Bioconjugate Chem. 2004, 15, 27−34
27
Effect of Limited Solid-State Glycation on the Conformation of Lysozyme by ESI-MSMS Peptide Mapping and Molecular Modeling Faustinus K. Yeboah,*,† Inteaz Alli,‡ Varoujan A. Yaylayan,‡ Konishi Yasuo,† Shafinaz F. Chowdhury,† and Enrico O. Purisima† Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P-2R2, and Department of Food Science and Agricultural Chemistry, Macdonald Campus, McGill University, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9. Received May 16, 2003; Revised Manuscript Received November 26, 2003
Although protein glycation has been implicated in the alteration of protein functionality, both in vivo (in biological systems) and in vitro (in food systems), the effect of the protein-bound glycan moiety on the structure/conformation of proteins that result in the modification of functionality is not clear. In this article, we report a study of the conformational changes of glycated lysozyme using LC-ESIMSMS peptide mapping, and molecular modeling. A comparison of the RP-HPLC of the tryptic digests of unglycated and glycated lysozyme showed markedly different chromatographic profiles. Analysis of the peptide composition of the chromatographic fractions of the tryptic digests revealed that glycation of lysozyme resulted in the modification of its conformation. Glycation-induced changes in the conformation of lysozyme resulted in the exposure of its active site region to increased proteolytic activity of trypsin. Molecular simulation of triglycated lysozyme also showed that limited glycation of lysozyme caused reorientation of the active site residues (Arg 45, Arg 68, Asn 44, and Trp 62) and increased solvent accessibility into the active site region of the protein. The results of the modeling experiment corroborated the results of the RP-HPLC and ESI-MSMS peptide mapping.
INTRODUCTION
The development of age- and diabetes-related microand macrovascular pathologies underlying nephropathy, neuropathy, retinopathy, and sclerosis have been associated, in part, with nonenzymatic glycosylation (glycation) of body proteins and the formation and the accumulation of advanced glycation end products (AGEs) in tissues under chronic hyperglycemia (1-4). Although the molecular basis of how protein glycation leads to the development of these pathologies is not well understood, it is generally accepted that (1) AGE-modified matrix proteins interact abnormally with other matrix components and with cell surface receptors such as integrins, and (2) age-modified plasma proteins bind to AGEspecific receptors on macrophages and mesangial cells. These interactions induce receptor-mediated production of reactive oxygen species, thereby increasing the oxidative stress status of cells (4, 5). Alteration of the biological properties of glycated or AGE-modified proteins may derive from changes in their structure and conformation, with a consequent modification to their physicochemical and biochemical properties. The effects of glycation on protein structure and conformation have not been clearly established. Early-stage protein glycation, involving the formation of Amadori rearrangement products, and the initial non-cross-linking reactions of the protein bound sugar moieties, have been reported to enhance the physicochemical properties of several proteins. For example, the emulsifying properties * Corresponding author. Tel: (514) 496 2593. Fax: (514) 496 5143. e-mail:
[email protected]. † Biotechnology Research Institute. ‡ McGill University.
of carp myofibrillar proteins (6), whey proteins (7), casein (8), plasma proteins (9), and lysozyme (10) have been shown to improve with limited glycation. The gel forming properties of dried egg white (11) and bovine serum albumin (12) have also been reported to improve with limited glycation. Limited glycation has also been shown to enhance the enzymatic activity of lysozyme (13, 14) and trypsin (15). The thermal and conformational stability of several proteins, including bovine β-lactoglobulin (16, 17) and bovine lens Crystallin (18) have also been shown to improve with limited glycation. These reports show that the effect of glycation on protein structure and functionality may depend on several factors, including the type of protein, the type of reducing sugar used for glycation, the glycation site on the protein, and the extent of glycation. The reports also suggest that, depending on the protein involved, limited glycation may cause a global conformational change or a localized structural change in the vicinity of the glycation site. Glycation-induced changes in the structure and functional properties of proteins may be due, in part, to the direct covalent attachment of sugar moieties to the basic amino acid residues of proteins, the glyco-oxidative reactions of the protein-bound sugar moieties, and to glycation-induced charge modification of proteins (19-21). Understanding the nature of the structure and conformational changes that accompany protein glycation will help in elucidating the functional consequences of protein glycation in both food and biological systems. Glycation-induced modification of proteins at the secondary, tertiary, and quaternary structural levels are yet to be elucidated. This paper describes the effect of glycation on conformational changes in the structure of lysozyme.
