Non-enzymatic glycation of antithrombin III in vitro

340

Biochimica et Biophysica Acta 964 (1988) 340-347 Elsevier

BBA 22887

N o n - e n z y m a t i c g l y c a t i o n of a n t i t h r o m b i n I I I in vitro T a m i k o S a k u r a i *, J e a n - P a u l Boissel a n d H. F r a n k l i n B u n n Laboratory of the Howard Hughes Medical Institute, Hematology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (US.A.) (Received 13 April 1987) (Revised manuscript received 6 November 1987)

Key words: Nonenzymatic glycation; Glycation; Antithrombin III; (Human plasma)

Non-enzymatic glycation of antithrombin III (AT-Ill) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37°C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 moi glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [t4C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14Clglucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.

Introduction Glucose and other reducing sugars react nonenzymatically with proteins in vivo to form stable covalent adducts [1-4]. The aldehyde (or ketone) function condenses with amino groups on proteins via a Schiff-base linkage. This aldimine product

* Present address: T. Sakurai, Tokyo College of Pharmacy, Tokyo, Japan. Abbreviations: AT-III, antithrombin III; TPCK-trypsin, L(tosylamido-2-phenyl)ethyl chloromethylketone-treated trypsin. Correspondence: H.F. Bunn, Laboratory of the Howard Hughes Medical Institute, Hematology Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, U.S.A.

can then rearrange to a highly stable ketoamine linkage. The extent to which a given protein is modified in vivo is a function of the local concentration of glucose as well as the lifespan of the protein [5,6]. Accordingly, a large number of proteins in patients with diabetes mellitus undergo increased non-enzymatic glycation, in direct proportion to the elevation in blood glucose. One of the central questions in diabetes research is the extent to which these modifications alter protein function and whether such changes contribute significantly to the long-term complications of the disease. Antithrombin III (AT-III) is the principal physiological inhibitor of blood coagulation [7]. Its activity is increased as much as 4000-fold by heparin [8]. Hereditary deficiency of AT-III [9] and certain functionally abnormal AT-III mutants

0304-4165/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

341 [10-12] are associated with a markedly increased risk of developing thromboses. Patients with diabetes are also prone to a significantly increased incidence of thromboses. Some investigators have proposed that the hypercoagulable state in diabetes is due in part to a functional impairment of AT-Ill resulting from non-enzymatic glycation [13-18]. However, experimental support for this hypothesis has not been persuasive. Published measurements of the levels of AT-Ill in diabetics vary widely [19-23]. Furthermore, there are conflicting reports as to whether AT-Ill activity is impaired in diabetics [22-24]. We have addressed this issue by studying the structural and functional properties of AT-Ill that has been glycated in vitro.

lide (Boehringer-Mannheim)) and et-thrombin (Sigma) were used. The absorbance at 405 nm was monitored. Heparin-binding activity was measured by the maximum enhancement of fluorescence (AF) of tryptophan residues when AT-Ill was titrated with heparin (Calbiochem, porcine mucosa, 168 U/mg) [27]. The excitation wavelength was 295 nm and the emission wavelength was 330 nm. Heparin-binding activity was also determined by chromatography on a heparinSepharose column (1.5 x 6 cm) at 4°C. The column was eluted first with 50 mM Tris (pH 7.7)/0.1 M NaC1 and then with the same buffer in 1.0 M NaC1. Protein concentration was estimated by absorbance at 280 nm utilizing an absorption coefficient (1%) of 6.5 [27].

Materials and Methods

Incubation conditions Initial experiments compared three incubation solutions (pH 7.7): 0.5 M phosphate; 0.5 M phosphate + 0.1 M NaC1; and 0.1 M phosphate + 50 mM NaC1. As explained in Results, the third buffer was selected for all subsequent experiments. Solutions of AT-III in this buffer containing 0-0.5 M glucose were sterilized by passage through a 0.45 /~m Millipore filter and then incubated for up to 3 days at 37°C. Following dialysis, the extent of non-enzymatic glycation of the incubated AT-Ill was determined by phenylboronate affinity chromatography (PBA 10, Amicon). The column was developed first with 25 mM phosphate (pH 8.5) and then with the same buffer which also contained 200 mM sorbitol. When incubations employed labeled glucose, [U14C]glucose (New England Nuclear) was first purified as previously described [28]. [laC]glucose (1.68 mCi) and unlabeled glucose were added to a solution of AT-Ill (100 mg/10 ml) so that the glucose concentration was 100 mM (comparable to a physiological concentration of 5 mM). Following incubation for 3 days at 37 ° C, the solution was dialysed against 20 mM ammonium formate (pH 7.7) and lyophilized prior to structural studies. Approximately 0.7 mol of glucose were incorporated per mol of AT-Ill.

