In vitro and in vivo reactions of nucleic acids with reducing sugars

Mutation Research, 238 (1990) 185-191 Elsevier

185

MUTREV 02803

In vitro and in vivo reactions of nucleic acids with reducing sugars Annette T. Lee and Anthony Cerami The Rockefeller University, 1230 York Ave., New York, N Y 10021 (U.S.A.) (Accepted 12 October 1989)

Keywords: Nucleic acids, reactions with reducing sugars; Sugars, reducing, reactions with nucleic acids; Glucose; Glucose 6-phosphate; Proteins, amino groups, nonenzymatic reactions; DNA bases, amino groups, reactions; Glycosylation, of DNA, nonenzymatic

Summary Reducing sugars such as glucose and glucose 6-phosphate have been shown to nonenzymatically react with the amino groups of proteins. The modification of proteins by reducing sugars can alter both physical characteristics and biological functions. Analogous to the reaction observed with proteins, the amino groups of DNA bases are also able to react nonenzymaticaUy with reducing sugars. The modifications of DNA by reducing sugars result in the time- and sugar-concentration-dependent changes in biological properties. In this communication we review data describing in vitro and in vivo models we have used to investigate the biological consequences of the nonenzymatic glycosylation of DNA.

Since 1912 when the food chemist L.C. Maillard (Maillard, 1912) first described the reaction of proteins with reducing sugars, the nonenzymatic glycosylation of proteins has gained increased recognition by food chemists in the study of food preservation. This reaction is responsible in part for the golden brown color of cooked foods and the change in taste and texture of foods during long-term storage. It has only been within the past two decades that the importance of this reaction with biologically relevant macromolecules has been investigated. The nonenzymatic glycosylation of long-lived proteins may play an important role in some of the complications associated with diabetes mellitus and aging. The Maillard reaction initially begins with the formation of a Schiff base between the aldehyde

Correspondence: Dr. Annette T. Lee, The Rockefeller University, 1230 York Ave., New York, NY 10021 (U.S.A.).

of a reducing sugar such as glucose and an amino group of a protein. Within a relatively short period of time the Schiff base reaches an equilibrium with a more stable but still reversible Amadori product. The Amadori product can then undergo a series of further rearrangements and dehydrations to form irreversible stable endproducts, collectively referred to as advanced glycosylation endproducts (AGE) (Fig. 1). These endproducts are characteristically fluorescent, yellow brown in color and are able to crosshnk proteins inter- and intra-molecularly. Since reducing sugars are found ubiquitously throughout the body, molecules with long half-lives have a higher probabihty to undergo nonenzymatic glycosylation. The amount of AGE formed is dependent on both sugar concentration and length of exposure. The nonenzymatic glycosylation of proteins has been observed in older individuals and is accelerated in patients with diabetes. Proteins which have undergone nonenzymatic glycosylation are

0165-1110/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

186

H,,c¢O CH=OH Glucose

+

NH2 Protein

~NH.

,'-.

"-'T'--

NH CH= ,C=0 (CHOH)s CH=OH

C,H (,CHOH), CH=OH ~¢hiff Base (Aidim~ne)

~

and concentration dependent loss in transfection capability of the phage D N A (Fig. 3). In these experiments, G-6-P resulted in a more dramatic

1.0

pLR~ ~_1~( Ketoom;ne}

~,Kn

0.75

ADVANCED GLYCOSYLATIONENDPRODUCTS (BROWN FLUORESCENTPIGMENTS WHICH CROSSLINK PROTEINS)

Fig. 1. Nonenzymatic reaction of glucose and proteins.

