Carnosine protects against the inactivation of esterase induced by glycation and a steroid

Biochimica et Biophysica Acta 1741 (2005) 120 – 126 http://www.elsevier.com/locate/bba

Carnosine protects against the inactivation of esterase induced by glycation and a steroid Hong Yan1, John J. Harding* Nuffield Laboratory of Ophthalmology, University of Oxford, Walton St., Oxford OX2 6AW, UK Received 20 August 2004; received in revised form 17 November 2004; accepted 18 November 2004 Available online 8 December 2004

Abstract Carnosine, an endogenous histidine-containing dipeptide, protects protein from oxidation and glycation, which may contribute to a potential treatment for some conformational diseases including cataract. Glycation, the non-enzymic reaction of sugars with proteins, promotes cross-linking and further aggregation. Prolonged use of glucocorticoids is a risk factor for cataract, as is diabetes. Esterase activity in the lens is decreased in senile cataract and diabetes. Previously, we reported that glycation and a steroid inactivate esterase. Here we tested the inactivation of esterase with fructose, fructose 6-phosphate (F6P) and ribose as model glycation reactions and prednisolone-21hemisuccinate (P-21-H) as a model steroid and investigated the ability of carnosine to protect esterase against inactivation. The activity of esterase was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. The modified esterase was examined electrophoretically. The esterase was progressively inactivated by F6P, fructose, ribose and P-21-H. P-21-H was more effective than the sugars. Carnosine significantly inhibited the inactivation of esterase induced by all four compounds. Carnosine decreased the extent of the cross-linking. These results provide further evidence for carnosine’s role as an anti-glycation compound. It is also proposed that carnosine may be an anti-steroid agent. D 2004 Elsevier B.V. All rights reserved. Keywords: Carnosine; Esterase; Glycation; Steroid

1. Introduction Carnosine has been known for over a century and is present at high concentrations in some mammalian tissues [1]. It has anti-glycation, anti-oxidant and free-radical scavenging roles and has been proposed as a potential therapeutic agent for some conformational diseases including cataract and Alzheimer’s disease [1,2]. Carnosine, an antiglycation agent, inhibits cross-linking of proteins and protects their native functions. In vitro, carnosine inhibited the inactivation and cross-linking of enzymes, including superoxide dismutase, by glycation [3,4] and oxidation [5,6] Abbreviations: F6P, fructose 6-phosphate; P-21-H, prednisolone-21hemisuccinate * Corresponding author. Fax: +44 1865 794508. E-mail address: [email protected] (J.J. Harding). 1 Present address: Department of Ophthalmology, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, China. 0925-4439/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2004.11.008

and of aspartate aminotransferase by glyceraldehyde 3phosphate [7]. It also protected against both hydrogen peroxide- and 12-O-tetradecanoylphorbol-13-acetateinduced apoptosis through mitochondrial pathways [6]. Glycation, the non-enzymic reaction of sugars with protein, occurs normally but to an increased extent in ageing and diabetic complications [8]. Cross-linked proteins interfere with tissue function and act as trapping material causing aggregation. Glycation inhibits enzyme activity [9] and causes a decrease in chaperone function of a-crystallin [10,11]. Glucocorticoids are steroid hormones that play a role in many physiological processes and are used clinically as antiinflammatory agents in treatment of diseases such a rheumatoid arthritis and asthma. Prolonged use of glucocorticoids leads to the formation of cataract by an unknown mechanism. In vitro, glucocorticoids inactivate important metabolic enzymes in the lens [12–14], bind to lens