Preparation of A T-III Recovered plasma, anticoagulated in citrate, was obtained from the local collecting center of the American Red Cross. AT-III was purified according to the method of Jorgensen et al. [25]. Affinity chromatography on heparin-Sepharose CL-6B (Pharmacia Fine Chemicals) was followed by ion-exchange chromatography on DEAE-Seph: adex A-50 (Pharmacia Fine Chemicals). The main peak, which eluted with 50 mM Tris-HC1 (pH 7.7)/0.25 M NaC1, corresponded precisely with thrombin-inhibitory activity. This peak, when concentrated, showed one M r 59000 band on SDSpolyacrylamide gel electrophoresis. In contrast, multiple bands appeared on isoelectric focusing (LKB ampholine polyacrylamide-gel plates, pH range 4-6). The pI of the main band was 5.3. The main DEAE-Sephadex peak was dialysed against 0.1 M phosphate/50 mM NaC1 and stored at 4°C for no more than 30 days. An average yield of 140 mg was obtained from 2 1 of plasma. Functional studies Thrombin-inhibitory activity was assayed according to Handeland [26] in the absence of heparin, with modifications that minimized protein adsorption to glass. All solutions for assay were prepared in 0.1% poly(ethylene glycol) 6000 (Sigma). A chromogenic substrate, 0.6 mM Chromozym TH (tosylglycylprolylarginine p-nitroani-

Structural analysis Lyophilized protein was reduced and carboxymethylated as follows. After dissolving (20 mg/ml)

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in 0.5 M T r i s / 6 M guanidinium chloride/2 m M EDTA, the solution was adjusted to p H 8.1 and flushed with N 2 at 50 ° C for 30 min. After the addition of dithiothreitol at a 50-fold molar excess over the disulfide content of the AT-III, the solution was again flushed with N 2 and incubated for 4 h at 50°C. In the dark, a 1.1 M solution of monoiodoacetate, adjusted to p H 8.1 with N a O H , was added in 2-fold molar excess to the dithiothreitol. The p H of the A T - I l l solution was again adjusted to 8.1 with N H 4 O H . After the addition of 2-mercaptoethanol (1% final concentration), the solution was dialyzed against a m m o n i u m acetate (pH 7.7) and lyophilized. A 250 m M solution of cyanogen bromide in 70% formic acid was added at a 100-times molar excess of methionine residues at AT-III, and incubated for 24 h at 4 ° C . To remove the cyanogen bromide, the solution was applied to a G-15 Sephadex column having a void-volume M r exclusion of greater than 1500. Only two cyanogen bromide peptides of A T - I l l have an M r lower than 1500, and these are irrelevant since they lack lysine residues. The void fraction containing 30 mg peptide and 80% of the 14C radioactivity was concentrated to 5 ml and analysed on a 2.5 × 90 cm column of G-50 Sephadex (fine) equilibrated with 0.1 M formic acid at a flow rate of 6 m l / h . Absorbance at 230 nm and 14C radioactivity were measured on 1.9 ml fractions. Isolated cyanogen bromide fragments were digested with trypsin. Following lyophilization, 1 mg aliquots were dissolved in 300 /~1 of ammonium acetate buffer ( p H 7.5). 25 #g of T P C K trypsin (1 m g / m l in 0.001 M HC1) were added and the p H was adjusted to 7.5. After 1 h, another 25/~g of trypsin were added and the solution was stirred overnight at room temperature. Digestion was stopped by heating for 5 min at 100 ° C. After 200 ~tl of digested fragment had been dried under nitrogen, it was dissolved in 0.25 M a m m o n i u m acetate buffer (pH 8.5) and applied to Affigel 601 equilibrated with the same buffer. The proportions of fragments II, III, IV and V that adhered to this boronate affinity resin were 70%, 65%, 43% and 80%, respectively. These glycated peptides were eluted with 0.1 M formic acid, dried, dissolved in 0.1% trifluoroacetic acid and applied to an H P L C reverse-phase column (C18, 4 × 250 m m )

and developed with an acetonitrile gradient at a flow rate of 1.5 m l / m i n . Results and Discussion In order to modify AT-III by in vitro glycation under physiologic conditions, it was necessary to subject solutions to prolonged incubations at 37°C. Initial experiments tested the effect of incubations for up to 9 days on the stability and

