In vitro non-enzymatic glycosylation of myofibrillar proteins

Inr. J. Biochem. Vol. 25, No. 6, pp. 941-946, Printed in Great Britain. All rights reserved

1993 Copyright

IN VITRO NON-ENZYMATIC MYOFIBRILLAR I.

SYROVY*

0

0020-71 IX/93 $6.00 + 0.00 1993 Pergamon Press Ltd

GLYCOSYLATION PROTEINS

OF

and Z. HODNY

Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic (Received 26 October 1992) Abstract-l.

Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered. 2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered. 3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated. 4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation. 5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

INTRODUCTION

Non-enzymatic glycosylation or glycation is a post-translational modification where the carbonyl group of sugars or their derivatives reacts with c-amino group of lysine or other free amino groups of proteins. Several plasma and tissue proteins have been shown to undergo glycation in uiuo (Van Boekel, 1991). Modification of amino acid side chain groups leads to conformational changes of proteins, crosslinking etc. and due to it also functional properties of proteins are altered. Proteins with long half-lives in vivo are more intensely subjected to this modification process. Glycation is responsible for many pathological changes in individuals with elevated blood-sugar levels, such as patients with diabetes mellitus or galactosemia; moreover, glycation products were found to accumulate with increasing age of various organisms (Baynes, 1991; Miksik and Deyl, 1991). Glycation has received attention because it is probably related to the development of some diabetic complication, pathological ageing, catharacta and atherosclerosis (Harding, 1985; Kohn et al., 1984). Non-enzymatic glycosylation and its biological significance has been studied mainly in long-lived proteins like collagens, lens crystallins, myelin or hemoglobin. The question, how the glycation alters cellular function of intracellular proteins with relatively short half-lives is practically unsettled (see Shilton and Walton, 1991).

*To whom correspondence should be addressed: Institute of Physiology, Czech Academy of Sciences, Videnska 1083, Prague 4, 14220 Czech Republic.

It was shown that glycation of myosin is slightly higher in diabetic human myosin when compared with healthy subjects (Yudkin et al., 1989). The purpose of this study is to report on the effect of in vitro glycation of myofibrillar proteins on ATPase activity. In order to evaluate this further the influence of 2-mercaptoethanol, aminoguanidine and DETAPAC was also studied. MATERIALS AND METHODS Material

or=glyceraldehyde, diethylenetriaminepentaacetic acid (DETAPAC), nitroblue tetrazolium (NBT), and l-deoxy-lmorpholino-D-fructose were obtained from Sigma; aminoguanidine bicarbonate from Koch-Light; ATP, 2-mercaptoethanol and 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) from Serva; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) from Calbiochem and D-ribose from Lachema. All other reagents were of highest analytical quality. Preparation of myofibrils

The myofibrils were prepared as described previously (Potter, 1974) from skeletal muscles of adult rats. The final pellet was resuspended in 80mM KCl, 1 mM HEPES (pH 7.0) to a final concentration of 5.0 mg protein/ml. In vitro glycation Suspension of myofibrils was glycated by incubation in 80 mM KCl, 1 mM HEPES (pH 7.0) 3 mM sodium azide, 2 mM phenylmethylsulfonylfluoride at 25°C for 48 hr. The amount and type of sugar is described in the legend for individual experiments. Samples were dialyzed extensively against 80mM KCl, 1mM HEPES (pH 7.0) for 48 hr at 4°C to remove unbound sugar prior to the glycation assay. 941

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SYROVY

Table I. Glycation of myofibrillar proteins and myofibrillar ATPase activity (n = 6) Sugar concentration Control Glucose 0.5 M Fructose 0.5 M Galactose 0.5 M Ribose 0.5 M twglyceraldehyde

Glycation (ketoamine pmol/g protein)

ATPase activity (pm01 P,/min/mg)