0.50 physically altered and in some instances, function is also compromised. Advanced glycosylation endproducts have been observed on proteins in vivo such as lens crystallins and collagen as well as other long lived molecules (Schnider and Kohn, 1980; Monnier and Cerami, 1981; Brownlee, Vlassara and Cerami, 1984). The reaction of reducing sugars with proteins is dependent on the formation of a Schiff base. Several years ago it was hypothesized that the aldehyde of reducing sugars could also react with the amino groups of D N A bases in a manner analogous to the reaction with proteins. In vitro incubations of D N A or nucleotides with glucose 6-phosphate (G-6-P) resulted in the formation of pigments which had absorbance and spectral properties that were similar to those of proteins which have been nonenzymatically glycosylated (Bucala, Model and Cerami, 1984). These spectra showed increased absorbance in the 300-400 nm range. It was observed that the reaction occurred at a faster rate with single-stranded D N A than with double-stranded DNA. Furthermore, there was no significant reaction of the reducing sugar with thymidine, which demonstrated the requirement for a free amino group on the nucleotide for the reaction to occur. These results suggested that the free amino groups of nucleotides or D N A were able to react with G-6-P in a manner similar to that observed with proteins (Fig. 2). The biological effects of the reaction of glucose and G-6-P were tested by studying the capacity of fl phage DNA to transfect E. coil (Bucala, Model and Cerami, 1984). The incubation of fl phage D N A with either glucose or G-6-P led to a time

G6P

~C°

Go

0.2! -

u l-

1.0 o 09 ..o

B

<

0.75

0.50 ~\

G6P

0.2!

ds DNA~ ss DNA,

ds D N A / ~

250

300

~'~" ..SS DNA~ .

350

400

450

Wavelength, nm Fig. 2. UV and visible spectra of nucleotides and ss and ds DNA after 4 days of incubation at 37 o C. (A) G-6-P alone (1 M G-6-P dissolved in 50 mM Hepes/0.5 mM EDTA, pH 8.0); A, C, G, T, nucleotides alone (10 mM); Ag, Cg, Gg, Tg, nucleotides incubated with G-6-P. (B) G-6-P alone (1 M G-6-P dissolved in 50 mM Hepes/0.5 mM EDTA pH 8.0); ss and ds DNA (2 mg/ml); ss DNAg and ds DNAg, ss and ds DNA incubated with G-6-P. (From Bucala et al., 1984.)

187

104/

,

i

i

,

i

,

",

+"'',,,t

o"

10z

}

lo

~

~

~

~

1'o

t~

Incubation time, days

Fig. 3. Rate of fl DNA inactivation. Each transfection assay contained 2 /~g of fl phage DNA containing: 25 mM G-6-P (o), 25 mM glucose ([n]), 25 mM G-6-P/5 mM N-a-tertbutoxycarbonyl(boc)-lysine (o), 25 mM glucose/5 mM boclysine ([liD, boc-lysine alone ( × ) dissolved in 50 mM Hepes/0.5 mM EDTA, pH 8.0. Following the incubation period, duplicate samples were transfected into E. coil (from Bucala et al., 1984).

decrease in transfection capacity than incubations with glucose, reflecting the increased rate of reaction of G-6-P compared to glucose. Similar results have also been observed with the reaction of these sugars with proteins. Presumably, this faster rate of reaction of G-6-P is due to the fact that G-6-P is more likely to be in the open-chain form, thus exposing the aldehyde and allowing for Schiff base formation and subsequent rearrangements. Glucose is more often in a stable cyclic structure, and the aldehyde is unable to react with the amino groups to form the necessary Schiff base. The inclusion of lysine in the incubations with glucose or G-6-P led to an additional decrease in transfection potential. Lysine was included in the incubations because the primary groups of lysine should be more reactive than the amino groups of D N A and accelerate the Maillard reaction. The inclusion of lysine in the incubations of glucose and fl phage D N A initially had little effect, but, following a lag period, there was a dramatic decrease in transfection capability. Similarly, incuba-

tions of G-6-P in the presence of lysine first protects the phage DNA, but then accelerates the reaction of the reducing sugar with the D N A and reduces the transfection efficiency of the phage D N A (Fig. 3). These results suggested that G-6-P first reacts with the amino groups of lysine to form an intermediate which can then react with DNA. Further investigations of the reaction of G-6-P, lysine and D N A were performed. It was found that following a lag period, the incubation of DNA, [3H]lysine and G-6-P led to the time- and sugar-concentration-dependent crosslinking of [3H]lysine to D N A (Lee and Cerami, 1987a). These results also suggested the presence of an intermediate formed during the incubation of G-6-P and lysine. To determine if an intermediate was formed during these incubations, G-6-P and [3H]lysine were preincubated in the absence of DNA, which was then added followed by an additional incubation period. The amount of [3H]lysine associated with trichloroacetic acid (TCA) precipitable D N A was used as an indicator of crosslink formation. These studies revealed that the preincubation of G-6-P and [3H]lysine results in the time-dependent accumulation of a reactive intermediate which can react completely within 1 h after addition to the DNA. This demonstrates the ability of the preformed intermediates to react rapidly with the D N A to form acid-stable complexes. Additions of solutions of DNA, G-6-P or [3H]lysine which had been preincubated separately for 4 days did not lead to incorporation of radiolabel when followed by an incubation for 1 h with the missing components (Table 1). Only preincubations of G-6-P and [3H]lysine led to the formation of an intermediate which could crosslink to DNA. Further characterization of these intermediates showed that they are capable of rapidly reacting with both double- and single-stranded D N A and require the presence of an amino group on the nucleotide for significant adduct formation. The formation of these reactive intermediates occurs in a concentration- and time-dependent manner. Inhibition studies have shown that aminoguanidine, which has been shown to prevent protein crosslinking (Brownlee et al., 1986) can prevent the formation of the reactive intermediate if it is present at the onset of the preincubation

