Characterization of a blue fluorophore isolated from in vitro reaction of N-proportional to-acetyllysine and 3-deoxyglucosone.

With the objective to investigate 3-deoxyglucosone (3-DG) mediated lysine crosslinks in vivo, we have isolated a lysine-3-DG-lysine crosslink from in vitro reaction of 3-DG and N-proportional to-acetyllysine (NAL). This crosslink, named as furopyrrolopyridine crosslink (FPPC), has intense blue fluorescence with absorption maxima at 235, 270 and 370 nm and emission maximum at 470 nm. The absorption and fluorescence spectra of FPPC were not altered in pHs ranging from 2-12, but the characteristic spectrum of FPPC (at pH 7.0) disappeared when it was reduced with sodium borohydride. FAB-MS showed that FPPC has a molecular mass of 611, equivalent to the reaction of two molecules each of NAL and 3-DG with the concomitant loss of 5 molecules of water. NMR data showed that FPPC has a pyridinium ring and four free hydroxy groups. Since acid hydrolyzed FPPC can be detected by amino acid analysis, we have determined its levels in the acid hydrolyzates of proteins glycated by 3-DG or in the acid hydrolyzates of normal aged, cataractous, diabetic and brunescent human lens proteins as well as in the acid hydrolyzed glycated hemoglobin, A0.

Immunochemical localization of the Batten disease (CLN3) protein in retina.

PURPOSE: Batten disease, also known as juvenile ceroid-lipofuscinosis and CLN3, is an autosomal recessively inherited disorder that results in blindness due to retinal degeneration. The CLN3 gene has been identified, but the function of the protein that this gene encodes is unknown. Experiments were conducted to determine where the CLN3 protein is localized in the mouse retina. Localization should provide a clue in evaluating potential functions of this protein. METHODS: Using oligonucleotide primers based on the reported human CLN3 cDNA sequence, the mouse cDNA nucleotide sequence was determined from products of the reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends. A synthetic 20-amino-acid peptide corresponding to an internal hydrophilic region of the predicted amino acid sequence of the mouse CLN3 protein was used to immunize rabbits. The resulting antiserum was used in immunoblot analysis of mouse retina homogenates and in electron microscopic immunocytochemical labeling of mouse retina sections. RESULTS: The peptide antibody labeled a single protein band of approximately 50 kDa on immunoblots of mouse retina homogenates. No labeling was detected with homogenates from human retinas. The antibody specifically labeled mitochondria of Müller cells and inner retinal neurons. Little labeling was observed in mitochondria of the photoreceptor cells. Mitochondria of other cell types, including the retinal pigment epithelium and choroidal cells, were not labeled. CONCLUSIONS: The retinal CLN3 protein appears to be localized almost exclusively in the mitochondria, but was detected only in certain cell types. Batten disease is characterized by massive lysosomal accumulations of a small inner mitochondrial membrane protein (subunit c of ATP synthase). The mitochondrial localization of the CLN3 protein suggests that it may play a role in the normal processing of subunit c.

The relative UV sensitizer activity of purified advanced glycation endproducts.

The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L-Threose, the most active of these oxidation products, was reacted with N-acetyl lysine and six AGE peaks were isolated by RP-HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 micrograms/mL) generated 5-10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100-fold less than the superoxide anion generated by kynurenic acid and 400-fold less than riboflavin. Ultraviolet irradiation generated from 1.2 to 2.7 mumol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM-1 and Ac-FTP) and with kynurenine and 3-OH kynurenine. This considerable singlet oxygen formation, however, was still 40-fold less than that obtained with kynurenic acid and 100-fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated 20-60-fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water-insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.

Rapid assessment of early glycation products by mass spectrometry.

In order to detect the early glycation products, we have reacted a model peptide (t-boc-lys-ala-ala) with L-threose (a degradation product of ascorbic acid) and analyzed the reaction products by a combination of HPLC and mass spectrometry. Amino group modification, as observed by a fluorescamine assay, indicated complete modification after 3 days of incubation with a 10-fold excess of threose. As much as 60% of the adducts were acid labile and only 4% of the adducts could be observed by amino acid analysis. However, Fast atom bombardment mass spectrometry (FABMS) of the samples incubated for 6 hr showed relative molecular masses consistent with the formation of adducts corresponding to the addition of one and two molecules of L-threose to the peptide. Likewise, samples incubated for 12 hr showed peptide adducts with two and three L-threoses. The number of threose molecules added to the peptide was also confirmed from the FABMS analysis by using [1-13C]-threose as the glycating agent.

