Distinct energy requirement for nuclear import and export of importin beta in living cells

Carbohydrates with reactive aldehyde and ketone groups can undergo Maillard reactions with proteins to form advanced glycation end products. Oxalate monoalkylamide was identified as one of the advanced glycation end products formed from the Maillard reaction of ascorbate with proteins. In these experiments, we have analyzed human lens proteins immunochemically for the presence of oxalate monoalkylamide. Oxalate monoalkylamide was absent in most of the very young lenses but was present in old and cataractous lenses. The highest levels were found in senile brunescent lenses. Incubation experiments using bovine lens proteins revealed that oxalate monoalkylamide could form from the ascorbate degradation products, 2,3-diketogulonate and L-threose. These data provide the first evidence for oxalate monoalkylamide in vivo and suggest that ascorbate degradation and its binding to proteins are enhanced during lens aging and cataract formation.     

Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo

The advanced stage of the glycation process (also called the "Maillard reaction") that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. AGEs elicit a wide range of cell-mediated responses that might contribute to diabetic complications, vascular disease, renal disease, and Alzheimer's disease. Recently, it has been proposed that AGE are not only created from glucose per se, but also from dicarbonyl compounds derived from glycation, sugar autoxidation, and sugar metabolism. However, this advanced stage of glycation is still only partially characterized and the structures of the different AGEs that are generated in vivo have not been completely determined. Because of their heterogeneity and the complexity of the chemical reactions involved, only some AGEs have been characterized in vivo, including N-carboxymethyllysine (CML), pentosidine, pyrraline, and crosslines. In this article, we provide a brief overview of the pathways of AGE formation and of the immunochemical methods for detection of AGEs, and we also provide direct immunological evidence for the existence of five distinct AGE classes (designated as AGE-1 to -5) within the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis. We also propose pathways for the in vivo formation of various AGEs by glycation, sugar autoxidation, and sugar metabolism.     

Nonenzymatic glycation of collagen in aging and diabetes

Considerable progress has been made in our understanding of nonenzymatic glycation of collagen, and the relationship between glycation of collagen and changes in connective tissue associated with aging and diabetes. Recent studies surveyed in this review suggest the following conclusions: 1. Collagen content of early glycation products does not appear to increase throughout the life span in normal human subjects, although small increases may occur that are linked to glycemic changes. These products are increased, relative to age-matched controls, in experimental diabetes and in diabetes mellitus in collagen from virtually all tissues analyzed. 2. Collagen content of browning products increases with aging and appears to be higher in diabetic subjects than in age-matched controls. Rates of accumulation may be accelerated in subpopulations of diabetic subjects at high risk for developing complications. 3. Increases in early glycation products do not appear to be associated with alterations in collagen solubility, thermal rupture time, or mechanical strength, nor is there an association with most diabetic complications. Alterations in these products may, however, affect conformation, ligand binding, lysyl oxidase-mediated cross-linking, and interactions between collagen and other macromolecules in the extracellular matrix. 4. Increased content of browning products is associated with many physicochemical changes in collagen as well as with long-term complications in diabetes mellitus. 5. Regulatory mechanisms have been identified in vivo that may serve to control or limit the formation of glycation products. 7. Pharmacologic agents have been identified that may be able to reduce collagen content of late glycation products. Despite the progress that has been made in this field, many areas of uncertainty and controversy exist. For example, there is not yet a consensus that the browning products associated with collagen exclusively comprise advanced Maillard products derived from nonenzymatically glycated residues. There is evidence that oxidative reactions involving lipids also play a role in generating fluorophores and chromophores that may alter properties of collagen. Thus, in the extracellular matrix collagen may be continuously modified by at least three very different processes: Maillard reactions, interactions with oxidizing lipids, and enzymatically mediated cross-linking. The interrelationships between these and possibly other posttranslational modifications remain a poorly understood area of great complexity.     

Immunochemical evidence for the presence of advanced glycation end products in human lens proteins and its positive correlation with aging.

Prolonged incubation of protein with reducing sugar proceeds through a series of reactions involving early stage products to the advanced glycation end products with fluorescence, brown color, and cross-linking. Known collectively as the Maillard reaction, these changes have been suggested as factors in diabetic complications and the aging process. The early stage products have been demonstrated in vivo, but evidence for the presence in vivo of the advanced glycation end products has been limited. We sought to provide immunochemical evidence by the preparation and use of polyclonal and monoclonal antibodies to these end products (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332) as probes to identify and quantitate such compounds in human lens crystallins. Neither of the antibodies reacted with extracts from infant lenses, but fractions from adult lenses showed a significant reactivity, correlating with lens age. Our findings provide the first immunochemical evidence that human lens crystallins contain advanced glycation end products and that these products increase with tissue age.     