188 TABLE 1 THE PRESENCE OF GLUCOSE 6-PHOSPHATE AND LYSINE IN THE PREINCUBATION MIXTURE IS NECESSARY FOR THE FORMATION OF REACTIVE INTERMEDIATES From Lee and Cerami, 1987a. 4-day prein- 60-minincubation cubation at 37 ° C b at 37 ° C

(#moles [3H]lysine per/~mole DNA bases) )< 1 0 9

G-6-P

+ (DNA + [3H]lysine)

3.6

[3H]lysine

+ (G-6-P + DNA)

2.2

DNA

+ (G-6-P + [3H]lysine)

2.8

G-6-P+ [3H]lysine

+ (DNA)

100.0

A 1-rnl solution of 1 M G-6-P, 20 #Ci of [3H]lysine in 0.1 M Hepes, E. coli DNA or 1 M G-6-P and 20/~Ci of [3H]lysine were incubated at 37 °C for 4 days. b 1 ml of E. coli DNA and 20/~Ci of [3H]lysine, E. coli DNA in 1 M G-6-P, 20/~Ci of [3H]lysine in 1 M G-6-P, or E. coli DNA was added as indicated to the 4-day preincubation mixture for 60 re.in prior to TCA precipitation.

a

period, but it is ineffective once the intermediate has formed. Reduction of the reactive intermediate with sodium borohydride does not affect its further reaction with DNA, suggesting that the intermediate lacks a carbonyl moiety and has ad~ vanced beyond the Amadori product stage. At present the chemical nature of the reactive intermediate is unknown. Currently, the structures of only a few of the rearrangement products of glucose or G-6-P with proteins have been identified, one of which is 2(2-furoyl)-4(5)-(2-furanyl)1H-imidazole (FFI). This product presumably arises by the condensation of two Amadori products to form an imidazole with subsequent ring closure and dehydrations to form the two furan rings (Pongor et al., 1984). Further investigations are in progress to determine the structure of the reactive intermediate and the adducts formed upon reaction with DNA. • The discovery of the reactive intermealates formed by the reaction of G-6-P and lysine led to the proposal that the nonenzymatic reaction in vivo of reducing sugars with D N A directly or through a protein-bound equivalent could ad: versely affect the functioning of D N A and may

