70% of virus–infected cells displayed cytopathic effect (CPE). The antiviral activity of MG was judged by assessing virus replication using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and median tissue culture infectious dose (TCID50) assays. Results: MG inhibited virus replication as measured by PCR and CPE of SARS-CoV-2 in vitro, with TCID50 increasing with increasing MOI. MG was most potent at MOI 0.02 and 0.01 where EC50 of MG was 49.6 ± 4.7 μM and 28.5 ± 1.3μM; respectively. Similar findings were also found for a shorter incubation period (3 hours) of MG and virus. Conclusion: MG has inhibitory activity against wild-type SARS-CoV-2 for virus exposure in the cell-free systems at low MOI. However, the antiviral activity of MG against other SARS-CoV-2 variants including alpha- and beta-variants is being investigated. Drugs increasing cellular concentration of MG to viricidal levels may have anti-COVID-19 activity.

Development of a conventional immunochemical detection system for determination of Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine in methylglyoxal-modified proteins.

Methylglyoxal (MGO) produced during glycolysis is known to react with arginine residues on proteins to generate advanced glycation end products, such as Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1). Since the production of MGO is increased during hyperglycemia or metabolic disorders in vivo, it is considered that the measurement of MG-H1 is useful for evaluating abnormalities in carbohydrate metabolism. Thus, we prepared a monoclonal antibody against MG-H1 to develop a conventional measurement system for MG-H1. Reactivity and specificity of the antibody to MGO-modified protein were confirmed by enzyme-linked immunosorbent assay and western blotting, respectively. The measurement of MG-H1 content by the antibody was positively correlated with that by electrospray ionization-liquid chromatography-tandem mass spectrometry and the ratio of modified arginine residues by amino acid analysis. Our results demonstrated that immunochemical methods could be useful for the estimation of MG-H1 content in modified proteins.

Vitamin C is a source of oxoaldehyde and glycative stress in age-related cataract and neurodegenerative diseases.

Oxoaldehyde stress has recently emerged as a major source of tissue damage in aging and age‐related diseases. The prevailing mechanism involves methylglyoxal production during glycolysis and modification of arginine residues through the formation of methylglyoxal hydroimidazolones (MG‐H1). We now tested the hypothesis that oxidation of vitamin C (ascorbic acid or ASA) contributes to this damage when the homeostatic redox balance is disrupted especially in ASA‐rich tissues such as the eye lens and brain. MG‐H1 measured by liquid chromatography mass spectrometry is several fold increased in the lens and brain from transgenic mice expressing human vitamin C transporter 2 (hSVCT2). Similarly, MG‐H1 levels are increased two‐ to fourfold in hippocampus extracts from individuals with Alzheimer's disease (AD), and significantly higher levels are present in sarkosyl‐insoluble tissue fractions from AD brain proteins than in the soluble fractions. Moreover, immunostaining with antibodies against methylglyoxal hydroimidazolones reveals similar increase in substantia nigra neurons from individuals with Parkinson's disease. Results from an in vitro incubation experiment suggest that accumulated catalytic metal ions in the hippocampus during aging could readily accelerate ASA oxidation and such acceleration was significantly enhanced in AD. Modeling studies and intraventricular injection of 13C‐labeled ASA revealed that ASA backbone carbons 4–6 are incorporated into MG‐H1 both in vitro and in vivo, likely via a glyceraldehyde precursor. We propose that drugs that prevent oxoaldehyde stress or excessive ASA oxidation may protect against age‐related cataract and neurodegenerative diseases.

Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways*

Strains of Salmonella utilize two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, whereas ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.

Arginine-Selective Chemical Labeling Approach for Identification and Enrichment of Reactive Arginine Residues in Proteins.