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crystallins [15,16] and inhibit chaperone activity of acrystallin [11]. Esterases are a complex family of enzymes. Porcine liver carboxyl esterase belongs to a large group of enzymes hydrolyzing ester bonds of carboxylic acids as carboxyl esterases, aryl esterases, lipases, acetyl esterases, cholinesterases and cholesterol esterases, which are widely distributed in animals, plants and microorganisms and show wide substrate tolerance with a preference for soluble substrates. Esterases preferentially break ester bonds of shorter-chain fatty acids [17]. The four main types of esterase activity have been demonstrated in mammalian serum [18]. Swanson and Truesdale [19] first identified esterase in normal human lens, and Kamei [20] separated it into two fractions. The activity of esterase decreased with ageing in the lens [20], in diabetic patients [21,22] and in experimental diabetic rats [23]. A significantly increased butyrylcholinesterase activity was found in the serum of type IIb hyperlipidaemic patients, suggesting that butyrylcholinesterase activity may responsible for an abnormal lipid metabolism [24]. Together with the esterase-like activity demonstrated in one of the hamyloid fragments in vitro, these observations might have implications as regards the pathogenesis of both Alzheimer’s disease and vascular dementias and the cholinesterase inhibitor therapy of dementing disorders [24,25]. More recently, neuropathy target esterase and similar esterases have been reported to play a central role in membrane lipid homeostasis [26]. Carboxylesterase 3 may mediate some hormone-sensitive lipase-independent lipolysis in adipocytes [27]. Therefore, inactivation of esterase may play a role in ageing, diabetic complications and possibly Alzheimer’s disease. The purpose of this study was to further explore the anti-glycation role of carnosine and examine whether it can prevent the inactivation and cross-linking of enzyme by a steroid. Based on our previous esterase reports [14,28], we thus used this target enzyme in the present study to investigate the protective effect of carnosine on the inactivation and cross-linking of the enzyme by glycation and a steroid. The results showed that exposure of esterase to sugar, sugar phosphate and a steroid led to the protein fragmentation and cross-linking and enzyme inactivation. Carnosine inhibited the cross-linking and inactivation, but not the fragmentation. The concentrations of sugars, steroid and carnosine used were higher than would be achieved in vivo but we are trying to simulate changes that may take years in the human tissues during the slow development of late life disease.

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fructose 6-phosphate (F6P), glucose, prednisolone-21-hemisuccinate (P-21-H), p-nitrophenyl acetate (PNPA), acetonitrile and all other chemicals were obtained from Sigma (Poole, Dorset, UK). Lower molecular weight protein markers were purchased from BioRad Laboratories (Hemel Hempstead, UK). 2.2. Incubation conditions Esterase (5 U/30.3 Ag) was incubated in a 50 mM sodium phosphate buffer (pH 6.8) with or without 20 mM fructose, F6P, ribose and 1 mM P-21-H in a final volume of 10 ml for 0–7 days, with the addition of various concentrations of carnosine (1, 10 and 50 mM). Stock solutions were divided into separate small sterilised glass vials with rubber tops (Whatman) through a sterilised Millipore filter (0.2-Am pore size). Individual vials were removed at zero time and various times thereafter for assay in triplicate. Additionally, esterase (2 mg/ml) was incubated with 20 mM fructose, F6P, ribose and 2 mM P21-H in the phosphate buffer. The solutions were analysed by SDS-PAGE after 6 days. 2.3. Esterase assay Inactivation of carboxylesterase by fructose, ribose, F6P and P-21-H in the presence and absence of carnosine was measured spectrophotometrically. The enzyme activity was assayed by monitoring the rate of PNPA hydrolysis catalysed by porcine liver esterase using a Hitachi U-2001 spectrophotometer [14,29]. Briefly, to assay esterase, the PNPA was dissolved in acetonitrile to make a stock solution (60 mM). Then, 1 ml of PNPA/ acetonitrile was added to 19 ml of buffer (50 mM sodium phosphate and ionic strength as 0.5 with NaCl, pH 6.8) just before the assay. For each assay, 1 ml of this diluted PNPA solution and 0.2 ml of enzyme solution were mixed in a 1.4 ml cuvette for reaction compared against a sodium phosphate buffer blank reference to subtract non-enzymatic background hydrolysis at 28 8C. The absorbance of released p-nitrophenol was measured at 400 nm for a standard period of 100 s with a lag of 30 s at the beginning of the reaction. The p-nitrophenol concentration was calculated using its absorbance beginning 30 s after the reaction had started, with measurements at 5-s intervals for 70 s. Activity is expressed relative to the control activity at each incubation time, set at 100%. 2.4. Analysis of electrophoresis gels

2. Materials and methods 2.1. Materials Esterase (porcine liver, carboxylic ester hydrolase from porcine liver, EC 3.1.1.1), carnosine, ribose, fructose,

Electrophoresis was performed on slab gels (BioRad Laboratories) with a Tris glycine buffer (pH 8.3) containing SDS (0.5%, w/v). A Mini-Protean II Dual Slab Cell Electrophoresis Units/PowerPac 200 apparatus (BioRad Laboratories) was used.