10.1021/bc034083v CCC: $27.50 © 2004 American Chemical Society Published on Web 01/21/2004
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MATERIALS AND METHODS
(LZM)1
Materials. Hen egg white lysozyme (Sigma cat. # L-6786), D-glucose, D-fructose, and bovine trypsin (Sigma cat. # T-8003) were obtained from Sigma Chemicals (St. Louis, MO). All other chemicals and reagents were of analytical grade from Fisher Scientific (Nepean, ON, Canada). Ultrapure water from the “Nanopure” water purification system (Barnstead, Thermolyne, Dubuque, IA) was used throughout the study. Protein Glycation. Glycated lysozyme was prepared according to the method described by Yeboah et al. (23). Briefly, lyophilized lysozyme or lyophilized mixtures of lysozyme and D-glucose or D-fructose (1:1 molar ratio of -amino group:sugar carbonyl groups) were incubated at 50 °C and 65% relative humidity. The temperature and humidity condition were chosen to accelerate the rate of glycation. The incubation temperature of 50 °C is below the denaturation temperature of lysozyme (∼76 °C) (22), so thermal denaturation of lysozyme was not expected to occur in the incubated samples. Samples were incubated for 1, 2, 5, 10, or 14 days. Lyophilized lysozyme was incubated alone (without any reducing sugar) under the same temperature and humidity conditions as a control. After 1, 2, 5, 10, or 14 days of incubation, samples were taken and immediately dialyzed exhaustively against water for 48 h at 4 °C (1 kDa MW cutoff) to remove free unreacted sugar and other low molecular weight reaction products. After dialysis, the samples were lyophilized and stored at -20 °C prior to further analysis. Tryptic Hydrolysis. Solutions (25 mL) of lysozyme that had been incubated alone for 14 days (LZM) or lysozyme that had incubated with D-glucose (LZM-G) or D-fructose (LZM-F) (100 mg/mL, pH 8.5), and of bovine trypsin (4 mg/mL, pH 8.5) were mixed together (lysozyme:trypsin; 25:1) and incubated at 37 °C. After 6 h incubation, the hydrolysis reaction was terminated by rapidly heating the reaction mixture to boiling. Samples were then cooled over ice, filtered (0.22 µm Millipore syringe filter), and lyophilized. The lyophilized tryptic digests were stored at -20 °C prior to further analysis. Reversed Phase HPLC. Quantities (5 mg) of the lyophilized tryptic digest of LZM, LZM-G, or LZM-F (prepared as described above) were dissolved in 0.1% aqueous trifluoroacetic acid (TFA) (1 mL), vortexed, and sonicated for 15 min. The resulting solutions were filtered through 0.22 µm syringe filters, and 50 µL of the filtrate was injected for RP-HPLC analysis. RP-HPLC analysis was performed on a Beckman Gold HPLC system, equipped with a UV diode-array detector (Beckman Instruments, Inc., Fullerton, CA). The mobile phase consisted of 0.1% TFA in water (solvent A) and in acetonitrile (solvent B), and the elution gradient was 0% to 60% solvent B in 40 min. Separation was achieved on a reversed phase analytical column (Vydac C18 column, 218TP54) at a flow rate of 1 mL/min. Eluting peaks (monitored 220 nm) were collected for electrospray tandem mass spectrometric analysis. ESI-Mass Spectrometry. Lyophilized HPLC fractions were reconstituted in 70% aqueous acetic acid and analyzed by ESI-MSMS using an API-III MSMS system (SCIEX, Thornhill, Ontario, Canada). Samples were infused into the electrospray ion source through a fused silica capillary (100 µm i.d.) at a rate of 1 µL/min using 1 Abrreviations: ESI/MS, electrospray mass spectrometry; ESI/MS/MS, electrospray tandem mass spectrometry; LZM, lysozyme incubated alone; LZM-F, lysozyme glycated by Dfructose; LZM-G, lysozyme glycated by D-glucose; TFA, trifluoroacetic acid.