2.0 f 0.3 3.0 f 0.5 9.5 f 0.6 9.4 * 0.5 15.9 +_1.0 30.2 + 2. I

0.52 f 0.025 0.52 f 0.020 0.50 f 0.019 0.41 f 0.01 I 0.15 * 0.008 0

0.05 M

RESULTS

Glycation assay procedure To determine the amount the protein, the fructosamine

of sugar

covalently

bound

to

assay was used with I-deoxyas standard (Johnson et al.,

I-morpholino-o-fructose 1982). Assay method for ATPase

Md+activated ATPase of myofibrils was determined in a reaction mixture containing 8 mM KCl, I mM MgCI,,

1mu ATP, 0.1mu &cl,, 2 mu NAN,, 20mM T~~_Hc] (PH 7.4) and 0.3 mg of myofibrillar protein/ml. Inorganic phosphate was assayed according to Fiske and Subbarow (Fiske and Subbarow, 1925). Estimation

of sulfbydryl

groups

Protein-bound sulfhydryl groups were determined with the use of Ellman’s reagent (Sedlak and Lindsay, 1968). Protein concentration was determined after digestion with a catalyst mixture (Chibnall et al., 1943) by the Conway microdiffusion technique (Conway, 1957). SDS-PAGE was performed according to Laemmli (Laemmli, 1970) on 12% gels. The gels were stained with Coomassie brilliant Blue R-250. Parallel gels were stained with silver staining procedure sensitive for glycation changes (Hodny et al., 1992).

Myofibrils were incubated with various sugars and glycation of proteins and ATPase activity were determined (Table 1). The level of glycation and inhibition of ATPase was dependent on the sugar used, the most efficient were ribose and glyceraldehyde. Increasing the ribose concentration from 0.1 to 0.5 M increased the glycation of myofibrillar proteins and reduced myofibrillar ATPase activity (Fig. 1). The addition of DETAPAC, aminoguanidine or 2-mercaptoethanol to incubation mixture of ribose and myofibrils lowered the NBT-reducing activity and the ATPase activity was partially or completely protected (Table 2). In the presence of 10 mM 2-mercaptoethanol incubation with ribose did not influence ATPase activity at all. The content of SH groups in myofibrillar preparation decreased as the result of glycation, and in the presence of 2-mercaptoethanol SH groups were partially protected. To show what component of myofibrillar proteins is preferentially glycated, a silver staining

0.5

h

g 0.4

0.4 Ribose

concentration

(M)

Fig. I. Glycation of myofibrillar proteins and myofibrillar ATPase activity after incubation with various concentrations of ribose at 25°C for 48 hr (mean of three experiments). (0) glycation, (0) ATPase activity.

In vitro non-enzymatic

glycosylation

1

2

of myofibrillar

3

proteins

4

Fig. 2. Electrophoretic pattern of control and glycated myofibrillar proteins. Myofibrils were treated with 0.5 M ribose, 25”, 48 hr. 1, 3Gcontrol; 2, 4-myofibrils treated with ribose; I, 2-silver staining; 3, 4--CBB staining; 50 pg of myofibrillar proteins per sample well. MHC, myosin heavy-chain; AA, cc-actinin; AC, actin; TM, tropomyosin; LC, myosin light chain.

943

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944 Table 2. Glycation

Incubation Myofibrils Myofibrils Myofibrils Myofibrils

+ Myofibrils Myofibrils Mvofibrils

of myot’ibrillar proteins, myofibrillar ATPase activity and protein SH groups content. DETAPAC, aminoguanidine and 2mercaptoethanol (n = 5) Glycation (ketoamine pcmol/g protein)

mixture + + + + + + + + + +

HODNY

ribose 0.5 M ribose 0.5 M DETAPAC 5 mM ribose 0.5 M aminoguanidine 0. I M ribose 0.5 M 2mercaptoethanol 0.1 mM ribose 0.5 M 2mercaptoethanol I mM ribose 0.5 M 2-mercaptoethanol 10 mM