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Table 1 The effect of carnosine on activity of esterase after 9 days incubation Preparations

0 days

2 days

4 days

6 days

9 days

E E+1 mM C E+10 mM C

97.9F5.7 94.2F2.9 76.6F6.1

95.0F4.0 98.7F6.7 93.7F7.0

91.9F7.5 95.7F4.7 95.3F6.0

92.0F5.4 98.0F2.7 97.0F5.6

90.0F5.5 92.4F6.3 88.5F7.5

The table represents the percentage of original activities over time incubation. Esterase (5 U/30.3 Ag) was incubated in a 50 mM sodium phosphate buffer (pH 6.8) with or without 1, 10 and 50 mM carnosine for 0–9 days in a final volume of 10 ml. The activity of esterase was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate.

The proteins were denatured by heating the samples for 4 min at 95 8C in a 0.5 M Tris buffer (pH 6.8) containing SDS (0.2%) and 2-mercaptoethanol (5%, v/v) prior to applying to gel. Protein bands were visualized by 0.1% Coomassie Brilliant Blue R-250 staining in fixing solution. 2.5. Statistics All measurements were made in triplicate. Statistical analysis was performed using Student’s paired t-test.

any effect of 10 mM carnosine on the pH of the incubation solution, but it seems that at this concentration carnosine interferes slightly with colour development. Possibly carnosine binds initially to the enzyme substrate site. After prolonged incubation time, the activity then went up to 95% of control with 10 mM carnosine after 6 days, but fell slightly to 88% after 9 days. High concentrations of carnosine may inhibit esterase activity and affect the esterase assay. Thus, 1 and 10 mM carnosine were used in further incubations. 3.2. Effects of carnosine on glycation-induced inactivation

3. Results

The inactivation of esterase has been previously reported [14,28]. Here we first tested whether carnosine itself has an effect on the esterase assay. Initially, esterase incubations were carried out with carnosine at 1, 10 and 50 mM for 9 days. The activity of esterase on its own was stable falling only 8% after 9 days incubation (Table 1). In the presence of 1 mM carnosine, there was no significant change in activity over the incubation period and at 9 days it was similar to the control. However, with 10 mM carnosine, apparent activity was about. 21% lower at time zero. This was not caused by

Esterase was inactivated by F6P, fructose and ribose (Figs. 1–3). After 2 days, only 54.0F4.3% ( P=0.0004) of control enzyme activity remained in the incubation containing 20 mM F6P. By 5 days, esterase activity had fallen further to 35.8F7.9% ( Pb0.0001) of control activity (Fig. 1). Fructose (20 mM) inactivated esterase less rapidly, activity falling to 75.7F1.0% ( P=0.0001), 64.3F5.0% ( P=0.0007) and 44.3F9.0% ( P=0.0008) of that of the control by 2, 3 and 5 days, respectively (Fig. 2). Only 54.2F0.7% ( P=0.00002) of control enzyme activity remained after 2 days incubation of esterase with ribose (Fig. 3). These observations of rapid inactivation are similar to rates in our previous report [28].

Fig. 1. Effect of carnosine on F6P-induced inactivation of esterase. Esterase (5 U/30.3 Ag) activity was monitored during incubation in a 50 mM sodium phosphate buffer (pH 6.8) with or without 20 mM F6P for 0–5 days in a final volume of 10 ml, with the addition of 1 and 10 mM carnosine. F6P: fructose 6-phosphate; E: esterase.

Fig. 2. Effect of carnosine on fructose-induced inactivation of esterase. Esterase (5 U/30.3 Ag) activity was monitored during incubation in a 50 mM sodium phosphate buffer (pH 6.8) with or without 20 mM fructose for 0–5 days in a final volume of 10 ml, with the addition of 1 and 10 mM carnosine. E: esterase.

3.1. Effects of carnosine on assay of esterase

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3.3. Effects of carnosine on inactivation by a steroid

Fig. 3. Effect of carnosine on ribose-induced inactivation of esterase. Esterase (5 U/30.3 Ag) activity was monitored during incubation in a 50 mM sodium phosphate buffer (pH 6.8) with or without 20 mM ribose for 0– 6 days in a final volume of 10 ml, with the addition of 1 and 10 mM carnosine. E: esterase.