a low-pressure infusion pump (Model 22, Harvard Apparatus, South Natick, MA). The sequence of tryptic peptides was determined from their collision-induced dissociation (CID) spectrum. The composition of glycoforms of lysozyme in the incubated samples was determined from the area of their corresponding peaks in the electrospray ionization (ESI) mass spectrum. The relative composition of individual glycoforms in the incubation mixture is proportional to the relative area their corresponding peaks. (23) Molecular Modeling of Glycated Lysozyme. The AMBER force field (24, 25) was used to simulate the effect of glycation on the conformation of lysozyme. The parameters had to be supplemented for glycated lysine residues. Missing equilibrium bond and angle parameters of the lysine-bound glucose moiety (Amadori product of lysine in the pyranose for) were obtained from the model compounds in the Cambridge Structural Database (26). Force constants were derived from similar atom types in the AMBER force field, and partial charges for glycated lysine were obtained by fitting to the 6-31G* electrostatic potential as calculated in GAMESS (27, 28). A triglycated model of chicken egg white lysozyme was developed, by replacing Lys-1, Lys-33, and Lys-97 with glucose-derived Amadori-lysine (in the pyranose form) that is glycated at the -amino group. These three residues were chosen for modification in the glycated lysozyme model because they were the most reactive residues in the protein. Another reason for using a triglycated model of lysozyme in the molecular modeling studies was that the average sugar load, in the form of Amadori product (average number of Amadori-products molecules per molecule of lysozyme) was approximately 2.4, so the next highest whole number was chosen. Molecular models of native lysozyme and of the triglycated lysozyme model were subjected to conjugate gradient energy minimization using a distance-dependent dielectric function ( ) 4r) and an 8 Å nonbonded cutoff. Solvation effects were included, using an implicit solvation model with continuum electrostatics (29, 30). A conformational search using Monte Carlo minimization (31-33) was carried out to explore the effect of glycation on the structure of lysozyme. The side chain dihedral angles of glycated Lys 1, 33, 97 and of 18 amino acids (Val 2, Arg 21, Phe 34, Asn 37, Gln 41, Thr 43, Asn 44, Arg 45, Asp 52, Gln 57, Trp 62, Trp 63, Arg 68, Leu 75, Ser 81, Ser 86, Val 109, Trp 123) present in and around the active site of lysozyme were searched for a total of 20 000 Monte Carlo-minimization cycles. During minimization, all atoms in the protein were allowed to move. As a control, a similar conformational search was carried out on the same set of amino acids for native enzyme. RESULTS
Glycation. Incubation of lysozyme with D-glucose or resulted in the formation of a mixture of glycoforms of lysozyme. The ESI-mass spectrum of lysozyme incubated alone for 1 day (A), lysozyme incubated with fructose (B), and lysozyme incubated with glucose (C) for 1 day are presented in Figure 1. The mass spectra show the profile of glycoforms of lysozyme formed after 1 day of incubation. Only one peak, corresponding to lysozyme can be observed when lysozyme was incubated alone for 1 day. Incubation of lysozyme with D-fructose resulted in the formation of two glycoforms, mono- and difructated lysozyme (Figure 1B), while the incubation of lysozyme in the presence of glucose resulted D-fructose
Limited Solid-State Glycation on Lysozyme Conformation
Bioconjugate Chem., Vol. 15, No. 1, 2004 29
Figure 1. Reconstructed ESI-MS spectra of lysozyme incubated alone for 1 day and of lysozyme incubated with D-fructose and D-glucose for 1 day, showing the distribution of glycoforms of lysozyme formed during incubation.