Glycation mediated crosslinking between alpha-crystallin and MP26 in intact lens membranes.

With advancing age, progressive crosslinking occurs between lens crystallin proteins and other lenticular components. This crosslinking may be involved in the development of senile cataracts. Experiments were conducted to determine whether non-enzymatic glycation could be involved in the crosslinking between lens alpha-crystallin and MP26, an abundant lens fiber cell membrane intrinsic protein. In vitro crosslinking of alpha-crystallin and MP26 of bovine lens membranes was observed in presence of two degradation products of ascorbic acid (ASA), dehydroascorbic acid (DHA) and threose. Alkali-washed bovine lens membranes, isolated after glycation with DHA and threose, contained both alpha-crystallin and MP26, as determined by immunoblot and double immunocytochemical labeling studies. In contrast, membranes incubated without these glycating compounds contained only MP26. SDS-PAGE analysis of [125I] alpha-crystallin incubated with lens membranes in the presence of threose showed a higher amount of radioactivity in high molecular weight aggregates than in the aggregates produced when alpha-crystallin and threose were incubated without membranes. A slot-blot immunoassay of alkali-washed human lens membranes showed a higher amount of covalently bound alpha-crystallin in aged, cataractous or diabetic lens membranes than was present in lens membranes from young normal donors. Based on the in vitro results, we hypothesize that non-enzymatic glycation is one of the vivo mechanisms in the crosslinking of alpha-crystallin to lens membrane proteins, such as MP26. This crosslinking may contribute significantly to the development of age-related and diabetic cataracts.

Ascorbic acid glycation: the reactions of L-threose in lens tissue.

L-Threose is a significant degradation product of ascorbic acid at pH 7.0 in the presence of oxygen. When compared to several other ascorbate-derived degradation products, it had the greatest ability to glycate and crosslink lens proteins in vitro. To determine whether L-threose was formed in the lens, the sugars in a TCA-soluble extract from human lenses were reduced to polyols with NaBH4, acetylated and analysed by gas-liquid chromatography. The threitol levels measured were 3.4 +/- 0.8 micrograms per lens (n = 4). GC-MS measurements made after reduction with NaBD4 indicated that threitol, but little or no threose, was originally present in the human lens. Rat lenses were incubated with [1-13C]D-threose for 24 hr, and considerable D-threitol formation was seen by NMR spectroscopy. Analysis of the lenses after medium removal showed that only [1-13C]threitol was present within the lenses indicating a rapid reduction of threose within the lens, presumably by aldose reductase. Assays with human recombinant aldose reductase and with human lens cortical and nuclear extracts all exhibited sorbinil-inhibitable aldose reductase activity with L-threose as substrate. This was confirmed by incubating a preparation of [1-14C]L-tetrose (a mixture of 40% L-threose and 45% L-erythrose) with both the pure aldose reductase and crude lens extracts followed by the subsequent identification of the [1-14C]L-threitol formed by thin layer chromatography. L-Threose degrades very slowly in 0.1 M phosphate buffer at pH 7.0, but the addition of a four-fold excess of N alpha-acetyl-L-lysine accelerated the rate of disappearance of threose 30-fold, indicating a rapid glycation reaction. When [1-14C]L-tetrose was incubated with a complete bovine lens homogenate, a linear incorporation into protein was observed over a 24 hr period. Increasing levels of lens extract exhibited increasing incorporation into protein. These data confirm a rapid reactivity of L-threose with lens protein and argue that glycation would occur in vivo in spite of the presence of aldose reductase.

Suppression of pentosidine formation in galactosemic rat lens by an inhibitor of aldose reductase.

Recent work from our laboratory revealed a correlation between the degree of protein pigmentation in human cataractous lens and the advanced Maillard reaction as reflected by pentosidine formation. Although the data suggested a role for ascorbate in pentosidine formation in senile cataractous lenses, elevated pentosidine levels in diabetic cataracts suggested that glucosylation may be involved directly in pentosidine biosynthesis. To clarify this issue, we quantified pentosidine in lenses from rats with experimental galactosemia with and without aldose reductase inhibitor treatment. At 12 months, pentosidine-like fluorescence (335/385 nm) was three to six times higher (P < 0.0001) in water soluble and insoluble crystallins of galactosemic compared with nongalactosemic rats. Actual pentosidine levels increased shortly after onset of galactosemia. Contents in water-insoluble crystallins were 6.32 +/- 2.2 and 1.40 +/- 0.66 pmol/mg protein in galactosemic and control lenses, respectively (P < 0.001). Fluorescence and pentosidine were suppressed to almost control levels upon treatment with sorbinil. Incubation experiments showed that pentosidine could form slowly from galactose, but much more rapidly from ascorbate and its oxidation products. Its formation could be inhibited partly by both reduced and oxidized glutathione or epsilon-aminocaproic acid. The requirement of oxygen for pentosidine formation suggests that oxidative stress associated with glutathione depletion and ascorbate oxidation are plausible mechanisms for rapid pentosidine formation upon onset of galactosemia. In contrast, Maillard reaction by glycoxidation products may account for the sustained increase in pentosidine. Both these events may be linked to the newly recognized pseudohypoxic state of cells exposed to high sugar concentrations.