detection technique after electrophoretic separation of myofibrils was applied. Fig. 2 (lane 1,2) shows that myosin heavy chain, actin, tropomyosin and LC, are more intensively stained by technique which is sensitive for glycation-derived chemical changes of protein (Hodny et aI., 1992). CBB staining (lane 3,4) resulted in a lower staining of glycated myofibrillar proteins, especially myosin heavy chains and tropomyosin. DISCUSSION When proteins are incubated with simple sugars the acyclic form of sugar reacts with end a-NH, or c-NH, group of lysine, guanidine amino group of arginine is further potential target for glycation intermediates such as dicarbonyls (Kato et al., 1987a). Such modified proteins have altered structural and biological properties. Various studies have investigated the effects of non-enzymatic glycosylation on proteins, but few have examined how this post-translational modification modulates enzymatic activity (Arai et al., 1987; Kondo et al., 1987). Theoretically, non-enzymatic glycosylation could be studied with the use of myosin, instead of myofibrillar proteins. This is, however, very difficult, as in the course of glycation, myosin solution becomes very viscous and even gelatinous. We therefore used myofibrillar preparation instead, where these problems do not exist. When myofibrillar ATPase is measured at high Ca*+ concentration, the values obtained correspond to actin-myosin interaction, whereas at low Ca*+ regulatory proteins are also involved. In the current study, myofibrillar glycation was found to decrease ATPase activity. Glycation of myofibrillar proteins and also the lowering ATPase was dependent on the sugar used; sugar with a high amount of the reactive open form in solution [ribose and glyceraldehyde (Angyal, 1984)] were more efficient (Table 1). It was demonstrated (Brown et al., 1990) that the decrease of actin activated myosin ATPase activity by non-enzymatic glycosylation is lower with fructose than with glucose, in spite of the fact that fructose is more reactive than glucose in Schiff base formation (Bunn and Higgins, 1981). Brown et al. have, however, incubated myosin in

ATPase activity (pm01 PJmin/mg)

The influence of SH groups (omoWg)

2.1 + 0.3 15.2 + 1.0

0.52 f 0.02 0. I5 * 0.008

68 + 6 52 5 4

10.4 * 0.9

0.35 f 0.03

12.3 f 1.1

0.35 f 0.04

14.5 * 1.2

0.40 * 0.05

55 * 5

7.5 f 0.7

0.43 f 0.05

61 +5

6.5 i 0.6

0.57 + 0.03

63 + 5

the presence of 1 mM DTT. Under these conditions myosin SH groups may be protected (see our results with 2-mercaptoethanol), It is thus not obvious whether in the absence of DTT the influence of glucose and fructose on actin activated myosin ATPase would be the same as in DTT presence. With increasing ribose concentration the level of glycation was also increased and the ATPase activity of myofibrils lowered (Fig. 1). In the course of glycation of many proteins, sugars react preferentially with t-NH2 of lysine and myosin especially contains high amount of lysine residues, often arranged in clusters or neighbouring histidine, which arrangement may facilitate amino group reactivity (Hunt and Wolff, 1991; Bai et aI., 1989). Glycation is a complex process in which a great number of reaction products are formed in several steps, such as intermediates of the reaction between sugar and amino residues, but also highly reactive products of sugar autoxidation or oxygen free radicals. The latter causes structural damage to proteins (Wolff et al., 1991). We have, therefore, examined the effect of DETAPAC, aminoguanidine and 2-mercaptoethanol, which may all exert an effect on protein modification due to glycation or oxidation. When myofibrils were glycated in the presence of DETAPAC, sugar attachment was lower than in its absence and the inhibition of myofibrillar ATPase activity was less pronounced. DETAPAC is a metal chelating agent and it is obvious that transition metals are involved in alteration of proteins exposed to sugar in uitro (Wolff and Dean, 1987) and that they catalyze generations of oxygen free radicals. Thus the inhibition of glycation in the presence of DETAPAC and the lower inhibition of myofibrillar ATPase can be explained at the same time by the assumption that trace amounts of transition metals present in used chemicals catalyze the oxidation reactions taking part in protein glycation. Aminoguanidine was also proved to lower glycation of myofibrils and inhibition of myofibrillar ATPase activity. It is proposed that aminoguanidine protects proteins indirectly by decreasing the concentration of the highly reactive dicarbonyl form of sugars (Lewis and Harding, 1990; Edelstein and Brownlee, 1992). It was also observed that