in extensive glycation, leading to the formation of a mixture of five glycoforms ranging from mono- to pentaglucated lysozyme (Figure 1C). Less than 30% of lysozyme was glycated in the lysozyme-fructose system after the 1st day of incubation, while over 90% of lysozyme was glycated in the lysozyme-glucose system in the same period of incubation. The extent of glycation, determined as the average sugar load per molecule of lysozyme, increased with time of incubation in both sugar systems, but the rate of sugar loading of lysozyme when incubated with glucose was 5 to 6 times higher than when lysozyme was incubated with fructose throughout the incubation period. Tryptic Digests of Unglycated and Glycated Lysozyme. The chromatographic profiles of the tryptic digest of unglycated and glycated lysozyme are presented in Figures 2 and 3. Figure 2A-D represents the chromatograms of the tryptic digests of lysozyme incubated alone for 14 days, and lysozyme incubated with D-fructose for 1, 5, and 14 days, respectively. Figure 3A-D represents the chromatograms of the tryptic digests of lysozyme incubated alone for 14 days, and lysozyme incubated with D-glucose for 1, 5, and 14 days, respectively. A comparison of the tryptic peptide profile of unglycated lysozyme and that of glycated lysozyme after different periods of incubation reveal marked differences between unglycated and glycated lysozyme. Several peaks (e.g. peaks 3 and 8) that were not observed in the peptide profile of the tryptic digest of unglycated lysozyme appeared in the tryptic digest of glycated lysozyme after only 1 day of incubation with D-fructose. After the 5th day of incubation, other peaks (e.g. peaks 2, 4, and 13) also appeared in the chromatogram in significant amounts. The relative proportion of peaks 7, 12, 14, and 15 decreased sharply after the 1st day of incubation, while at the same time the relative proportions of peaks 5 and 9 increased sharply. The HPLC peptide profile of lysozyme changed progressively with time of incubation. Similar results
Figure 2. Reversed phase HPLC chromatograms of tryptic digest of unglycated lysozyme incubated alone for 14 days (LZM) (panel A), and lysozyme incubated with D-fructose after 1, 5, and 14 days (panels B, C, and D, respectively), showing peaks (labeled 1 through 15) that were collected and analyzed with ESI-MSMS.
were observed for the tryptic digest of lysozyme glycated with D-glucose as with D-fructose, but the extent of the changes was more pronounced for lysozyme incubated with D-glucose especially after prolonged incubation (Figure 3). Identification of Tryptic Peptides. Electrospray tandem mass spectrometric was used to identify and sequence the peptide components of the tryptic digests. Table 1A,B shows the m/z (M + H)+ values and the sequence of the peptides identified in the HPLC fractions of the tryptic digests of unglycated and glycated lysozyme after 1, 5, and 14 days of incubation. The symbol “+” or “-” is used to indicated the presence or absence of the designated peptide in the HPLC fraction, and the symbol “++” is used to indicate the major peptide component of an HPLC fraction, if the fraction contains more than one peptide component. The symbol “)” is used to indicate peptide fragments that contain disulfide bonds, and the symbol “*” placed after an amino acid letter code indicates that that amino acid residue is the glycation site. Most of the identified peptides were trypsin-specific peptides (i.e., produced by the cleavage of peptide bonds at the carboxyl end of a lysine or arginine residue). Nontrypsin specific of peptides, such as peptide residues 109-112, 117-123, 97-108, and 98-108 produced by the cleavage of peptide bonds at the carboxyl end of Trp-108 and Trp123 were also identified. Residues 117-125 and 98-112, identified in HPLC fractions 12 and 15, respectively, of the tryptic digest of unglycated lysozyme were not detected in the digests of any of the glycated samples. The peptide components of HPLC fractions 2, 3, and 8 of the tryptic digest of glycated
30 Bioconjugate Chem., Vol. 15, No. 1, 2004
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detected in the digests of glycated lysozyme samples (both LZM-F and LZM-G) after the 5th day of incubation. Glycated peptides that had undergone both dehydration and deamination ([M + H]+ ) 1654.8 and 1493.6) were only detected in the digest of LZM-G after the 5th day of incubation. DISCUSSION
Figure 3. Reversed phase HPLC chromatograms of tryptic digest of unglycated lysozyme incubated alone for 14 days (LZM) (panel A), and lysozyme incubated with D-glucose after 1, 5, and 14 days (panels B, C, and D, respectively), showing peaks (labeled 1 through 15) that were collected and analyzed with ESI-MSMS.