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Incubation Time (Days) Fig. 1. Effect of incubation at 37 ° C on (A) thrombin inhibition and (B) heparin-binding activity. 2 ml of 17 # M AT-Ill was incubated for 0 - 9 days in three media: m, 0.1 M phosphate-buffered saline/50 m M NaC1; 12, 0.5 M phosphatebuffered saline; &, 0.5 M phosphate-buffered saline/0.1 M NaC1. After sampling each day, they were stored at 4 o C until assay. In (A), chromogenic substrate was used as mentioned in the text. In (B), AT-III was diluted to 0.1 /zM by 0.1 M phosphate-buffered saline/50 m M NaC1 and 2 ml of it was titrated by heparin (6.4 m g / m l , dissolved in water) at 37 o C. A F 0 is the m a x i m u m increase in fluorescence without incubation. A F is fluorescence obtained by each incubation.

343

function of AT-III. Fig. 1 shows thrombin inhibition in the absence of heparin (top panel) and heparin binding (bottom panel). Prolonged incubation resulted in a nearly linear decrease in both antithrombin activity and heparin binding. During the first 3 days of incubation, these properties were not significantly affected by the ionic strength of the buffer solution. Thrombin inhibition had fallen by only 10% while heparin binding had decreased by 30%. Since the half-life of AT-Ill in the human circulation is 2.8 days [29], we selected an incubation period of up to 3 days and utilized the most 'physiologic' of the three buffers tested, 0.1 M phosphate and 50 mM NaC1. The affinity of AT-Ill to heparin was examined by heparin-Sepharose affinity chromatography. It was affected both by the incubation per se and the presence of glucose in the incubation medium. Nearly all (96%) of the non-incubated AT-Ill adhered to the column. After 2 days' incubation in the absence of glucose, adherence dropped to 75%. This decrease agreed well with the fluorometric measurement of heparin binding, shown in Fig. lB. The presence of glucose in the incubation medium further decreased the adherence of AT-Ill

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to the heparin-Sepharose column. In 0.1 M glucose, the binding decreased to 69%, while in 0.5 M glucose, only 56% of the AT-III still adhered to the affinity column. These results suggest that incubation with glucose caused a modest decrease in heparin binding. In order to determine whether non-enzymatic glycation was responsible for this decreased binding to heparin, the incubated AT-III solutions were analysed by boronate affinity chromatography (as shown in Fig. 2). In an AT-III solution incubated for 3 days in 150 mM glucose, 24% of the protein adhered to the affinity resin. In contrast, when glucose was omitted from the incubation medium, only 3.5% adhered. When another enzymatically glycated protein, transferrin, was applied to the phenylboronate column, only 4% adhered. These findings strongly indicate that enzymatically linked carbohydrate does not bind significantly to phenylboronate. Thus, the phenylboronate affinity column is effective in distinguishing between enzymatic glycation, an early post-translational event, and non-enzymatic glycation, a much later post-translational event. Measurements employing [14C]glucose indicate

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344

that the phenylboronate column binds only a portion of the non-enzymatically modified AT-III. In the experiment depicted in Fig. 2, a total of 0.67 mol of glucose were incorporated into 1 tool of protein, and yet only 24% of AT-Ill remained adherent to phenylboronate. As shown in Fig. 2, the fraction of protein that failed to bind strongly was slightly retarded compared to the non-incubated AT-Ill. This was a highly reproducible finding and probably reflects a class of non-enzymatically glycated sites on AT-Ill that bind weakly to the resin. The proportion of glycated AT-III that bound strongly to the phenylboronate resin was increased over 2-fold by reductive alkylation of disulfide groups of AT-Ill. It is likely that this structural modification unfolds the protein and therefore allows more glycated sites on AT-III to have access to the phenylboronate groups. Measurements o f [ 1 4 C ] g l u c o s e incorporation showed that the material that adheres to the phenylboronate contained 0.6-0.8 mol of glucose per mol of AT-III. It is unclear why this ratio was less than the expected value of 1.0. In order to investigate the functional properties of glycated AT-III, the fraction of incubated ATIII that adhered to phenylboronate was applied to