Molecular basis of maillard amide-advanced glycation end product (AGE) formation in vivo

The Maillard reaction in vivo entails alteration of proteins or free amino acids by non-enzymatic glycation or glycoxidation. The resulting modifications are called advanced glycation end products (AGEs) and play a prominent role in various pathologies, including normoglycemic uremia. Recently, we established a new class of lysine amide modifications in vitro. Now, human plasma levels of the novel amide-AGEs N(6)-acetyl lysine, N(6)-formyl lysine, N(6)-lactoyl lysine, and N(6)-glycerinyl lysine were determined by means of LC-MS/MS. They were significantly higher in uremic patients undergoing hemodialysis than in healthy subjects. Model reactions with N(1)-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N(6)-formyl lysine from glucose responded considerably to the presence of oxygen, glucosone was identified as another precursor. Comparison of the in vivo results with the model experiments enabled us to elucidate possible formation pathways linked to Maillard chemistry. The results strongly suggest a major participation of non-enzymatic Maillard mechanisms on amide-AGE formation pathways in vivo, which, in the case of N(6)-acetyl lysine, parallels enzymatic processes.     

The role of glycation in aging and diabetes mellitus

One of the hypotheses trying to explain the process of aging is the idea of glycation of proteins. This reaction, also called the Maillard or browning reaction, may explain age-related symptoms such as cataract, atherosclerosis and modification of collagen-containing tissues. Diabetics, which posses elevated blood sugar levels, show signs of accelerated aging exposing similar complications. The Maillard reaction, which occurs on a large scale in vivo, may play a key role in the initiation of these symptoms.     

Targeting advanced glycation with pharmaceutical agents: where are we now?

Advanced glycation end products (AGEs) are the final products of the Maillard reaction, a complex process that has been studied by food chemists for a century. Over the past 30 years, the biological significance of advanced glycation has also been discovered. There is mounting evidence that advanced glycation plays a homeostatic role within the body and that food-related Maillard products, intermediates such as reactive α-dicarbonyl compounds and AGEs, may influence this process. It remains to be understood, at what point AGEs and their intermediates become pathogenic and contribute to the pathogenesis of chronic diseases that inflict current society. Diabetes and its complications have been a major focus of AGE biology due to the abundance of excess sugar and α-dicarbonyls in this family of diseases. While further temporal information is required, a number of pharmacological agents that inhibit components of the advanced glycation pathway have already showed promising results in preclinical models. These therapies appear to have a wide range of mechanistic actions to reduce AGE load. Some of these agents including Alagebrium, have translated successfully to clinical trials, while others such as aminoguanidine, have had undesirable side-effect profiles. This review will discuss different pharmacological agents that have been used to reduce AGE burden in preclinical models of disease with a focus on diabetes and its complications, compare outcomes of those therapies that have reached clinical trials, and provide further rationale for the use of inhibitors of the glycation pathway in chronic diseases.     

Non-enzymatic glycation of proteins: From diabetes to cancer

Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Non-enzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage, and kidney failure. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.     

Peptide and amino acid glycation: new insights into the Maillard reaction.

Nonenzymatic glycation of proteins, peptides and other macromolecules (the Maillard reaction) has been implicated in a number of pathologies, most clearly in diabetes mellitus. but also in the normal processes of aging and neurodegenerative amyloid diseases such as Alzheimer's. In the early stage, glycation results in the formation of Amadori-modified proteins. In the later stages, advanced glycation end products (AGE) are irreversibly formed from Amadori products leading to the formation of reactive intermediates, crosslinking of proteins, and the formation of brown and fluorescent polymeric materials. Although, the glycation of structural proteins has been attributed a key role in the complications of diabetes, recent attention has been devoted to the physiological significance of glycated peptide hormones. This review focuses on the physico-chemical properties of the Amadori compounds of bioactive peptides of endogenous and exogenous origin, such as Leu-enkephalin and morphiceptin, investigated under different conditions as well as on novel pathways in the Maillard reaction observed from investigating intramolecular events in ester-linked glycopeptides.     