lysozyme (glucated and fructated) were identified as residues 46-53, 35-45, and 60-68, respectively. These peptides were not observed in the tryptic digest of unglycated lysozyme, but they appeared in the digest of glycated lysozyme after the 1st day of incubation. The peptide components of HPLC fraction 13 were identified as residues 22-28, 46-59, 47-61, and 64-84. These peptides were also not released in the digest of unglycated lysozyme. The peptide defined by residues 22-28 was also released in the tryptic digest of glycated lysozyme after the 1st day of incubation in both sugar systems, but residues 46-59, 47-61, and 64-84 were detected in the digest of glucated lysozyme samples after the 5th day of incubation. These peptides were derived by the cleavage of nontrypsin specific bonds. Glycation of lysozyme with glucose had a more pronounced effect on the peptide composition of the tryptic digest of lysozyme than with fructose. For example, after just 1 day of incubation of LZM with glucose (LZM-G), the peptides defined by residues 1-5, 97-108, 2223)115-116, 97-112, and 98-112 could not be detected in HPLC fractions 5, 10, 14, and 15, respectively. These peptides were, however, observed in the digest of LZM-F even after the 5th day of incubation. Prolonged incubation of lysozyme with glucose (after 5 days) also resulted in the release of peptides defined by residues 46-59, 4761, and 64-84 in HPLC fraction 13. These residues were not observed in the tryptic digests of unglycated lysozyme or any of the LZM-F samples. Di- and triglycated peptides ([M + H]+ ) 936.4 and 2400.2; see Table 1), and glycated peptides for which the bound sugar moieties had undergone further dehydration (residues with (M + H)+ ) 750.4, 1654.8, and 1493) to form deoxyosones were
Tryptic Peptide Profile. Differences in the chromatographic profile of the tryptic digest of unglycated lysozyme (LZM) and glycated lysozyme (LZM-F or LZMG) (Figures 2 and 3) suggests that glycation of LZM resulted in changes to its conformation. A comparison of the chromatographic profile of the tryptic digests of unglycated and glycated lysozyme provided information about glycation-induced conformational changes in the structure of lysozyme. Monitoring of the peptide components of chromatographic peaks that appeared only in the tryptic digest of the glycated lysozyme samples also provided information about the regions or domains of unglycated lysozyme that had become more exposed to the proteolytic activity of trypsin after glycation. The peptide profile of peaks that disappeared, or reduced in relative intensity in the chromatogram of the tryptic digest of unglycated lysozyme after glycation provided information about the regions of lysozyme that had become less exposed, or more resistant to the proteolytic activity of trypsin. Information about the glycation sites of lysozyme, and the relative reactivities of the identified glycation sites could also be determined from changes in the chromatographic and peptide profiles of the tryptic digests of lysozyme after glycation. The detection of peptide residue 46-53, 35-45, 6068, and 22-28 in HPLC fractions 2, 3, 8, and 13, respectively (Table 1A), of the tryptic digests of glycated lysozyme after the 1st day of incubation indicate that the region(s) or domain(s) of lysozyme that contained these peptide fragments became more exposed to the proteolytic activity of trypsin after limited glycation. An inspection of the 3-D structure of native lysozyme showed that these peptide fragments are all located within or in the vicinity of the active site of lysozyme. A comparison of the chromatographic profile of the