In vitro non-enzymatic glycosylation of myofibrillar proteins

945

amino-guanidine inhibits the form&ion of AGES in vitro and in vivo (Nicholls and Mandel, 1989). Our results with aminoguanidine support data previously obtained (Lewis and Harding, 1990; Edelstein and Brownlee, 1992). The presence of 2-mercaptoethanol during incubation of myofibrils with ribose influenced the level of myofibrils glycation, their ATPase activity and also SH groups content (Table 2). The high concentration of 2-mercaptoethanol (10 mM) resulted in the marked decrease of glycation; the ATPase activity of myofibrils was much higher than in samples incubated with ribose only. The decrease of glycation (expressed as NBT reducing activity) can be explained by the reaction of 2-mercaptoethanol with dicarbonyls (according to Mira et al., 1991) formed either during sugar autoxidation (Wolff and Dean, 1987) or Amadori product decomposition (Reynolds, 1965; Kato et al., 1987b) thus lowering their concentration available for protein glycation. It is difficult to explain how ATPase activity is influenced during the complex set of Maillard reaction. It is known that SH groups are involved in myosin ATPase activity (Sreter et al., 1966). Less information is available on the role of lysine residues. In myosin the actin binding sequence GKGKKKG was found (Chaussepied and Morales, 1988). Its modification by glycation might influence the binding of actin to myosin and thus also myosin ATPase. Our results about the effect of 2-mercaptoethanol on ATPase are in accordance with observation that dithioerythritol or glutathione has a protector effect on oxidative inhibition of red blood cell ATPase by glyceraldehyde (Mira et al., 1991). They proposed that the sulhydryl groups are the critical target for dicarbonyl compounds and that the inactivation of ATPase is probably due to the thiol oxidation or the reaction of SH groups with dicarbonyl compounds. Our results strengthen this assumption, as ATPase activity is to a greater extent influenced in our experiments in the presence of 2-mercaptoethanol than glycation. The present eletrophoretic study shows that glycation in vitro concerns heavy chains of myosin and other myofibrillar proteins. No quantitative data are available but our results demonstrate that various myofibrillar proteins are glycated. Gels also show that no fragmentation or considerable polymerization occurs in the course of glycation. The mobility of some myofibrillar proteins after glycation is slightly lowered. This may be due to

It was shown (Yudkin et al., 1989) that human diabetic subjects have slightly higher levels of glycated myosin when compared with non-diabetic ones. We have observed that glycation of myosin (Syrovy and Hodny, 1992) and myofibrils (unpublished data) of rat and human subjects increases with age and that it is higher in diabetic muscles than in control ones. Although the difference in glycation was small (up to 20%) data nevertheless confirm that glycation in vivo has biological significance especially under pathological conditions.

lower binding of SDS; changes in molecular mass are a less probable explanation. The lower CBB stainability of glycated proteins may be explained by slower binding of CBB to glycated protein (Syrovy,

Hodny Z., Struzinsky R. and Deyl Z. (1992) Silver staining of collagen type I after sodium dodecylsulfate polyacrylamide gel electrophoresis: effect of Maillard reaction.

1992). In our experimental design non-physiological concentration of ribose was used. It is thus improbable that similar effects occur in vivo, though proteins are exposed in vivo to various sugars for a much longer time.

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