John J. Harding

Nonenzymatic Covalent Posttranslational Modification of Proteins In Vivo

Publisher Summary This chapter presents the in vivo analysis of non-enzymatic covalent post-translational modification of proteins. Enzyme-controlled post-translational modification of proteins, especially enzymes, has been demonstrated in many tissues and organisms. These post-translational modifications are of the utmost importance in the control of metabolism and have been discussed in many reviews. However, over the years evidence has also accumulated for non-enzymatic modification of proteins in vivo, which is the subject of the present review. Some of these changes are well known to workers in particular fields—for example, glycosylation in diabetes, but others are less well known. Based on such analysis this chapter is subdivided according to the nature of the chemical reactions and the types of compounds binding to proteins. It is possible to produce a modified hemoglobin indistinguishable hemoglobin AIc by incubation of hemoglobin A with glucose (Fluckiger and Winterhalter, 1976). The pH-dependence curve for this corresponds to the titration curve of the a-amino groups. Syntheses glucose labeled in either the 2- or the 5- position indicated that the major product was the ketoamine and that it can be produced within the red cell.

The lens in diabetes

This paper reviews the changes which occur in the human lens in diabetes. They include refractive changes and cataract and age-related increases in thickness, curvatures, light scattering, autofluorescence and yellowing. The incidence of cataract is greatly increased over the age of 50 years, slightly more so in women, compared with non-diabetics. Experimental models of sugar cataract provide some evidence for the mechanism of the uncommon, but morphologically distinct, juvenile form of human diabetic cataract, where an osmotic mechanism due to sugar alcohol accumulation has been thoroughly studied in diabetic or galactose-fed rats. The discrepancy between the ready accumulation of sugar alcohol in the lens in model systems and the very slow kinetics of aldose reductase (AR) has not been satisfactorily explained and suggests that the mechanism of polyol formation is not yet fully understood in mammalian systems. The activity of AR in the human lens lies mainly in the epithelium and there appears to be a marginal expectation that sufficient sorbi-tol accumulates in cortical lens fibres to explain the lens swelling and cataract on an osmotic basis. This is even more so in the cataracts of adult diabetics, which resemble those of age-related non-diabetic cataracts in appearance. The very low levels of sorbitol in adult diabetic lenses make an osmotic mechanism for the increased risk of cataract even less likely. Other mechanisms, including glycation and oxidative stress, are discussed. The occurrence of cataract is a predictor for increased mortality in the diabetic.

Glutathione-related enzymes and the eye

Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive exygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and administration of some drugs.

Viewing molecular mechanisms of ageing through a lens

Many late-life diseases are conformational diseases in tissues where there are unfolded or misfolded proteins which can form aggregates. These diseases have other common features in their aetiology. Cataract is one such disease and post-translational modifications of proteins in the lens during cataract formation are described as a possible guide to the changes in other age-related conditions. Delineation of common pathways in these diseases could lead to common treatment regimes, and in this respect, there are promising results for aspirin-like drugs in Alzheimers disease, cataract, myocardial infarction, stroke and various cancers.

Aspirin prevents carbamylation of soluble lens proteins and prevents cyanate-induced phase separation opacities in vitro: a possible mechanism by which aspirin could prevent cataract

The carbamylation of lens proteins by cyanate causes conformational changes, and cyanate causes cataract. There is some evidence that aspirin is beneficial to cataract patients, so its effect on the carbamylation of lens proteins and on opacification produced by cyanate in vitro was studied. Aspirin decreased the phase separation temperature in lenses exposed to cyanate, and was found to reduce the rate of carbamylation of most, if not all, soluble lens proteins. Studies with radiolabelled aspirin lead to the conclusion that the drug achieves this protection by chemically modifying the proteins. The nature of this modification and the relevance of these results to human cataract is discussed.