Carnosine protected esterase against glycation-induced inactivation. During incubation with F6P alone, only 54.0F4.3%, 47.6F7.1% and 35.8F7.9% of activity were retained after 2, 3 and 5 days without carnosine, whereas 73.4F1.9% ( P=0.0020), 75.3F8.5% ( P=0.0122) and 85.8F6.8% ( P=0.0011) of activity remained in the presence of 1 mM carnosine, 83.4F5.8% ( P=0.0021), 95.1F6.8% ( P=0.0011) and 83.3F7.4% ( P=0.0016) of activity remained in the presence of 10 mM carnosine (Fig. 1). After 5 days, the protective effect was approximately 48–50%. There was no statistically significant difference between 1 and 10 mM carnosine ( P=0.6949) after 5 days, whereas the protective effect was greater in 10 than 1 mM carnosine ( P=0.0341) after 3 days. Additionally, the inhibition of enzyme with 10 mM carnosine was greater than that with 1 mM carnosine at time zero, indicating the higher concentration of carnosine affects the esterase assay, so the results with 1 mM carnosine are more easily interpreted. The incubation of esterase with 20 mM fructose for 3 and 5 days lowered the activity to 64.3F5.0% and 44.3F9.0%, respectively, whereas the remaining activity was 81.8F3.3% ( P=0.0071) and 83.0F5.6% ( P=0.0035) in the presence of 1 mM carnosine, 89.7F5.1% ( P=0.0035) and 85.0F5.0% ( P=0.0024) in the presence of 10 mM carnosine (Fig. 2). The protection by both concentrations of carnosine was approximately 40% after 5 days. Ribose inactivated esterase slightly faster than fructose (Fig. 3). After 2 days, the activity that remained was 80.2F1.8% ( P=0.00002) and 94.2F5.8% ( P=0.0003) of the native enzyme in the presence of 1 and 10 mM carnosine, respectively, whereas 54.2F0.7% was retained with ribose alone (Fig. 3). After 6 days, there was a statistically significant difference ( P=0.0067) in the protective effect of carnosine between 1 mM (40%) and 10 mM (56%), but even 1 mM carnosine had a major protective effect.

The esterase was progressively inactivated by P-21-H at a lower concentration [14,28]. We investigated the effect of carnosine on P-21-H-induced inactivation of esterase (Fig. 4). During incubation with P-21-H, only 62.4F4.0% and 38.2F2.4% of the activity remained after 4 and 7 days, but with 1 mM carnosine and P-21-H, 78.9F12.4% ( P=0.0928) and 79.8F5.1% ( P=0.0002) of control activity remained; with 10 mM carnosine and P-21-H, 65.2F15.9% ( P= 0.7766) and 82.6F14.7% ( P=0.0067) of control activity remained. After 7 days, the protective effect was increased to approximately 42% (Fig. 4). There was no significant difference in protective effect between these two concentrations of carnosine. 3.4. Effects of carnosine on cross-linking by glycation and a steroid The effect of carnosine on cross-linking induced by glycation and a steroid was examined by SDS-PAGE after 6 days (Fig. 5). The esterase from porcine liver (EC 3.1.1.1) appeared as an approximately 60-kDa molecular mass band (Fig. 5, lanes E) as we previous reported [28]. Incubation of esterase with F6P resulted in a time-dependent inactivation (Fig. 1), in parallel with a decreased intensity of the original esterase band with the appearance of cross-linked protein on top of the gel after 6 days incubation (Fig. 5A, lanes FP). Carnosine protected the molecular structure against crosslinking as shown by weakening of cross-linked protein on top of the gel (Fig. 5A, lanes FP+C). A similar protective effect was observed in the SDS-PAGE band pattern of esterase incubated with P-21-H after 6 days (Fig. 5B), suggesting that carnosine prevents cross-linking of esterase by F6P and P-21-H. There was no noticeable cross-linked protein in the presence of ribose or fructose after 6 days incubation, whereas a few lower 60-kDa molecular weight

Fig. 4. Effect of carnosine on P-21-H-induced inactivation of esterase. Esterase (5 U/30.3 Ag) activity was monitored during incubation in a 50 mM sodium phosphate buffer (pH 6.8) with or without 1 mM P-21-H for 0– 7 days in a final volume of 10 ml, with the addition of 1 and 10 mM carnosine. P-21-H: prednisolone-21-hemisuccinate; E: esterase.

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Fig. 5. SDS-PAGE analysis of a solution of esterase (2 mg/ml) incubated with 20 mM F6P, fructose, ribose and 1 mM P-21-H in the absence and presence of 10 mM carnosine for 6 days. Lane M: molecular weight marker; E: esterase; FP: F6P and esterase; FP+C: esterase incubated with F6P and carnosine; R: esterase and ribose; R+C: esterase incubated with ribose and carnosine; F: esterase and fructose; F+C: esterase incubated with fructose and carnosine; P: esterase and P21-H; P+C: esterase incubated with P-21-H and carnosine.

bands appeared, suggesting that this phenomenon might have been associated with acceleration of the fragmentation of the enzyme molecule induced by glycation. Carnosine did not significantly prevent this fragmentation (Fig. 5A, lanes R, R+C; B lanes F, F+C).