a heparin-Sepharose column. As shown in Fig. 3, non-enzymatically glycated AT-III clearly has heparin-binding activity. 56% of this protein bound to the heparin affinity column, compared to 65% of the material which did not adhere to phenylboronate. Nearly the same percentage (66%) was obtained when material that was incubated in a glucose-free medium and passed through phenylboronate was analysed on a heparin affinity column. These measurements suggest that only a small proportion (approximately 9%) of heparin binding sites on AT-III are lost because of nonenzymatic glycation. The AT-III activities of the non-enzymatically glycated and the non-glycated preparations was assessed by the inhibition of thrombin cleavage of chromogenic substrate. No difference was found (data not shown). Taken together, these results indicate that non-enzymatic glycation causes no significant impairment in the function of AT-III. In assessing whether non-enzymatic glycation of AT-III is likely to affect its function, it is important to determine whether specific sites are modified. Fig. 4 shows the gel-filtration elution profile of cyanogen-bromide-cleaved carboxymethylated fragments of AT-III following a 3-day

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incubation in 100 mM [14C]glucose. Eight peaks were obtained, in good agreement with the analysis of Koide et al. [11]. The primary structures of each peptide are shown in Fig. 5. A significant amount of 14C radioactivity was found in every peptide. Considerable attention has focussed on the heparin-binding sites on AT-III [12,31-36]. An a-helical segment containing lysines 290, 294 and 297 appear to interact electrostatically with three sulfate groups on the octasaccharide [31]. These residues are located in cyanogen-bromidecleaved fragment V. On the other hand, the region around Trp-49 near the N-terminus also plays a role in heparin binding [12,31,32]. This region is located in cyanogen-bromide-cleaved fragment III.

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Atha et al. [33] proposed that the 3-O-sulfate and 6-O-sulfate groups of glucosamine residues within the heparin octasaccharide interact with this region in fragment III and an a-helical segment in fragment V, inducing a conformational change in AT-Ill. The proportion of total 14C activity in fragments III and V were 13% and 14%, respectively, Fig. 6 shows the HPLC analyses of the boronate-absorbed tryptic peptides of cyanogenbromide-cleaved fragments II, III, IV and V. In each case, the radioactivity was distributed rather evenly over the lysine residues in each fragment. No highly favored sites of non-enzymatic glycation were detected. Modifications on fragment V may account for the above-mentioned modest decrease in heparin binding that can be attributed to non-enzymatic glycation. However, any conclusions about the effect of glycation of specific sites on heparin binding are very tenuous. The lack of site-specificity of glycation of ATIII poses an interesting contrast to other proteins in which there are favored sites. In human serum albumin, Lys-525 is by far the most frequently glycated residue both in vivo [37,38] and in vitro [39]. In hemoglobin, the most favored sites are the N-terminal amino groups of the a and/3 chains as well as a-Lys-61 and/3-Lys-66 [40]. It is of interest that Lys-525 in albumin as well as the two lysine residues in hemoglobin are all on the carboxyl side of Lys-Lys sequences. (AT-Ill has a Lys-Lys sequence at positions 28-29, but our analyses indicate that it is not a favored site for non-enzymatic glycation.) In addition, when ribonuclease A is glycated in vitro, a restricted group of lysine residues is modified [41]. In all three proteins, it is likely that these favored sites arise because local acid-base catalysis favors the Amadori rearrangement. Our structural studies of AT-Ill indicate that the sites of non-enzymatic glycation are distributed non-specifically over the entire molecule. Therefore, it is not surprising that glycated AT-Ill, having one mol glucose per mol protein, has nearly normal heparin binding. (In the AT-III preparation containing 0.67 mol glucose per mol protein, a small proportion of molecules would have 2 mol glucose bound non-enzymatically.) Rosenberg and Damus [42] have shown that extensive modification of lysine c-amino groups is required to ablate

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