Synthesis and quantitation of glucitollysine, a glycosylated amino acid elevated in proteins from diabetics

ANALYTICAL

BIOCHEMISTRY

119, 330-334

(1982)

Synthesis and Quantitation Amino Acid Elevated BEAT TR~EB, GRAHAM Laboratorium

fiir

of Glucitollysine, a Glycosylated in Proteins from Diabetics

J. HUGHES,

AND

KASPAR H. WINTERHALTER

Biochemie I, Eidgeniissische Technische ETH-Z, CH-8092. Ziirich, Switzerland Received

Hochschule.

June 25, 1981

A method for preparative synthesis of glucitollysine in high yield is described. It is shown that this compound is useful as a standard for the determination of nonenzymatic glycosylation of proteins. .

The demonstration that glucose binds nonenzymatically to a variety of proteins, including hemoglobin ( 1 ), albumin (2) lens crystallins (3), and collagen (4) has established that nonenzymatic glycosylation is a common posttranslational modification of proteins in viva. Prolonged elevation of blood glucose in diabetes mellitus is known to cause an increase in the levels of glycosylated hemoglobin (1) and albumin (5,6). Exact and unequivocal quantitation of nonenzymatic glycosylation is of great importance; this modification may induce changes in the biological properties of crucial proteinse.g., in the wall of blood vessels-and thus contribute to the progressive complications of diabetes. The amount of glycosylated hemoglobin is routinely measured by column chromatography, thiobarbituric acid color reaction, or isoelectric focusing (7,8). A radioimmunoassay has also been described (9). For other proteins, radioactive labeling techniques are commonly utilized to estimate the extent of nonezymatic glycosylation. Radioactivity is incorporated either by reduction of the ketoamine bond between glucose and protein with tritiated NaBH4 (3) or by incubation of the isolated polypeptides with radioactive sugars (2,3,10). Both methods have serious disadvantages, because incorporated radioactivity does not directly cor0003-2697/82/020330-05502.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

relate with nonenzymatic glycosylation. In the latter case, radioactively labeled sugars from several manufacturers have been shown to contain radioactive contaminants, which bind strongly to proteins and thus simulate nonenzymatic glycosylation ( 11,12); in the former case, NaBH,, was reported also to reduce other sites in proteins and even cleave peptide bonds leading to the formation of a-aminoalcohols ( 13,14). Therefore, a good way to evaluate nonenzymatic glycosylation is direct chemical determination of the amino acid glucitollysine’ resulting from binding of glucose to the c-amino group of lysine.2 We report here synthesis and characterization of glucitollysine as a useful standard in amino acid analysis of glycosylated proteins. It may permit exact determination of the extent of nonenzymatic glycosylation at c-amino groups of lysine in any protein. ’ Abbreviations used: Glucitollysine, c-N-( I-deoxy-Dglucitol-1 -yl)L-lysine or I-L-lysino1-deoxy-o-sorbitol (Lysinodeoxysorbitol is probably a more appropriate abbreviation. We pefer, however, the abbreviation glucitollysine, because this term is widely used in the literature, e.g., Refs. (2,4,11)); Z-, benzyloxycarbonyl-. 1 Reaction of glucose with the t-amino group of lysine yields, after Amadori rearrangement, c-N- l( l-deoxyfructosyl)lysine. Glucitollysine is obtained upon reductive stabilization of this compound. 330

QUANTITATION

OF NONENZYMATIC

331

GLYCOSYLATION

identified as Z-glucitollysine and was lyophylized. The third small peak eluted near Materials. Chemicals were of the highest the total volume of the column and was idenpurity available from Fluka or Merck. D-[ U- tified as unreacted Z-lysine. “C]glucose (336 mCi/mmol, batch 111) Removal of the protecting group was perwas obtained from the Radiochemical Centre formed by catalytic transfer hydrogenation (Amersham). (15). Under nitrogen, 10% palladium-carSynthesis of glucitollysine (Fig. 1). (Y-N- bon ( 10 mg) and 1,4-cyclohexadiene ( 1.2 ml, 12.7 mmol) were added to a solution of ZZ-L-Lysine (1.5 g, 5.4 mmol), D-glucose (4.9 g, 27 mmol), and NaBH,CN (1.7 g, 27 glucitollysine (1 .O g, 2.3 mmol) in 5% (v/v) mmol) were dissolved in 55 ml 0.2 M sodium acetic acid (40 ml). Incubation was perphosphate, pH 8, and incubated for 17 days formed at 25°C with vigorous agitation (viat room temperature. Dowex 50 WX8 (75 bromixer). A constant stream of nitrogen g, wet resin) was then added and the sus- was passed through the reaction mixture. pension mixed overnight in a hood (Hz and After 2 and 4 h, respectively, the initial HCN evolve). The resin was packed into a amounts of catalyst and diene were again glass column, washed with 4 liters of water added. The reaction was allowed to proceed and then eluted with 0.6 N NH40H. The for a total of 6 h and the mixture was then effluent was continuously monitored at 254 filtered through Celite and subsequently through Millipore and lyophilized. nm, the absorption maximum of the Z group. Z-Glucitollysine eluted as soon as the resin Pure glucitollysine was obtained with a was completely converted to its ammonium yield of 7990 based on weight after extensive form and changed its color from orange to drying over PzOs in vacua. The compound brown. The eluate was concentrated to 10 consisted of yellowish, honeylike crystals, ml by rotary evaporation and chromatowhich were extremely hygroscopic. graphed in aliquots on Sephadex G-l 5 ( 1.6 Glucitollysine appeared pure by standard X 100 cm, equilibrated with water at room amino acid analysis (see Results and Distemperature). The chromatogram showed cussion) and by high-performance liquid three peaks. The first minor one had an chromatography on a sulfonated polystyrene elution volume compatible with the disubcolumn (4.6 X 250 mm, DC 6A resin, Durstituted amino acid I*l-N-Z-t-N-di( 1-deoxy- rum, Calif.) with a linear gradient of 0.53 glucitol- 1-yl)lysine. The major peak was M formic acid, 0.125 M pyridine, pH 3.0, to EXPERIMENTAL