Determination of glycation crosslinking by the sugar-dependent incorporation of [14C]lysine into protein.

A simple, quantitative assay has been established to determine the glycation-dependent crosslinking ability of any sugar by measuring the incorporation of [14C]-lysine into protein. The assay was shown to be both sugar-dependent and protein-dependent and was completely inhibited by sodium cyanoborohydride, 2-aminoguanidine, and semicarbazide. A typical 1.0-ml reaction mixture contained 5 mg lysozyme, 20 mumol threose, and 5 microCi of [14C]lysine and exhibited an incorporation of 8 x 10(5) cpm (1.6 nmol of lysine) after 7 days of incubation. A comparison of the crosslinking ability of a variety of sugars showed glyceraldehyde and dihydroxyacetone to be twice as active as erythrose and threose and eight times more reactive than ribose. Little or no crosslinking could be demonstrated with three different hexoses as well as their phosphorylated derivatives. The dicarbonyl sugars 3-deoxyglucosone and xylosone were at least as effective as ribose in crosslinking, as were the oxidation products of ascorbic acid. Several amine-containing compounds were tested as inhibitors of crosslinking; however, 2-aminoguanidine was the most effective. The rate of synthesis of Lys-Lys, Lys-Arg, and Lys-His crosslinks was determined by measuring the incorporation of [14C]lysine into specific amino acid homopolymers. The relative incorporation was polylysine > polyarginine > polyhistidine with threose, but polyarginine > polyhistidine > polylysine with dehydroascorbic acid, suggesting a different crosslinking mechanism for these two compounds.

Chemical modification of alpha crystallin.

Calf lens alpha-crystallin was isolated and the lysine residues were extensively modified with a variety of chemical agents. The effect of these modifications on elastase inhibitor activity, apparent molecular size, antibody reactivity and solubility were determined. The addition of either a methyl group or a threose residue did not alter the charge on the lysine residues and had little or no effect on either inhibitor activity or apparent molecular size. The introduction of a negative charge by either carboxymethylation or citraconylation caused a marked decrease in size and an almost complete loss of inhibitor activity. The introduction of a hydrophobic residue by reaction with either a trinitrobenzenesulfonic acid or Bolton Hunter reagent caused a slight increase in size, but a 70% increase in elastase inhibitor activity. Reaction with fluorescamine resulted in the dissociation of alpha-crystallin in a 200-kDa species, yet caused a two to four-fold increase in elastase inhibitor activity, which was similar to the activity of the water-insoluble fraction isolated from aged human lens and cataract. Several of these modified alpha-crystallins were compared for reactivity with a polyclonal alpha-crystallin antiserum using a quantitative slot blot assay. Charge neutral modifications resulted in a two to three-fold loss of antibody recognition, whereas the other preparations showed an almost complete loss of antigenic activity. None of the modifications caused the alpha-crystallin to precipitate at higher salt concentrations (0.3 M) with the exception of threose which caused a 30% decrease in soluble protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The glycation and cross-linking of isolated lens crystallins by ascorbic acid.

Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both alpha-crystallin and beta H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta L-crystallin was somewhat less reactive, but gamma-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: (a) the binding of protein to a boronate affinity column; (b) the incorporation of 3H from NaB3H4 into protein; (c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and (d) the incorporation of [1-14C]ASA into individual crystallins. When the separated crystallins were combined with [125I]gamma-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with gamma-crystallin itself. Gel filtration chromatography of a mixture of [125I]gamma-crystallin and alpha-crystallin showed the formation of a complex between gamma- and alpha-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Glycation of MP26 and MP22 in bovine lens membranes.