4. Discussion Carnosine prevented the inactivation of esterase and also protected the molecular structure against cross-linking by glycating agents. Incubation with F6P produced crosslinked protein that appeared on top of the gel. Carnosine significantly inhibited this cross-linking with protection against inactivation. Carnosine can react with small carbonyl compounds, aldehydes and ketones [30] and glyceraldehyde 3-phosphate [31], and protect macromolecules against their cross-linking actions [30]. Our preliminary observations have shown that carnosine reacts with F6P and ribose in a time-dependent fashion. The initially clear solution became bright yellow upon incubation, even brown with ribose, resulting in the increase in fluorescence and absorbance (H. Yan and J.J. Harding, unpublished results). This indicates that a glycation product is formed between carnosine and sugar or sugar phosphate, so that although some of the colour in esterase incubations was due to protein adducts some is the result of glycation of carnosine itself to give a low molecular mass product. The product exhibits similarities to an imidazole structure linked via conjugation to one or more aromatic rings such as pyrrole or pyrazine rings [31]. The protective effect of carnosine against enzyme inactivation by glycating agents can be explained by their function as a sacrificial amino sink, based on its high reactivity with carbonyl compounds. Carnosine also has hydroxyl radical scavenging activity [4]. At first, it may seem surprising that 1% carnosine can protect against 20 mM sugar, but it must be remembered that only the open chain form of

each sugar takes part in glycation and this can be much less than 5% of the sugar [32,33]. Ribose, shown to be most damaging in these experiments, has a greater proportion of the sugar in the open chain form and has been found more damaging in earlier studies [14,34]. P-21-H contains a reactive carbonyl group that inactivates enzyme more rapidly than the sugars. In the present study, P-21-H led to significant inactivation of enzyme at a lower concentration compared with sugars. Based on the rapid inactivation, even at a concentration lower than 2 mM, P-21-H may attach to the amino group of lysine residues, or react elsewhere on the enzyme surface, resulting in the destabilisation of the enzyme structure. The remarkable protection by carnosine was shown during 7 days incubation. Furthermore, carnosine clearly exhibited the ability to inhibit cross-linking of esterase by P-21-H. Our previous study showed that carnosine may react with P-21-H resulting in adduct formation. A concentration- and time-dependent colour change was observed in the presence of carnosine with P-21-H, and a significant absorbance at about 250 nm appeared over time (H. Yan and J.J. Harding, unpublished results), suggesting the adducts may be formed between carnosine and P-21-H. Glucocorticosteroid–protein adducts have been identified by radioimmuno-assay in lenses of patients treated with steroids and in animal models. Corticosteroids react with protein amino groups to form an Schiff base through a reactive carbonyl group at the C-20 position (similar to glycation); this rearranges to form a more stable adduct [35]. Therefore, it is speculated that carnosine may intracellularly suppress the deleterious effects of a reactive carbonyl group by reacting with P-21-H to form protein– carbonyl–carnosine adducts similar to the reported aldehyde adducts [30] and carnosine-glyceraldehyde 3-phosphate adduct [31]. This work points to a novel property of carnosine acting as an anti-steroid compound. Its potential protection against steroid-induced inactivation of enzymes in vivo needs to be further demonstrated.

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The results obtained show that all four insults progressively inactivate esterase. Higher concentrations of carnosine inactivate slightly more activity at zero time than thereafter, indicating that higher concentrations of carnosine might have a deleterious effect on protein function. Further investigation will be required to clarify this possibility. Inactivation of esterase may contribute to complications of diabetes and some conformational diseases including cataract and Alzheimer’s disease [20–24]. It is increasingly apparent that carnosine not only has crucial anti-glycation and anti-oxidation roles, but that carnosine-derived products have potential effects on a wide range of processes within the cell. Cross-linking or fragmentation of protein is associated with a large number of cellular malfunctions that give rise to disease. This present investigation clearly indicates that carnosine protected against the glycation and a steroid induced inactivation of esterase and suppressed the extent of the cross-linking. Further investigation into the possible role of carnosine in ageing and other conformational diseases in vivo is required.

Acknowledgements The authors gratefully acknowledge the Wellcome Trust for a Travelling Research Fellowship to H Yan.

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