contribute to D N A damage and mutations. Previous work done by Bucala et al. (1985) demonstrated that the incubation of plasmid pBR322 D N A and G-6-P led to a time- and concentrationdependent decrease in transformation efficiency, analogous to the decreases observed with incubations of fl phage D N A . In addition, it was found that a number of the transformants recovered had various plasmid mutations. These mutations were primarily the result of insertions and deletions of the plasmid DNA. Of particular interest was the finding that plasmid mutations occurred when glycosylated plasmid D N A was transformed into a repair-proficient E. coli host strain. N o plasmid mutations were found when plasmid D N A which had been glycosylated in vitro was transformed into a repair-deficient host ( u v r C - ) suggesting that the plasmid mutations observed were due to the error prone attempt of the bacteria to repair the damaged DNA. To further study the in vivo effects of D N A glycosylation we developed a model system which took advantage of two E. coli glycolytic mutants, DF40 and DF2000 which lack phosphoglucose isomerase or phosphoglucose isomerase and G-6-P dehydrogenase, respectively (Lee and Cerami, 1987b). These mutations affect the utilization of glucose as a carbon source. Mutations which affect phosphoglucose isomerase interfere with the conversion of G-6-P to fructose 6-phosphate which prevents further steps in glycolysis. G-6-P dehydrogenase converts G-6-P to 6-phosphogluconolactone, which is necessary if G-6-P is to be metabolized by the E n t e r - D o u d o r o f f pathway. Thus, these two mutants grow well with gluconate as their sole carbon source but accumulate intracellularly the reducing sugar G-6-P, when glucose is also present in the medium. Table 2 shows the concentration of intracellular G-6-P levels found in each strain when grown for a 24-h period in minimal medium containing ratios of 9 : 1, 7 : 3 and 1 : 1 of glucose to gluconate. At the 9 : 1 ratio the DF40 strain accumulated approx. 20 times more G-6-P than the control K10 strain, while DF2000 accumulated approx. 30 times more. When the concentration of G-6-P was analyzed in the K10, DF40 and DF2000 strains after growth on gluconate minimal medium no significant accumulation of G-6-P was detected.

189 TABLE 2 G L U C O S E 6 - P H O S P H A T E LEVELS F O U N D IN G L U C O S E / G L U C O N A T E OR G L U C O N A T E A L O N E

CELLS

GROWN

IN

MINIMAL

MEDIUM

CONTAINING

From Lee and Cerami, 1987b. Strain

Glucose 6-phosphate,/tmoles per 5 × 109 cells

Gluconate alone b

Glucose/gluconate 9:1 a

7:3 b

1:1 b

K10

0.028 + 0.005

0.018 + 0.003

0.018 + 0.003

0.030 + 0.003

DF40

0.553 + 0.072

0.348 + 0.023

0.164 + 0.059

0.005 + 0.002

DF2000

0.864 + 0.011

0.632 + 0.037

0.323 + 0.080

0.004 + 0.001

Overnight cultures of each strain grown in either gluconate or glucose/gluconate minimal medium were diluted to 108 ceils per ml. Diluted culture (50 mi) was treated with perchloric acid and then assayed for glucose 6-phosphate content. a The results are means of triplicate experiments + S.D. b The results are means of duplicate experiments + S.D.

It was postulated that a target plasmid carried in the strains which accumulated elevated levels of G-6-P would be susceptible to D N A damage by the reducing sugar and result in D N A mutations in an analogous manner to plasmid D N A glycosylated in vitro. To investigate this possibility, these strains were transformed with the plasmid pAM006 which carries the genes for ampicillin resistance, fl-galactosidase and lactose permease production. Following a 24-h growth period minimal medium containing gluconate alone or a 9 : 1 ratio of glucose to gluconate, plasmid D N A was isolated from each strain and used to transform a L a c - indicator strain. The transformants were selected for ampicillin resistance and screened for fl-galactosidase production on MacConkey lactose indicator plates supplemented with ampicillin. Those colonies which were ampicillin resistant but exhibited a L a c - phenotype were scored as mutants. The relative rate of mutations is given by the ratio of the number of mutants found in the test strain (DF40 or DF2000) divided by the number of mutants found in the control strain (K10) per 103 transformants. As shown in Table 3, the number of mutants increased proportionately in those strains which accumulate G-6-P. The relative number of mutants increased approx. 7-fold with plasmid D N A isolated from the DF40 strain and approx. 13-fold with plasmid D N A isolated from the DF2000 strain. This relationship is dependent on the elevated intracellular

levels of G-6-P found in the mutant strain since no increase was observed when the strains were grown in the absence of glucose. Analysis of the mutated plasmids isolated from the K10, DF40 and DF2000 strains demonstrated that the mutations were the result of various large insertions and deletions ( > 1 kb) as well as smaller

TABLE 3 RELATIVE M U T A T I O N RATES OF CELLS G R O W N IN GLUCONATE ALONE OR GLUCOSE/GLUCONATE MINIMAL MEDIUM From Lee and Cerami, 1987b. Strain

Relative lac--mutagenesis per 105 transformants Glucose/gluconate ~

Gluconate alone b

K10

0.67+0.47

0.5!0.5

DF~

4.84+0.65 c

1.0+1.0

DF2000

8.71+1.24 c

1.5+0.5

Plasmid D N A (50 ng) isolated from cultures grown in gluconate or glucose/gluconate minimal medium was used to transform SB4288 competent cells. Colonies that were Amp R but had a L a c - phenotype were scored as mutants. Relative mutagenesis was determined by the ratio of mutants found in the mutant strains (DF40 or DF2000)/control strain (K10). a The results are means of triplicate experiments + S.D. The results are means of duplicate experiments + S.D. c The difference in mutation rate between K10 and the mutant strains DF40 and DF2000 is statistically significant ( P <

0.0001).