Non-enzymic glycosylation (glycation) of lens proteins by galactose and protection by aspirin and reduced glutathione

Radioactive galactose becomes attached covalently to lens proteins in the same way as glucose. Simultaneous incubation with aspirin inhibits the reaction with galactose in a dose-related manner. Incubation with aspirin before incubation with galactose in the absence of aspirin showed that aspirin can modify crystallins permanently to prevent the binding of galactose. The galactosylation was also inhibited by glutathione at physiological concentrations. All major groups of lens proteins reacted with galactose but a higher level of modification of protein in the material of high molecular weight may indicate that galactosylation has induced aggregation of the proteins. The modification of all major crystallin groups was confirmed by isolating the galactosylated proteins by affinity chromatography. The results are discussed in relation to glycosylation of lens proteins in diabetes and galactosaemia and the role of glycosylation in cataract.

Prevention of cataract in diabetic rats by aspirin, paracetamol (acetaminophen) and ibuprofen

Evidence from epidemiological, in vitro and animal studies has accumulated to support the idea that aspirin, ibuprofen and paracetamol protect against cataract. In this study rats made diabetic with streptozotocin were given these drugs in their drinking solution for up to 160 days. All three drugs delayed cataract formation assessed by slit-lamp examination for a large part of this time. Blood glucose levels were a little lower in diabetic rats treated with aspirin and ibuprofen than in untreated diabetic rats although all groups remained diabetic. Similarly, the increased glycation (non-enzymic glycosylation) of lens proteins caused by diabetes was less in the diabetic rats treated with aspirin and ibuprofen. The fall in glutathione induced by diabetes was also alleviated by aspirin and ibuprofen. Paracetamol appeared to afford similar protection against the biochemical changes but its effect was not statistically significant. The decrease in glutathione and increase in glycation were related to the progression of lens opacification. The greatest loss of glutathione occurred at an early stage, whereas glycation had its greatest change at the later stages--nuclear and mature cataract. These results encourage the view that ibuprofen, aspirin and paracetamol could protect against cataract in man: a hypothesis that could be tested in a properly-conducted clinical trial.

Carbamylation of lens proteins: A possible factor in cataractogenesis in some tropical countries

It has been suggested that severe and repeated diarrhoea may be a major factor in cataracto-genesis in some tropical countries and account for the high prevalence of cataract in these countries. At least four effects of the diarrhoeal syndrome could lead to cataract and one of these is the high concentration of urea in the blood. Urea comes to equilibrium with cyanate and in this paper we investigate the reaction of cyanate with lens proteins and report preliminary evidence that carbamylation has occurred during cataractogenesis in Pakistan.

The Unusual Links and Cross-Links of Collagen

Publisher Summary This chapter concerns the work on the unusual links and cross-links of collagen. The unusual linkages may exist as branching points or cross-links or may be built into the peptide backbone itself. This chapter discusses the cross-links of collagen. It also reviews the evidence for different unusual links in collagen. Certain of these linkages appear to be involved in the cross-links. This chapter illustrates that it must be stressed that the glutamic acid residues in collagen that are involved in the γ-peptide linkages are not responsible for the cross-linking of collagen. They are present solely as an alternative mode of linkage of the amino acids. Such linkages constitute the greatest deviation from the α-peptide theory of protein structure yet established. The suggestion that є-amino lysine peptide linkages occur in collagen is based entirely on the evidence of the tripeptide. This chapter concludes by describing the nature of the protein–carbohydrate link in collagen, which seems to resemble that of most glycoproteins in that it involves aspartic acid.

The influence of some post-translational modifications on the chaperone-like activity of alpha-crystallin

We investigated the influence of phosphorylation, glycation, carbamylation and oxidative modification on the capacity of alpha-crystallin to protect beta-crystallins against heat denaturation. Simple modification of lysine residues by early glycation or carbamylation had no effect. However, late (cross-linking) glycation products and oxidative modifications decreased the chaperone-like activity of alpha-crystallin. Homopolymers of alpha A-crystallin had a higher protecting capacity compared with those of alpha B-crystallin. The in vivo phosphorylated forms of especially alpha A- but also alpha B-crystallin revealed a somewhat better protecting ability than the respective non-phosphorylated forms.