Evidence for a relationship between protein glycation and red blood cell membrane fluidity

This study examines the relationship between protein glycation and membrane fluidity in RBC membranes. Incubation of RBC membranes of healthy subjects with 25mM glucose or galactose at 37 degrees C induced a 38% (p less than 0.02) increase in protein glycation (using furosine determination by HPLC) and higher fluidity (p less than 0.05) in DPH polarization ratio). However, incubation of RBC membranes from diabetic subjects under the same conditions did not modify either membrane fluidity or protein glycation; protein glycation was above normal before incubation because of the high diabetic plasma glucose. There was no difference in the membrane fluidities of 21 healthy subjects and 32 diabetic subjects, despite a significantly elevated protein glycation in diabetics. Furthermore, there was no change with respect to age in either population. We conclude that other in vivo factors, such as membrane lipid changes (increase in CL/PL ratio) or formation of advanced Maillard products and peroxidation in the diabetic subjects, could be responsible for the difference between these in vitro results and the in vivo situation.     

Production and characterization of antibodies to advanced glycation products on proteins

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.     

Vitamin C degradation products and pathways in the human lens

Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p < 0.05). In water-insoluble proteins, only 3-deoxythreosone and threosone were detected, whereby the level of 3-deoxythreosone was ～20 times higher than the level of threosone. The identification of 3-deoxythreosone as the major degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo.     

Formation of the molten globule-like state during prolonged glycation of human serum albumin

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0-630 mg/dl): the early, intermediate and late stages, which occurred after 7-14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.     

A study on human serum albumin influence on glycation of fibrinogen

Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [¹³C₆] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen.     

Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins

Proteins can be chemically modified by sugars by glycation, or the Maillard reaction. The Maillard reaction produces irreversible adducts on proteins that are collectively known as advanced glycation end products, or AGEs. Recent studies indicate that several alpha-dicarbonyl compounds, including glyoxal (GXL), are precursors of AGEs in vivo. We developed antibodies against a GXL-modified protein (GXL-AGE) and purified a mixture of GXL-AGE-specific antibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human serum albumin-carboxymethyllysine (HSA-CML)-NHS-Sepharose to remove CML-specific antibodies. We used the resulting purified antibody in a competitive ELISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater antibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incubation experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incubated with N-alpha-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine-lysine crosslinking structure, GOLD was found to be one of the antigenic epitopes for the antibody. Analysis of human plasma proteins revealed significantly higher levels of GXL-AGE antigens in type II diabetic subjects compared with normal controls (P<0.0001). We also found GXL-AGEs in human lens proteins. Bovine aortic endothelial cells cultured for 7 days with 30 mM glucose did not accumulate intracellular GXL-AGEs. These studies underscore the importance of GXL for extracellular AGE formation (except in lens where it is likely to be formed intracellularly) and suggest that changes associated with age and diabetes might be prevented by alteration of GXL-AGE formation.     

15N chemically induced dynamic nuclear polarization during reaction of N-acetyl-L-tyrosine with the nitrating systems nitrite/hydrogen peroxide/horseradish peroxidase and nitrite/hypochloric acid

While the Maillard reaction of amino acids and proteins as well as its consequences in vivo has been thoroughly investigated, little attention has so far been paid to the glycation of aminophospholipids such as phosphatidylethanolamine (PE) or phosphatidylserine (PS), which are essential for structure and functionality of biological membranes. PE-derived glucosylamines (Schiff-PEs) and aminoketoses (Amadori-PEs) have now for the first time been simultaneously identified and quantified in erythrocytes from diabetics and healthy individuals by liquid chromatography-electrospray mass spectrometry (LC-(ESI)MS). The amounts of glycated PE (gPE) were significantly higher in diabetics (0.18-34.2 mol% Schiff-PE and 0.047-0.375 mol% Amadori-PE) than in controls (0.12-3.99 mol% Schiff-PE and 0.018-0.055 mol% Amadori-PE). A positive correlation between fructosylated hemoglobin (HbA(1c)) and the gPE levels was established. No advanced glycation endproducts (AGEs) like 5-hydroxymethylpyrrole-2-carbaldehyde (pyrrole-PE), carboxymethyl (CM-PE), or carboxyethyl (CE-PE) derivatives were detected. To investigate the influence of gPE on lipid peroxidation of biological membranes, liposomes consisting of soy-PE and synthetically prepared Amadori-PE (16:0-16:0) were incubated for several days and the formation of oxidation products was monitored. It could be shown that Amadori-PE extensively promotes lipid peroxidation even in the absence of transition metal ions like Cu(2+) and Fe(3+). Oxidative damage to membrane lipids therefore is supposed to be at least partially caused by the glycation of aminophospholipids.     