H

PROCEDURES

,O ‘C’ H&F

YOOH Z-HN$-i-i

r FOOH z.HN-F-H

COOH Z-H,6-H

YOOH H>N-C-H

y2 p2 fH2 CHz h

+2

HZI HtOH HO& H&OH HtOH ~H~OH gl Luxe

Z-lyslne

Schlff

H$OH HOCH H&OH t&OH &OH

base

FIG. 1. Scheme for the synthesis of glucitollysine.

HYOH WOCH HtOH HkOH LH~~H

332

TRUEB,

HUGHES,

AND

0.5 M acetic acid. 1.0 M pyridine, pH 5.5. In both systems glucitollysine eluted as a sharp, single peak. Spectroscopic identification. The infrared spectrum of glucitollysine revealed several broad bands, all of which are found in the standard spectra of L-lysine and D-glucitol. The ‘H NMR spectrum corresponded largely to the spectra of L-lysine and D-glucitol. The resonances of the methylene hydrogens at carbon atom C-l’ occurred at 3.5 ppm as a new, complex signal. The “C NMR spectra fitted within 0.1 to 8 ppm to the spectra of L-lysine and D-glucitol. Only the signal for carbon atom C-l’, which does not carry a hydroxylic group in glucitollysine, differs largely from the resonance of the first C atom in D-ghCitOl (Table 1). Incubation. Bovine serum albumin (2 mg/ ml) was incubated at room temperature with 20 mh4 [ C’4]glucose (200 &/ml) in 50 mM Na2HP04, acetic acid, pH 7.4. Sodium azide (0.02%) was added to prevent bacterial growth. After 4 days the reaction was stopped by dialysis in the cold against five changes of a lOOO-fold volume of 0.1% acetic acid. TABLE

WINTERHALTER

Reduction of glycosylated albumin, determination of protein and radioactivity, and amino acid analysis were performed as previously described ( 11). RESULTS

AND DISCUSSION

Previous reports on the syntheses of glucitollysine have failed to describe fully the experimental conditions. Often the products were obtained in yields too low for characterization. Bailey and Robins prepared glucitollysine by incubating hexose and lysine in a 2:l molar ratio followed by reduction with NaBH4 (4). In our hands, the yields of this method were insufficient for spectroscopic identification. We also question whether the condensation of hexose selectively occurs at the e-amino group rather than the a-amino group of lysine, especially in view of the lower pK, of the latter. Bunn et al. obtained glucitollysine by incubating polylysine with glucose; after separation of the reaction product from free glucose by gel filtration, it was reduced with borohydride and subjected to acid hydrolysis (16). This 1

"C NMR SPECTRUM~OFGLUCITOLLYSINEIND~OATROOMTEMPERATURE~ Chemical Chemical shift 175.6 55.2 25.1 22.3 30.6 48.0 50.2 68.9 71.4’ 71.4’ 71.7 63.4

Intensity 14 74 99 114 104 103 89 138 298 162 149

Multiplicity 1 2 3 3 3 3 3 2 2 2 2 3

u 25.2 MHz, Varian XL-loo, 23.5 kG. b Chemical shifts are expressed in parts per million downfield ’ The signal at 71.4 ppm showed more than twice the intensity to correspond to the resonances of two C atoms.