tryptic digests (Figures 2 and 3) of unglycated and glycated lysozyme also reveal that the relative chromatographic response of peaks 6, 9, 10, and 11 increased markedly after the 1st day of incubation. The peptide component(s) of these chromatographic fractions were identified as residues 109-112, 117-123, 54-61, and 97-108, respectively. These residues were also found to be located in the vicinity of the active site of lysozyme. These results show that limited glycation of lysozyme caused an increased exposure of its active site residues to the proteolytic activity of trypsin. It also suggests that limited glycation of lysozyme increased solvent penetration into its active site. This observation may explain the enhanced enzymatic activity of lysozyme after limited glycation (13, 14). The active site residues of lysozyme include Phe 34, Glu 35, Ser 36, Asn 37, Asn 44, Asp 52, Leu 56, Gln 57, Trp 62, Trp 63, Leu 75, Asp 101, Asn 103, Ala 107, Trp 108, Val 109, Ala 110, and Arg 114. The sharp decrease in the relative chromatographic response of peaks 7, 12, 14, and 15 after the 1st day of incubation show that the regions of lysozyme that contain their peptide components had become resistant to the proteolytic activity of trypsin after glycation. The peptide component(s) of fractions 7, 12, 14, and 15 are residues 34-45, 117-125, 97-112, and 98-112 respectively, all
Limited Solid-State Glycation on Lysozyme Conformation
Bioconjugate Chem., Vol. 15, No. 1, 2004 31
Table 1. Peptide Composition of the Chromatographic Fractions of the Tryptic Hydrolysates of Lysozyme (LZM) and Its Glycated Analogues Lysozyme-Fructose (LZM-F) and Lysozyme-Glucose (LZM-G) after 1, 2, 5, 10, and 14 days of Incubation LZM-F HPLC fractions
(M + H)+
1 2 3 5 5 5 6 7 7 8 9 10 10 11 12 12 12 13 13 13 14 14 15
517.4 872.4 1281.7 874.6 478.4 606.4 531.6 1429.6 1474.8 1179.8 776.4 900.6 1292.6 1163.6 1045 1155.6 1755.2 796.4 1510.6 2205.8 1517.6 1805.1 1676.7
69-73 46-53 35-45 15-21 2-5 1-5 109-112 34-45 117-129 60-68 117-123 54-61 97-108 98-108 117-125 46-56 46-61 22-28 46-59 64-84 22-33)115-116 97-112 98-112
A. Unglycated Peptides R-TPGSR-N R-NTDGSTDY-G F-ESNFNTQATNR-N R-HGLDNYR-G K-VFGR-C KVFGR-C W-VAWR-N K-FESNFNTQATNR-N K-GTDVQAWIRGCRL N-SRWWCNDGR-T K-GTDVQAW-I Y-GILQINSR-W K-KIVSDGNGMNAW-V K-IVSDGNGMNAW-V K-GTDVQAWIR-G R-NTDGSTDYGIL-Q R-NTDGSTDYGILQINSR-W R-GYSLGNW-V R-NTDGSTDYGILQIN-S W-CNDGRTPGSRNLCNIPCSALL-S R-(GYSLGNMVCAAK)-F)R-(CK)-G K-KIVSDGNGMNAWVAWR-N K-IVSDGNGMNAWVAWR-N
5 5 5 9 10 13 13 14 14
768.6 750.4 936.4 1153.2 1454.6 1315.8 1654.8 1493.6 2400.2
(1-5)+G (1-5)+G-H2O (1-5)+2G (6-14)+G (97-108)+G (6-14)+2G (46-59)+G-H2O (46-59)-NH3 (1-14)126-128)+3G
B. Glycated Peptides K*VFGR-C K*VFGR-C K**VFGR-C R-CELAAAMK*R-H K-K*IVSDGNGMNAW-V R-CELAAAMK**R-H R-NTDGSTDYGILQIN-S R-NTDGSTDYGILQIN-S (K**VFGRCELAAAMK*R)-H)R-(GCR)-L
residues
sequence
LZM-G
LZM
day 1
day 5
day 14
day 1
day 5
day 14
+ + ++ + + + + + + + ++ ++ + +
+ + + ++ + + + + + + ++ + + + ++ + ++ + -
+ + + ++ + + + + + + + + + ++ + + -
+ + + ++ + + + + + + + + ++ + -
+ + + ++ + + + + + + + + ++ + -
+ + + + + + + + + + + + ++ + + ++ -
+ + + + + + + + + ++ + + ++ -
-
-
+ + + + +
+ + + + + +
+ + + +
+ + + + + + + + +
+ + + + -
"+" indicates that peptide was identified in fraction, and "++" is used to indicate the Major peptide component in cases where HPLC fraction contained more than one peptide. “-” indicates that peptide was not identified in fraction or is present below the detectable limit, and ")" is used to indicate disulfied bonds. A. unglycated peptides, and B glycated peptides identified in the HPLC fractions of the hydrolysates of the LZM-F and LZM-G systems.