Alkali treated membranes were isolated from mature bovine lenses and incubated with different sugars for 3 weeks to study the effect of glycation on the lens intrinsic membrane proteins, MP26 and MP22. The obtained results show that a) [1-14C] ascorbic acid (ASA) was able to glycate the intrinsic membrane proteins as rapidly as soluble lens proteins; b) on 15% acrylamide gels in SDS, glucose, fructose, galactose and ribose exhibited low activity for crosslinking membrane proteins; whereas ASA, dehydroascorbate (DHA), diketogulonate (DKG), xylosone and threose, all showed not only the formation of protein multimers, but also highly crosslinked products, which did not enter the spacer gel; c) except glycated MP22, all of the crosslinks of MP26 or MP22, and also the glycated MP26, showed cross reactivity with polyclonal MP26 antibody; d) the extent of crosslinking correlated with an equal loss of lysine and arginine contents by amino acid analysis.

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with alpha-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.

The glycation-associated crosslinking of lens proteins by ascorbic acid is not mediated by oxygen free radicals.

The reaction by which ascorbic acid (ASA) causes the glycation and crosslinking of lens proteins displays a rigid requirement for the presence of oxygen, and is inhibited by the presence of glutathione. Oxygen is required to oxidize ASA to dehydroascorbic acid (DHA) and other products which are the active glycating species. No evidence could be found to support a role for oxidative protein crosslinking by a free radical mechanism. Crosslinking was not inhibited by blocking protein sulfhydryl groups with iodoacetamide, nor were the protein crosslinks dissociated by boiling with 2% mercaptoethanol prior to SDS-PAGE. The addition of a variety of oxygen free radical quenchers had no effect on the extent of protein crosslinking. In fact, the removal of oxygen from the reaction mixture had no effect on either protein glycation, protein crosslinking or the modification of lysine residues, provided DHA was used as the glycating agent. All of these activities were inhibited, however, if ASA was the glycating agent. This confirms that oxygen is required only to convert ASA to DHA.

Interaction of caffeine and of theophylline with liver glutamate dehydrogenase.

Caffeine and theophylline inhibited the activity of rat liver glutamate dehydrogenase (GDH), but not that of beef liver GDH, in forward and reverse directions of the enzyme reaction. In the forward direction, approximately 16 mM caffeine or 16 mM theophylline inhibited 50 per cent of the rat liver GDH activity (I50); while in the reverse direction, the I50 of caffeine and theophylline was 15 mM and 8 mM, respectively. The inhibition produced by caffeine was cooperative in both directions, while that of theophylline was negatively cooperative in the forward direction and non-cooperative in the reverse. However, ADP reduced the inhibitory effect of caffeine and theophylline to the extent of 40% and 80%, respectively. The Ki values obtained for caffeine and theophylline were different in the presence of various concentrations of substrates and coenzymes. Based upon these data, we presume that certain subtle changes occurring in the conformation of the rat liver GDH (probably at the ADP/NADH site) in comparison with those of the beef liver GDH may be responsible for its inhibition by caffeine and theophylline.

Properties of purified liver glutamate dehydrogenase of aging rats.

Glutamate dehydrogenase was purified from the liver of immature (4-weeks), young (22-weeks) and old (116-weeks) female rats to study the effect of age on this enzyme. From 30 g of liver tissue of each age group, approximately 90-fold purified enzyme was obtained in crystalline form with an average yield of 4% of the original enzyme activity. This 4% enzyme was used extensively for analyzing its age-related properties. The elution profile on a Sephadex G-200 column, molecular weight (53,000 +/- 3,000), ratio of A280/260 (1.8), optimum pH (7.8), optimum temperature (25 degrees C), and Km values for different substrates/coenzymes of the purified enzymes did not show marked age-dependent variations. Similarly, remarkable differences were not observed in the effect of a variety of nucleotides or steroid hormones on the activity of the purified enzymes of the three ages. These findings may suggest that rat liver glutamate dehydrogenase is an unaltered enzyme with advancing age of the animal.

Effect of age on the crystalline rat liver lactate dehydrogenase.

Lactate dehydrogenase (LDH) was purified and crystallised from the liver of immature (4 weeks), young (22 weeks) and old (116 weeks) female rats and the kinetic, physical and chemical properties of the purified enzyme were studied. Even though slight differences existed in effect of pH, temperature, storage and ratio of A280/260, significant differences were not observed in kinetic properties, behaviour on polyacrylamide gels, ion-exchange chromatography column, and -SH groups among the three age groups. These data indicate that, unlike muscle LDH of old rats which had shown altered properties (earlier report from our laboratory), liver LDH is an unaltered, like LDH of heart. The possible reason for the unaltered nature of the liver LDH may be: (a) the tissue origin; (b) the gene responsible for its synthesis is expressing well even in old age; or (c) non-influence of post-translational modifications on the enzyme molecule.