190 TABLE 4 PLASMID SIZE CHANGES From Lee and Cerami, 1987b. Fraction of plasmid size changes K10 DF40 DF2000 >1 kb increase 3/12 (25%) 24/78 (31%) 75/153 (49%)

Size change

> 1 kb decrease 8/12 (67%) 54/78 (69%) 27/153 (18%) < 1 kb change in either direction 1/12 (8%) 0/78 (0%) 51/153 (33%) Plasmid DNA from phenotypic AmpR/Lac- colonies was isolated and linearized with Xbal. Samples were treated with RNAase A and visualized on a 1% agarose gel containing ethidium bromide at 1 #g/ml. Mutated plasmids from each strain were grouped as indicated, and the percentage of the detectable size changes is in parentheses.

size changes ( < 1 kb). Table 4 summarizes the distribution of plasmid size changes observed in the mutated plasmids from each strain. The background mutations in the K10 control strain and the plasmid mutations which originated from the DF40 strain show a predominance in plasmid size decreases. The mutations in plasmid D N A isolated from the DF2000 strain appear to be distributed mainly between minor size changes and plasmid size increases. The mechanism(s) behind the occurrence of plasmid size changes in the mutated plasmids is unknown. The possibility that these mutations may be the result of the induction of the SOS D N A repair/recombination system was investigated by comparing the amount of RecA protein present under conditions which produced plasmid mutations. Western blot analysis of the amount of RecA present in the 3 strains grown in minimal medium containing gluconate alone or in the presence of glucose, failed to demonstrate detectable differences in the amount of RecA protein present in cellular extracts. It is possible that D N A damage mediated by reducing sugars activates other error-prone repair systems or recombinational pathways. These possibilities are under current investigation. The formation and accumulation of glucose-derived D N A adducts may contribute to some of the

D N A dysfunctions observed with the onset of aging. Reducing sugars or reactive intermediates formed by reducing sugars and polyamines or protein bound equivalents could lead to genetic damage associated with aging. It is easy to imagine how G-6-P mediated crosslinking of amino groups of proteins could account for a portion of the increased amounts of proteins covalently attached to D N A in aged cells (Bojanovic et al., 1970). Other age-related changes in genetic material such as increase in DNA-strand breakage (Price, Modak and Makinodan, 1971) and decreases in D N A replication (Petes et al., 1974), D N A repair (Karran and Ormerod, 1973) and transcription (Berdyshev and Zhelabovskaya, 1972) and the increase in the incidence of cancer (Bennington, 1986) could be partly attributed to the reaction of reducing sugars with D N A . Recent studies have provided evidence that insertional mutations have resulted in the activation of cellular oncogenes (Rechavi, Givol and Cananni, 1982; Cohen et al., 1983; Morse et al., 1988). It will be an exciting prospect to determine whether D N A damage due to reducing sugars influences the increased incidence of cancer which has been associated with aging. Although D N A glycosylated by reducing sugars or reactive intermediates of reducing sugars and polyamines has not been identified in vivo, it seems possible that cells which have little to no turnover would accumulate these D N A adducts with time. Extrapolation of the results from in vitro studies with plasmid D N A has led to the postulation that the human genome would be subjected to 3 'hits' per day per diploid cell by G-6-P under physiological conditions (Bucala et al., 1985). The authors have pointed out that this is most likely an underestimate, since other reducing sugars would also contribute to D N A glycosylation. The elucidation of the reaction of reducing with the amino groups of DNA, in both in vitro and in vivo model systems, has revealed the potential of these otherwise innocuous molecules to influence the integrity of DNA. Further investigations will no doubt need to examine the induction of DNA mutations by reducing sugars in mammalian cells, and its relationship to D N A damage in general and those observed in aged cells.

191

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