Modification of arginine residues using dicarbonyl compounds is a common method to identify functional or reactive arginine residues in proteins. Arginine undergoes several kinds of posttranslational modifications in these functional residues. Identifying these reactive residues confidently in a protein or large-scale samples is a very challenging task. Several dicarbonyl compounds have been utilized, and the most effective ones are phenylglyoxal and cyclohexanedione. However, tracking these reactive arginine residues in a protein or large-scale protein samples using a chemical labeling approach is very challenging. Thus, the enrichment of modified peptides will provide reduced sample complexity and confident mass-spectrometric data analysis. To pinpoint arginine-labeled peptide efficiently, we developed a novel arginine-selective enrichment reagent. For the first time, we conjugated an azide tag in a widely used dicarbonyl compound cyclohexanedione. This provided us the ability to enrich modified peptides using a bio-orthogonal click chemistry and the biotin–avidin affinity chromatography. We evaluated the reagent in several standard peptides and proteins. Three standard peptides, bradykinin, substance P, and neurotensin, were labeled with this cyclohexanedione-azide reagent. Click labeling of modified peptides was tested by spiking the peptides in a myoglobin protein digest. A protein, RNase A, was also labeled with the reagent, and after click chemistry and biotin–avidin affinity chromatography, we identified two selective arginine residues. We believe this strategy will be an efficient way for identifying functional and reactive arginine residues in a protein or protein mixtures.

Site specific modification of the human plasma proteome by methylglyoxal

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300μM MG from a prodan–HSA complex (75μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients.

ADP‐ribosylargininyl Activity of Cholix Toxin Is Mediated Through Diffusible Intermediates

OBJECTIVEWe discovered and characterized the ADP‐ribosylargininyl activity of the cholix toxin catalytic fragment, which was first identified as a diphthamide‐dependent ADP‐ribosyltransferase.METHODSWe utilized genetic approach to construct direct and reverse mutant enzymes and substrates in conjunction with biochemical assays including fluorescence/non‐fluorescence based enzymatic assays, protein/peptide‐2D‐electrophoresis analysis.RESULTSOur studies show that the transfer of ADP‐ribose to toxin itself takes place by a predominantly intramolecular mechanism. Multiple arginine residues, located in the proximity of the catalytic site but also at distal sites, can be the ADP‐ribose acceptor in the auto‐reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially modifies arginine residues in proximity to the NAD+ binding pocket. ADP‐ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates, such as oligo arginine peptides and eEF2(H715R) mutant. Auto‐ADP‐ribosylation of cholix toxin appears to have negative regulatory effect on its enzymatic activities. However, at the presence of both endogenous and exogenous substrates, the ADP‐ribosylation of exogenous substrate occurred more efficiently than endogenous substrate.CONCLUSIONSThe lifetime of the hypothetical intermediate exceeds recorded and predicted lifetime for the cognate oxocarbenium ion. We therefore hypothesize a strained form of NAD+ intermediate generated through an error‐prone mechanism of the enzyme to react with arginine residues in a proximity dependent manner.

Specific tandem mass spectrometric detection of AGE-modified arginine residues in peptides.

Glycation is a non-enzymatic reaction of protein amino and guanidino groups with reducing sugars or dicarbonyl products of their oxidative degradation. Modification of arginine residues by dicarbonyls such as glyoxal and methylglyoxal results in formation of advanced glycation end-products (AGEs). In mammals, these modifications impact in diabetes mellitus, uremia, atherosclerosis and ageing. However, due to the low abundance of individual AGE-peptides in enzymatic digests, these species cannot be efficiently detected by LC-ESI-MS-based data-dependent acquisition (DDA) experiments. Here we report an analytical workflow that overcomes this limitation. We describe fragmentation patterns of synthetic AGE-peptides and assignment of modification-specific signals required for unambiguous structure retrieval. Most intense signals were those corresponding to unique fragment ions with m/z 152.1 and 166.1, observed in the tandem mass spectra of peptides, containing glyoxal- and methylglyoxal-derived hydroimidazolone AGEs, respectively. To detect such peptides, specific and sensitive precursor ion scanning methods were established for these signals. Further, these precursor ion scans were incorporated in conventional bottom-up proteomic approach based on data-dependent acquisition (DDA) LC-MS/MS experiments. The method was successfully applied for the analysis of human serum albumin (HSA) and human plasma protein tryptic digest with subsequent structure confirmation by targeted LC-MS/MS (DDA). Altogether 44 hydroimidazolone- and dihydroxyimidazolidine-derived peptides representing 42 AGE-modified proteins were identified in plasma digests obtained from type 2 diabetes mellitus (T2DM) patients.

