Non-enzymatic glycation of elastin

Non-enzymatic glycation of proteins is one of the key mechanisms in the pathogenesis of diabetic complications and may be significant in the age-related changes of tissues. We investigated thein vitro glycation of human aortic -elastin, and chose and adapted methods for evaluating the degree and kinetics of glycation. -Elastin was prepared from thoracic aortas of young accident victims and glycated by incubating with different glucose concentrations (25, 50, 75 and 100 mmol/l) in 0.2 M phosphate buffer, pH 7.8 for 30 days, at 37°C. The degree of glycation was measured by three colorimetric methods,i.e. Nitroblue tetrazolium, 2-thiobarbituric acid and hydrazine; by aminophenyl-boronate affinity chromatography which determines Amadori products; and by a fluorescence method which determines advanced glycosylation end products. The highest degree of glycation was found on day 3 after the beginning of incubation. Fluorescence, as an index of advanced glycation, consistently increased from days 5 to 24. Investigation of the properties of glycated elastin may help in understanding the importance of this long-lived protein for the age-related changes in tissues and for diabetic complications.     

Phosphate promotes glycation of antithrombin III which interferes with heparin binding

Nonenzymatic glycation of antithrombin III has been reported to cause the reduction of heparin-catalyzed thrombin-inhibiting activity in diabetes. The effect of in vitro nonenzymatic glycation of pure antithrombin III on heparin binding and heparin-potentiated activity under a variety of buffers and pH values was studied to further clarify the physiological significance of this reaction. The extent of glycation, measured by the fructosamine assay and [14C]glucose binding, was enhanced by the presence of phosphate ion (pH 7.45, 8.5 and 9.5) and increased linearly with increasing phosphate ion concentration from 0.01 to 0.2 M phosphate. Conversely, the heparin-catalyzed antithrombin activity decreased from 93.1% of controls for 0.01 M phosphate to 73.5% for 0.2 M phosphate as the extent of glycation increased. The increase in intrinsic fluorescence induced by binding of heparin to antithrombin III was also moderated by glycation of antithrombin III in a dose-dependent manner with a negative correlation coefficient of -0.94. Direct measurement of the heparin binding by affinity chromatography showed a decrease in the heparin-binding fraction which correlated with the degree of glycation and the decrease in heparin-catalyzed activity. These studies suggest that nonenzymatic glycation may be responsible for the reduction in antithrombin III activity observed in some diabetics.     

Non-enzymatic glycation induces structural modifications of myoglobin

Increased glucose concentration in diabetes mellitus causes glycation of several proteins, leading to changes in their properties. Although glycation-induced functional modification of myoglobin is known, structural modification of the protein has not yet been reported. Here, we have studied glucose-modified structural changes of the heme protein. After in vitro glycation of metmyoglobin (Mb) by glucose at 25°C for 6 days, glycated myoglobin (GMb) and unchanged Mb have been separated by ion exchange (BioRex 70) chromatography, and their properties have been compared. Compared to Mb, GMb exhibits increased absorbance around 280 nm and enhanced fluorescence emission with excitation at 285 nm. Fluorescence quenching experiments of the proteins by acrylamide and KI indicate that more surface accessible tryptophan residues are exposed in GMb. CD spectroscopic study reveals a change in the secondary structure of GMb with decreased α-helix content. 1-anilino-naphthaline-8-sulfonate (ANS) binding with Mb and GMb indicates that glycation increases hydrophobicity of the heme protein. GMb appears to be less stable with respect to thermal denaturation and differential calorimetry experiments. Heme-globin linkage becomes weaker in GMb, as shown by spectroscopic and gel electrophoresis experiments. A correlation between glycation-induced structural and functional modifications of the heme protein has been suggested.     

肌红蛋白的非酶糖化及其生物学效应

蛋白质的非酶糖化（non－enzymatic glycation）在糖尿病及其并发症的发生发展中起着重要作用。糖化血红蛋白（hemoglobin Alc,HbAlC）已作为糖尿病患者血糖控制的“金标准”。相对而言,同为血红素蛋白的肌红蛋白（myoglobin,Mb）,其非酶糖化的研究相对较少。研究发现：非酶糖化能改变Mb的空间构象,且对Mb血红素活性中心产生一定的影响,从而导致Mb各种生物学功能发生一定程度的改变。本文综述非酶糖化对Mb结构与功能的影响的研究现状,旨在阐明Mb非酶糖化后的结构．性质-功能之间的关系及其生物学意义,为糖尿病及其并发症的发病机制研究提供新的线索。     

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.     