Assignment C-l c-2 c-3 c-4 c-5 C-6 C-l’ C-2’ c-3 C-4’ C-5’ C-6

shift

of standard

L-Lysine

D-Glucitol

115.4 55.3 21.2 22.4 30.7 40.0

from external tetramethylsilane. of the neighboring signals and is therefore

65.8 74.5 12.9 14.3 76.1 66.1

believed

QUANTITATION

OF NONENZYMATIC

method favors condensation of hexose with t-amino groups of lysine, but the relative yield of glucitollysine is small because of steric hindrance. In addition, the acid hydrolysis necessary to liberate glucitollysine from the polypeptide causes partial decomposition of the product (see below). The condensation reaction involving the aldehyde group of the open chain form of glucose (17) and the uncharged amino group of lysine is very slow ( 12). The resulting Schiff base is unstable and may again decompose to lysine and glucose or rearrange to the more stable ketoamine adduct. In order to force the equilibrium to the side of product, we added NaBH,CN to the reaction mixture. At pH values higher than 7, NaBH3CN selectively reduces Schiff bases, thus removing the product from the equilibrium before it may rearrange or decompose, whereas unreacted glucose is barely affected (18). A similar approach to the one described here was reported by Schwartz and Gray for the synthesis of c-N- 1-( 1-deoxylactitol)lysine (19). Amino acid analysis of synthetic glucitollysine revealed one ninhydrine positive peak eluting near phenylalanine (Fig. ZA). The retention time relative to lysine (RLys) was 0.94, and the color yield was approximately 83% of that of lysine. Upon acid hydrolysis of glucitollysine, a time-dependent decrease of the peak at RLys 0.94 with a concomitant increase of a new peak at R,, 0.96 was seen (Fig. 2B). The integral of the two peaks together made up 100% of the starting material, suggesting the same color yield for both glucitollysine and its decomposition product. Acid hydrolysis with 6 N HCl under standard conditions (110°C 24 h) resulted in 40% conversion of glucitollysine to its decomposition product with RLys 0.96, while hydrolysis in 3 N HCI gave only 16% loss of glucitollysine. This acid decomposition product is also described by others and may represent an anhydroalditol of glucitollysine (4,19). Basic hydrolysis in 2 N NaOH did not affect the peak at R,,, 0.94 even after

333

GLYCOSYLATION

0

10

20

30

EFFLUENT.ml

FIG. 2. Amino acid analysis of glucitollysine. (A) Synthetic glucitollysine. The elution position of several standard amino acids is indicated by arrows. (B) Synthetic glucitollysine after hydrolysis for 24 h in 6 N HCI at 110°C. (C) Hydrolysate of bovine serum albumin after glycosylation with [‘4C]glucose and reduction with NaBH4 (see Experimental Procedures). Fractions (1.35 ml) were collected and radioactivity was counted in 15 ml of Biofluor (New England Nuclear). The elution position of three radioactively labeled amino acids is indicated by arrows.

heating to 110°C for up to 48 h; however, extensive destruction of most other amino acids was observed. The extent of nonenzymatic glycosylation can thus be determined by simple amino acid analysis of the reduced and hydrolyzed protein. If the amount of glucitollysine is too small to be detected by the ninhydrine color reaction or by fluorescence detection, it may be labeled by use of tritiated NaBH&N (reduction at pH 3.5, Ref. (18)) or, for in vitro studies, by incubation with labeled glucose. Albumin was reported to undergo nonenzymatic glycosylation in viva and in vitro (2,5,6). E-Amino groups of lysine, probably lysine 189, were reported to be modified (20). In this study incubation of albumin with 20 mM labeled glucose resulted in the incorporation of radioactivity corresponding

334

TRUEB, HUGHES,

AND WINTERHALTER

to 0.31 f 0.01 mol glucose/mol protein. This amount is somewhat smaller than reported by Day et al. (2). The difference may be explained by use of commercially available bovine albumin rather than purified rat albumin, by slightly different incubation conditions, and by the application of the purest glucose batch available (cf. Ref. (11)). Amino acid analysis after reduction and acidic hydrolysis revealed three radioactive peaks (Fig. 2C). No difference could be observed between two samples, one reduced with NaBH, and the other with NaBH$N. Fifty percent of the incorporated radioactivity migrated with retention times equal to glucitollysine (RLys 0.94) and its acid decomposition product (RLys 0.96); 18% of radioactivity eluted near alanine. The amount obtained was insufficient for further analysis. Thirty-two percent of the applied radioactivity eluted in the breakthrough fraction of the ion-exchange column, where hexoses and their decomposition products run as well as glucitolaspartic acid, a compound presumably also formed by interaction of glucose with the iv-terminal aspartic acid of bovine albumin. The fact that three radioactive peaks can be separated by amino acid analysis from a hydrolysate of glycosylated albumin again clearly demonstrates that nonenzymatic glycosylation cannot be determined solely by the incorporation of radioactivity from glucose solutions into polypeptides (11). It is mandatory to identify the individual modified amino acids. ACKNOWLEDGMENTS We thank Dr. H. F. Bunn and Dr. P. J. Higgins for making available two manuscripts of their work prior to publication. This study was supported by Grant 0.330.077.99/l from the Eidgenossische Technische Hochschule.

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