Structures of Neutrophil Serine Protease 4 Reveal an Unusual Mechanism of Substrate Recognition by a Trypsin-Fold Protease

Trypsin-fold proteases, the largest mammalian protease family, are classified by their primary substrate specificity into one of three categories, trypsin-like, chymotrypsin-like, and elastase-like, based on key structural features of their active site. However, the recently discovered neutrophil serine protease 4 (NSP4, also known as PRSS57) presents a paradox: NSP4 exhibits a trypsin-like specificity for cleaving substrates after arginine residues, but it bears elastase-like specificity determining residues in the active site. Here we show that NSP4 has a fully occluded S1 pocket and that the substrate P1-arginine adopts a noncanonical "up" conformation stabilized by a solvent-exposed H-bond network. This uncommon arrangement, conserved in all NSP4 orthologs, enables NSP4 to process substrates after both arginine as well as post-translationally modified arginine residues, such as methylarginine and citrulline. These findings establish a distinct paradigm for substrate recognition by a trypsin-fold protease and provide insights into the function of NSP4.

Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species.

Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increases of molecular weights of 72 and 54 Da, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact that cell culture conditions can have on the product quality of recombinant protein pharmaceuticals.

FRI0003 ACPA response against fibrinogen epitopes citrullinated in vivo in the synovial membrane in RA patients detected with an autoantibody microarray

BackgroundCitrullination is a post-translational modification of proteins catalyzed by specific peptidylarginine deiminases, which modify arginine residues to citrulline. Citrullinated proteins are frequently detected in various inflammatory states including rheumatoid arthritis,...

ACPA response against fibrinogen epitopes citrullinated in vivo in the synovial membrane in RA patients detected with an autoantibody microarray

Backgroundand objectivesCitrullination is a post-translational modification of proteins catalysed by specific peptidylarginine deiminases, which modify arginine residues to citrulline. Citrullinated proteins are frequently detected in various inflammatory states including rheumatoid...

Importance of Two Arginine Residues in Lapemis Postsynaptic Neurotoxins: Re-examination using Acetylcholine Receptor-neurotoxin Complex Instead of Free Toxin

The role of the three arginine residues in Lapemis neurotoxin has been re-examined using an acetylcholine receptor-toxin complex. The receptor-bound neurotoxin was treated with phenylglyoxal to modify available arginine residues. The positions of the modified arginine residues were then identified from the amino acid sequences of proteolytically digested fragments of the detached neurotoxin. The result was compared with the result from modification of the free Lapemis toxin (unbound). Arg-31 and Arg-34 were not modified when Lapemis toxin was bound to receptor although they were modified when the toxin was not bound to receptor. It was concluded that Arg-31 and Arg-34 residues are involved in the toxin-receptor interaction.

Reaction of the Butter Flavorant Diacetyl (2,3-Butanedione) with N-α-Acetylarginine: A Model for Epitope Formation with Pulmonary Proteins in the Etiology of Obliterative Bronchiolitis

The butter flavorant diacetyl (2,3-butanedione) is implicated in causing obliterative bronchiolitis in microwave popcorn plant workers. Because diacetyl modifies arginine residues, an immunological basis for its toxicity is under investigation. Reaction products of diacetyl with N-α-acetylarginine (AcArg) were determined as a model for hapten formation, with characterization by mass spectrometry, NMR, and HPLC with UV detection and radiodetection. Four products were identified by LC-MS, each with a positive ion of m/z 303 (diacetyl + AcArg); one pair displayed an additional ion at m/z 217 (AcArg), the other pair at m/z 285 (- H(2)O). Their (1)H-(13)C NMR correlation spectra were consistent with the addition of one or two of the guanidine nitrogens to form aminols. Open-chain pairs interconverted at pH 2, as did the cyclized, but all four interconverted at neutral pH. This is the first structural characterization of the covalent adducts between diacetyl and an arginine moiety.