Non-enzymatic glycation of IgG: an in vivo study.

The IgG glycation level of 30 healthy subjects and 60 type 2 diabetic patients with different degrees of metabolic control was evaluated by matrix-assisted laser desorption ionization mass spectrometry, a technique allowing the determination of mass increase of the IgG molecule. When applied to the digested mixture obtained by the action of papain on the plasma protein fraction, the same method established the mass increase of Fab and Fc fragments of IgG; for the former, a higher mass increase was found, possibly explained by its high reactivity to glucose. Experimental results were confirmed by molecular modeling calculations. Results suggest that the immunodeficiency observed in diabetic patients may be due to the inhibition of molecular recognition between antibody and antigen as a result of a change in functionality of the modified Fab fragment of IgG.     

Non-enzymatic glycation of bone collagen modifies osteoclastic activity and differentiation

Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.     

Non-enzymatic glycation of proteins: a cause for complications in diabetes

Diabetes mellitus is one of the most common non-communicable diseases, and is the fifth leading cause of death in most of the developed countries. It can affect nearly every organ and system in the body and may result in blindness, end stage renal disease, lower extremity amputation and increase risk of stroke, ischaemic heart diseases and peripheral vascular disease. Hyperglycemia in diabetes causes non-enzymatic glycation of free amino groups of proteins (of lysine residues) and leads to their structural and functional changes, resulting in complications of the diabetes. Glycation of proteins starts with formation of Shiff's base, followed by intermolecular rearrangement and conversion into Amadori products. When large amounts of Amadori products are formed, they undergo cross linkage to form a heterogeneous group of protein-bound moieties, termed as advanced glycated end products (AGEs). Rate of these reactions are quite slow and only proteins with large amounts of lysine residues undergo glycation with significant amounts of AGEs. The formation of AGEs is a irreversible process, causing structural and functional changes in protein leading to various complications in diabetes like nephropathy, retinopathy, neuropathy and angiopathy. The present review discusses about role of glycation in various complications of diabetes.     

Heterogeneity of mammalian antithrombin III

Affinity chromatography on heparin-Sepharose was used to isolate two forms of antithrombin III(AT) from human, bovine, rabbit and rat blood plasma. The two isolated forms of AT are the major form. AT alpha, making up to 90% of the whole inhibitor molecule, and the minor form, AT beta (10% of AT). The molecular mass of AT beta in all mammalian species under study is by 3-5 kDa lower than that of AT alpha. The isoelectric point for bovine AT alpha lies within the range of 4.95-4.5, whereas that for AT beta--at 5.28-4.76. No significant differences in the progressive antithrombin activity of the major and minor forms of the bovine inhibitor were observed. In contrast, the heparin-cofactor activity of the AT beta-heparin complex exceeds that of the AT alpha-heparin complex--3-fold. The functional differences in the AT forms are due to the differences in their affinities for heparin. It was shown that AT beta exhibits a higher affinity for free and bound heparin.     

Non-enzymatic in vitro DNA labeling and label immunoquantification

In the present work, a label immunoquantification procedure was developed in order to determine the number of markers introduced into DNA. A non-enzymatic, in vitro labeling method for introducing the p-bromobenzoyl radical (label), through transamination and acylation reactions of the cytidine nucleotides in calf thymus DNA, was carried out. Three spacer arms with different lengths were used for separating the label from the nucleotide and three labeled DNA were obtained. Anti-p-bromobenzoyl chicken IgY polyclonal antibodies were obtained. The antibodies detected the label, into three-labeled DNA, with different sensitivities, in relation to spacer arm length used. About 3-11 labels per 4 x 10(6) bases into thermally denatured DNA were immunoquantified.     

Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.     

Non-enzymatic glycation of type I collagen diminishes collagen-proteoglycan binding and weakens cell adhesion

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.     