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Possible evidence is presented for Maillard glycation of enzymes during oligosaccharide synthesis by reverse hydrolysis. In 70% (w/v) mannose solutions, 1,2-α-mannosidase from Penicillium citrinum lost 40% and α-mannosidase from almonds lost 60% activity at 55 °C over 2 weeks. Oligosaccharide yields were 15 and 45% respectively. Higher molecular weight glycation adducts were formed in a time-dependent manner as seen by MALDI-TOF. Inhibitors of the Maillard reaction were able to partially alleviate these effects resulting in reduced loss of enzyme activity and oligosaccharide yield increases of 27–53% relative to the control.     

Ovalbumin Modified with Pyrraline,a Maillard Reaction Product,shows Enhanced T-cell Immunogenicity

The Maillard reaction (also referred to as “glycation”) takes place between reducing sugars and compounds with free amino groups during thermal processing of foods. In the final stage of the complex reaction cascade, the so-called advanced glycation end products (AGEs) are formed, including proteins with various glycation structures. It has been suggested that some AGEs could have immunostimulatory effects. Here, we aimed to identify specific glycation structure(s) that could influence the T-cell immunogenicity and potential allergenicity of food allergens, using ovalbumin (OVA, an egg white allergen) as a model allergen. OVA was specifically modified with representative glycation structures: N^ϵ-carboxymethyl lysine (CM-OVA), N^ϵ-carboxyethyl lysine (CE-OVA), pyrraline (Pyr-OVA), or methylglyoxal-derived arginine derivatives (MGO-OVA). As well as AGE-OVA, a crude glycation product in thermal incubation of OVA with glucose, only Pyr-OVA, and not other modified OVAs, was efficiently taken up by bone marrow-derived murine dendritic cells (BMDCs). The uptake of Pyr-OVA was reduced in scavenger receptor class A (SR-A)-deficient BMDCs, but not in cells treated with inhibitors of scavenger receptor class B, galectin-3, or blocking antibodies against CD36, suggesting that pyrraline binds to SR-A. Compared with other modified OVAs, Pyr-OVA induced higher activation of OVA-specific CD4⁺ T-cells in co-culture with BMDCs. Furthermore, compared with native OVA, AGE-OVA and Pyr-OVA induced higher IgE production in mice. Pyrraline could induce better allergen uptake by DCs via association with SR-A and subsequently enhance CD4⁺ T-cell activation and IgE production. Our findings help us to understand how Maillard reaction enhances the potential allergenicity of food allergens.     

衰老研究的新纪元

在人类历史进入２１世纪的时刻,生命科学关于衰老原理的研究迎来了一个划时代的新纪元。多领域、多层次、跨学科的衰老研究,在分子水平上将遗传基因衰老学说、自由基氧化衰老学说、糖基化衰老学说和交联衰老学说汇聚到了一个新的焦点－－羰基毒化衰老学说。由于现代生命科学逐步认识到了羰－－氨反应是诸多生物副反应中的一个核心过程,该反应引起了衰老机理研究了极大重视。     

New insights into protein crosslinking via the Maillard reaction: structural requirements,the effect on enzyme function,and predicted efficacy of crosslinking inhibitors as anti-ageing therapeutics

Protein crosslinking via the Maillard reaction with alpha-dicarbonyl compounds has been the subject of intense literature scrutiny. We report here a systematic study of three previously-neglected aspects of the reaction. Firstly, structural requirements were probed. An arginine-free peptide that contains two lysine residues, and a lysine-free peptide that contains arginine, were reacted with glyoxal, methylglyoxal and biacetyl. Methylglyoxal was able to crosslink in the absence of arginine residues, but glyoxal and biacetyl were not. Glyoxal crosslinked the lysine-free peptide via the N-terminus, but methylglyoxal and biacetyl could not. In this study, crosslinking did not require the presence of arginine but did require a free amino group, from a lysine residue, or the N-terminus. Thus specificity in structural requirements for protein crosslinking by alpha-dicarbonyls has been demonstrated. Secondly, protein function following glycation was examined by treating ribonuclease A with the three alpha-dicarbonyls, which were shown both to crosslink the enzyme and impair enzymatic activity. Thirdly, the effects of two reported Maillard reaction inhibitors, aminoguanidine and 3,5-dimethylpyrazole-1-carboxamidine on the crosslinking reaction were assessed, with a parallel measurement of the effect on enzyme activity. The results demonstrate that preventing protein crosslinking does not necessarily preserve enzyme activity. These results cast doubt on the likely efficacy of some purported anti-ageing compounds in vivo.