Modification of Collagen IV by Glucose or Methylglyoxal Alters Distinct Mesangial Cell Functions

Diabetic nephropathy (DN) affects both glomerular cells and the extracellular matrix (ECM), yet the pathogenic mechanisms involving cell-matrix interactions are poorly understood. Glycation alters integrin-dependent cell-ECM interactions, and perturbation of these interactions results in severe renal pathology in diabetic animals. Here, we investigated how chemical modifications of the ECM by hyperglycemia and carbonyl stress, two major features of the diabetic milieu, affect mesangial cell functions. Incubation of collagen IV with pathophysiological levels of either the carbonyl compound methylglyoxal (MGO) or glucose resulted in modification of arginine or lysine residues, respectively. Mouse mesangial cells plated on MGO-modified collagen IV showed decreased adhesion and migration. Cells plated on glucose-modified collagen IV showed reduced proliferation and migration and increased collagen IV production. Inhibiting glucose-mediated oxidative modification of collagen IV lysine residues rescued the alterations in cell growth, migration, and collagen synthesis. We propose that diabetic ECM affects mesangial cell functions via two distinct mechanisms: modification of arginine residues by MGO inhibits cell adhesion, whereas oxidative modification of lysine residues by glucose inhibits cell proliferation and increases collagen IV production. These mechanisms may contribute to mesangial cell hypertrophy and matrix expansion in DN.

Substrate Profiling of PRMT1 Reveals Amino Acid Sequences That Extend Beyond the “RGG” Paradigm

Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm.

Synthesis of Proteins Containing Modified Arginine Residues

Unnatural amino acid mutagenesis provides the wherewithal to study protein function in great detail. To extend the repertoire of functionalized amino acids available for study by this technique, seven structural analogues of arginine were prepared and used to activate a suppressor tRNACUA. These included Ngamma-methylarginine, Ngamma-nitroarginine, citrulline, homoarginine, and three conformationally constrained analogues based on proline. These misacylated tRNAs were shown to be capable of introducing the arginine analogues into dihydrofolate reductase (position 22) and Photinus pyralis luciferase (positions 218 and 437). Most of the modified luciferases containing arginine analogues at position 218 emitted light with less efficiency and at longer wavelength than the wild type. This is consistent with the postulated role of this residue as essential for maintaining the polarity and rigidity of the luciferin-binding site. Interestingly, the luciferase containing Ngamma-methylarginine at position 218 emitted light at the same wavelength as the wild type and was at least as efficient. Alteration of the arginine residue at position 437 had no effect on the wavelength of emitted light but afforded analogues, all of which emitted light less efficiently than the wild type. This is altogether consistent with the putative role of Arg437, which is an invariant residue within the superfamily of enzymes that includes P. pyralis luciferase. This amino acid is part of the linker between the two structural domains of luciferase that is believed to be essential for efficient enzyme function but not part of the substrate-binding site.

Methylglyoxal inhibits glycation-mediated loss in chaperone function and synthesis of pentosidine in α-crystallin

α-Crystallin is a major protein in the eye lens and it functions as a molecular chaperone by preventing aggregation of mildly denatured proteins. Glycation, the reaction of sugars and ascorbate with proteins, causes covalent cross-linking and reduces the chaperone function of α-crystallin. We demonstrated that methylglyoxal (MGO), a metabolic α-dicarbonyl compound, modifies arginine residues in α-crystallin and enhances its chaperone function. We wanted to determine whether modification by MGO could protect α-crystallin from glycation-mediated cross-linking and loss of chaperone function. Our results show that MGO-modification of isolated bovine lens α-crystallin inhibits formation of pentosidine, a glycation-derived protein crosslink. Proteins in organ cultured rat lenses were similarly protected from pentosidine formation. Glycation by sugars and ascorbate resulted in almost complete loss of chaperone function of α-crystallin. Surprisingly, addition of MGO during or before glycation not only inhibited the loss of chaperone function, but it actually enhanced the chaperone function of α-crystallin. Together, these data suggest that in the aging lens, MGO inhibits glycation-mediated pentosidine synthesis and the loss of chaperone function of α-crystallin.