Ramanakoppa H. Nagaraj

Isolation and characterization of a blue fluorophore from human eye lens crystallins: In vitro formation from Maillard reaction with ascorbate and ribose

A blue fluorophore, named LM-1 was isolated from human eye lens crystallins. The fluorescence property of LM-1 (excitation/emission, 366/440 nm) is similar to the fluorescence originating during non-enzymatic glycation (Maillard reaction) of proteins with the reducing sugars. LM-1 accumulates linearly with age in highly cross-linked water insoluble crystallins and is present at higher levels in cataractous lenses. The fluorophore could be synthesized by incubation of bovine serum albumin (BSA) with ribose, but not with glucose or fructose. Incubation of bovine lens crystallins with ascorbic acid (ASA) and its oxidative products, dehydroascorbic acid (DHA) and 2,3-diketogulonic acid (DKG) in presence of oxygen resulted in LM-1 formation. When oxygen was removed from the system, only DHA and DKG could synthesize LM-1, but not ASA, suggesting that ASA oxidation is obligatory for LM-1 synthesis. Modification of lysine residues on BSA prior to incubation with ribose resulted in corresponding decrease in LM-1 formation. Since ASA concentration is unusually high in lens and has been found to be a powerful glycating agent of crystallins and since LM-1 does not form with hexoses, it is likely that ASA is the major precursor of LM-1.

Chromatographic evidence for pyrraline formation during protein glycation in vitro and in vivo.

Pyrraline (epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine) is an advanced Maillard reaction product formed from 3-deoxyglucosone in the non-enzymatic reaction between glucose and the epsilon-amino group of lysine residues on proteins. Although its presence in vivo as well as in in vitro incubations of proteins with sugars has been documented by immunochemical methods using polyclonal and monoclonal antibodies, its formation in proteins has recently been questioned by similar methodology. To clarify this issue, we investigated pyrraline formation in proteins following alkaline hydrolysis and quantitation by high-performance liquid chromatography on a C18 reverse-phase column. Time- and sugar concentration-dependent increase in pyrraline formation was noted in serum albumin incubated with either 100 mM glucose or 50 mM 3-deoxyglucosone. Formation of pyrraline from 3-deoxyglucosone was rapid at slightly acidic pH, confirming its synthetic pathway through this Maillard reaction intermediate. Low levels of pyrraline (< 10 pmol/mg protein) were also detected in a pool of human skin collagen by this method, but no age effect was apparent. Using a slightly different approach, pyrraline-like material was detected in human plasma proteins following enzyme digestion and analysis by high performance liquid chromatography. Plasma from diabetic patients showed a significant increase in pyrraline-like material compared to controls. The levels in diabetic and normal individuals were 21.6 +/- 9.56 and 12.8 +/- 5.6 pmol per mg protein, respectively (P = 0.005), reflecting thereby the elevated levels of the immediate precursor of pyrraline, 3-deoxyglucosone, in diabetic plasma.

Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer disease

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the hyperphosphorylation of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of tau generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.

Evidence of a Glycemic Threshold for the Formation of Pentosidine in Diabetic Dog Lens but Not in Collagen

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 ± 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 ± 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Urinary pyrraline as a biochemical marker of non-oxidative maillard reactions in vivo

The presence of pyrraline, a non-oxidative glucose-derived Maillard reaction product in plasma proteins has been established previously. In this study we have investigated the presence of pyrraline in human urine to determine whether pyrraline-containing proteins are metabolized or selectively retained. Pyrraline was detected by means of HPLC, and its presence was confirmed by UV and electrospray-mass spectrometry. The quantification of pyrraline in urine from healthy individuals showed 1.21 +/- 0.4 micrograms/mg creatinine. In urine from diabetic patients, pyrraline levels varied considerably, although the mean level was higher than in healthy subjects (1.37 +/- 0.6 micrograms/mg creatinine). These data further support the presence of a catabolic pathway for advanced non-oxidative Maillard reaction products in vivo and suggest their role in the pathogenesis of diabetes.