Kinetics of thermal denaturation of antithrombin III

Purified antithrombin III (AT III), a single-chain human plasma glycoprotein, molecular weight 58,000 daltons, and one of the major serine protease inhibitors, was heated in the 60-70 degrees C range for inactivating possible contaminations by hepatitis B virus (HBV). Loss of inhibitory activity, unfolding of tertiary structure, and the rate of aggregate formation of AT III were monitored experimentally during heatig. Sucrose and sodium citrate were demonstrated to stabilize the protein. From the rate data the calculated activation energies (E) showed E(tert. struct.) < E(biol. act.) < E(aggreg.) indicating the order (lower activation energy process first) in which heat causes these changes in the protein molecule. The activation energy corresponding to denaturation of HBV was estimated to be at least fourfold lower than that associated with the unfolding of the tertiary structure of the protein. Purified AT III, thus stabilized and pasteurized, should be therapeutically effective, and the risk for transmission of hepatitis B should be decreased significantly.     

Inhibition of factor XIa by antithrombin III

The inactivation of human factor XIa by human antithrombin III was studied under pseudo-first-order reaction conditions (excess antithrombin III) both in the absence and in the presence of heparin. The time course of inhibition was followed by using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. After electrophoresis, proteins were blotted onto nitrocellulose and stained either for glycoprotein or for antithrombin III using antibodies against antithrombin III. Concomitant with factor XIa inactivation, two new slower migrating bands, one of which represented the intermediate complex consisting of one antithrombin III complexed with factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two antithrombin III molecules. Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity toward the chromogenic substrate S2366. Pseudo-first-order reaction kinetics were observed throughout. The rate constant of inactivation was found to be 10(3) M-1 s-1 in the absence of heparin and 26.7 X 10(3) M-1 s-1 in the presence of saturating amounts of heparin. From the kinetic data, a binding constant (Kd) of 0.14 microM was inferred for the binding of antithrombin III to heparin. The time course of inactivation and the distribution of the reaction products observed upon gel electrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e., independent of each other) with the same rate constant of inhibition.     

Re-formation of disulphide bonds in reduced antithrombin III.

The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.     

Non-enzymatic glycosylation induced changesin vitro in some molecular parameters of collagen

Non-enzymatic glycosylation of rat tail tendon collagen was examined by incubation with D-glucosein vitro. The changes in molecular parameters such as viscosity, thermal stability, electrophoretic mobility and solubility were determined on nonenzymatically glycosylated collagenin vitro. Tendons incubated with 8 and 24 mg glucose/ml showed an increase in dissolution temperature and a l.6-3-fold increase in thermal isometric tension respectively when compared to tendons incubated in the absence of glucose, indicating the formation of new intermolecular bonds. This conclusion was further supported by the decreased solubility of glycosylated collagen in 0.5 N acetic acid and the change in sub-unit composition as measured from the sodium dodecyl sulphate Polyacrylamide gel electrophoresis pattern. Glycosylated collagen gave a characteristic absorption spectra λ_max 248 nm) as distinct from that of control (λ_max 242 nm). Denaturation temperature of glycosylated collagen, as determined from temperature dependent viscosity measurements, was reduced. These studies indicate that glycosylation affects the molecular interactions as well as the crosslinking of collagen.     

Alterations of antithrombin III after acute myocardial infarction

The antithrombin III (AT III) activity and the AT III concentration were investigated in 62 consecutive patients with acute myocardial infarction (AMI). To identify the reactant pattern of AT III in postaggressive situations, we also determined labile and acute phase proteins. Firstly, 29 patients were nourished orally and then 33 patients were fed by i.v. hyperalimentation (additional caloric intake of approximately 1,000 cal). AT III activities and concentrations as well as prealbumin and retinol-binding protein decreased concomittantly and significantly whereas haptoglobin, C-reactive protein and fibrinogen increased significantly after AMI. The changes cannot be interpreted as being alterations of the haematocrit. The alterations of AT III correlated significantly with the changes of labile proteins but not with the acute phase reactant proteins. The AT III decrease in the postinfarctional phase may promote a prethrombotic state. In In addition it can be concluded from our results that AT III reacts as (nutritive-dependent) labile protein, which is lowered in postaggressive situation and does not increase as an acute phase reactant. This is in accordance with results from recent animal experiments.     

Preparation and characterization of an antithrombin III concentrate

Tri-calciumphosphate was found to have not only a known adsorption capacity for factors of the prothrombin complex, but also for antithrombin III. Depending on the inserted blood stabilizer the human plasma fractions Cohn I, PPSB and antithrombin III may be isolated from the same initial material in the area of the transfusion service. Enriching antithrombin III is achieved by a three-stage procedure under aseptic conditions in a closed system. Liberating antithrombin III from calciumphosphate is made with care without using any concentrated salt solutions.