The Role of α- and ε-Amino Groups in the Glycation-mediated Cross-linking of γB-crystallin STUDY OF THREE SITE-DIRECTED MUTANTS

In the previous report we demonstrated that γB-crystallin is glycated predominantly at the N-terminal α-amino group (Casey, E. B., Zhao, H. R., and Abraham, E. C. (1995)J. Biol. Chem. 270, 20781–20786). To investigate the possible role of α- and ε-amino groups of γB-crystallin in glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were mutated to threonine by site-directed mutagenesis in bovine γB-crystallin cDNA. Wild type and mutant γB-crystallins were expressed in Escherichia coli cells. Cross-linking studies were performed by incubating wild type and mutant γB-crystallins with glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions. When both of the lysines of γB-crystallin were mutated to threonines (γB-K2T/K163T), the quantity of cross-linked products was greatly reduced, indicating that, despite the fact that the α-amino group is a major glycated site, ε-amino groups play a predominant role in cross-linking. Therefore, cross-linking ability depends not only upon the level of glycation but also upon which amino group is glycated. Steric hindrance may decrease the cross-linking ability of the α-amino group. Our results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of γB-crystallin. By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel “no lysine” γB (γB-K2T/K163T) with sugar, we were able to show for the first time that significant cross-linking occurs between lysines and non-lysine sites. The fact that pentosidine and imidazolysine, formed from ribose and methylglyoxal, respectively, were present in the cross-linked γB-crystallins revealed the existence of Lys-Arg and Lys-Lys cross-linking.

Relevance of the Early and Advanced Maillard Reaction in Diabetic Neuropathy

A number of cellular, metabolic, and morphological changes which are thought to play a role in diabetic neuropathy in the human have been first detected in the diabetic and galactosaemic rat model. These include an increase in nerve glucose, sorbitol, and fructose concentration as a result of an activation of the sorbitol pathway and a concomitant depletion in myo-inositol. The extent of nerve protein glycation is increased both in the rat and in the human. Na+,K+-ATPase appears to be impaired early on, thus probably explaining the intracellular accumulhtion of Na+, the axonal swelling, and axo-glial dysjunction which have been observed in peripheral nerves. Impaired anterograde and retrograde axonal transport, loss of axonal neurofilaments, reduction in myelinated fibre size and fibre loss eventually lead to irreversible lesions characterized by segmental demyelination. Most of the changes described above can be prevented by treating the animal with aldose reductase inhibitors or by adding myo-inositol to the diet. A clearcut loss of myo-inositol has not been demonstrated in the human and therapy with aldose reductase inhibitors had mixed result^,^,^ the latter being in part attributed to the irreversibility of the lesions. Mechanisms by which the early (protein glycation) and the advanced Maillard reaction may participate in the pathogenesis of diabetic neuropathy are discussed. These include the inhibition of Na,K+-ATPase, the inhibition of axonal transport, and the crosslinking of neuronal proteins. The possible role of advanced Maillard products in the segmental demyelination process is then discussed. Finally, the similarities between aldose reductase inhibition and aminoguanidine therapy in the amelioration of experimental diabetic neuropathy are considered. Since, to date, few data on the role of glycation on diabetic neuropathy per se are available, concepts presented below are in part derived from data obtained from other tissues such the lens or the kidney.

Protocol for quantitative analysis of paired helical filament solubilization: A method applicable to insoluble amyloids and inclusion bodies

Biochemical studies of amyloidoses have been plagued by the sparing solubility of most amyloids in denaturant solvents. Consequently often only a subclass of amyloid protein is analyzed, a fact that is omitted in most studies. This means that there is often no evaluation of the chemical basis for amyloid insolubility, a factor that may provide valuable information concerning amyloid pathogenesis. We have devised a protocol to quantitatively evaluate the solubilization of insoluble amyloid proteins. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents that dissolve all protein. Here we describe the application of this protocol to quantitatively establish complete solubilization of the paired helical filaments (PHFs) from Alzheimer disease. PHFs are distinct from the other amyloid that defines Alzheimer disease (AD), i.e., extracellular amyloid-beta deposits of senile plaques, nonetheless, PHFs share all the properties of, and are defined as, an amyloid, i.e., binding Congo red; beta-pleated sheet conformation and, most significantly, sparing solubility. PHFs of neurofibrillary tangles are the most striking intraneuronal change seen within the brains of patients with AD. Despite intense efforts to understand the molecular composition of this amyloid, quantitative biochemical analyses have been severely hampered by the extreme insolubility of PHF and by difficulties obtaining a homogeneous PHF fraction. Therefore, to date, all of the published studies on the biochemical composition of insoluble PHFs (SDS-insoluble) are qualitative and have provided little or no quantitative data on the proportion of material assayed. Using the solubilization protocol described herein, we found that only high pH was effective in solubilizing PHF while a variety of denaturants and chaotropes resulted in only partial release of component protein. Significantly, the approach is analytical because it allows direct assessment of the significance of two posttranslational modifications in mediating PHF insolubility, i.e., phosphorylation and glycation. Further this protocol provides solubilized protein that can be readily characterized. For example, coupling the method to immunoblotting, ELISA, microsequencing or other analytical techniques would identify components as well as provide a quantitative measure.