of which are produced by the cleavage of a peptide bond at the carboxyl end of a lysine residue (Lys-33, -96, -97, and -116). The decreased rates of release of these fragments indicate that the -amino groups of the respective lysine residues had undergone glycation. This does not mean that every molecule of lysozyme is glycated at all four lysine residues, but that different populations of glycoforms of lysozyme with varying degrees of glycation at Lys-33, -96, -97, and -116 had been formed in the glycation mixture (Figure. 1). The major peptide component of fraction 5 of unglycated lysozyme was residue 2-5, which is formed by the cleavage of peptide bonds at the carboxyl end of Lys-1 and Arg-5. After the 1st day incubation of lysozyme with fructose, the major peptide component of fraction 5 was no longer residue 2-5, but residue 15-21. In addition, peptide fragment 1-5 was not observed in any of the tryptic digests of LZM-G samples, even after only 1 day of incubation, due to the extensive nature of the glycation of lysozyme in the when incubated with glucose. These results indicate the glycation of either the N-terminal R-amino group and/or the -amino group of Lys-1. The fact that the peptide fragments defined by residues 117-125, and 98-112 could not be detected in the tryptic digest of even the least glycated sample (LZM-F after 1 day incubation) also indicates the glycation of Lys-97 and -116. The results of this study show that all seven lysine residues of
lysozyme were involved in the glycation reaction and that Lys-1, -33, and -97 were the most susceptible residues to the glycation reaction. The list of identified glycated peptides (Table 1B) show that diglycation occurred at Lys-1 and -33. The diglycation at Lys-1 could be due to glycation of the N-terminal R-amino and -amino group or diglycation at the -amino group. The results of this study are consistent with the report of previous studies (where the lysine residues of lysozyme were modified with acetic anhydride) (33), where the order of reactivity of the -amino groups of the lysine residues of lysozyme was reported as Lys-97 > -33 > -1> -13 > -116 > -96. Effect of Glycation on the Structure Lysozyme. Changes in the chromatographic profile of the tryptic digest of lysozyme, and in the peptide composition of the HPLC fractions after glycation, show that the glycation of lysozyme resulted in a modification of its structure and conformation. Differences between in the chromatographic and peptide profile of the tryptic digests of lysozyme glycated with glucose and fructose (Figures 2 and 3) can be explained by the differences in the extent of glycation in the two sugar systems. The most dramatic change in the peptide profile of the tryptic digest of lysozyme occurred after the 1st day of incubation with either glucose or fructose. These changes were accompanied by the release of new peptides (residues 4653, 35-45, 54-61, 46-56, and 22-28) that were not
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Figure 4. Superimposed structures of native hen egg white lysozyme and its triglycated model. Residues that showed conformational change after glycation are highlighted in yellow (in the glycated model) and purple (in the native enzyme). Secondary structures colored green (in the glycated model) and orange (in the native enzyme) are displayed. Glycated residue Lys 1, Lys 33, and Lys 97 are displayed for clarity.
Figure 5. Solvent-accessible surface area of the triglycated model of lysozyme (A) and of the native lysozyme (B). The solventaccessible surface area of the active site residues is highlighted in red for comparison (see text for detail).
observed in the digest of unglycated lysozyme. The amino acid residue of the peptide fragments defined by residues 46-53, 35-45, 54-61, and 46-56 constitute the active site amino acid residues of lysozyme. The increased rate of release of the peptide fragments from the active site of lysozyme as a result of glycation show that the glycation of lysozyme modified its conformation to expose the active site to the proteolytic activity of trypsin.
It has been reported that reductive methylation of all the -amino groups of lysozyme resulted in only minor overall structural change of the protein, but caused significant modifications to the surface loops defined by residues 69-72 and 101-103, as well as the C-terminal residues 126-129 (34). The minor structural changes effected by the reductive methylation of lysozyme may be due to the small size and hydrophobic nature of the
Limited Solid-State Glycation on Lysozyme Conformation
methyl group. The larger size of D-fructose or D-glucose, with their numerous hydroxyl groups, on the other hand, may cause a more significant change to the structure and hydrodynamic properties of lysozyme as observed in this study. Modeling. Monte Carlo simulations of models of native and glycated lysozyme were carried out to determine structural perturbation caused by limited glycation, given that limited glycation of lysozyme (after the 1st day of incubation) resulted in significant changes in the chromatographic and peptide profile of the tryptic digest of lysozyme. A superposition of the lowest-energy structures of native lysozyme and a model of triglycated lysozyme is presented in Figure 4. Overall, only minor structural differences were observed between the model of native lysozyme and the triglycated model. However, there was significant reorientation of the side chains of glycated Lys-1, -33, and -97, Gln 41, Thr 43, Asn 44, Arg 45, Arg 68, Trp 62, and Val 109. With the exception of the glycated lysine residues (Lys-1, -33, and -97), the common feature between these amino acid residues is that they are all located in the active site region of lysozyme. A comparison between the solvent-accessible surface area of native lysozyme (6487.11 Å2) and of the triglycated lysozyme model (6894.38 Å2) shows a significant net increase of 407.27 Å2 in the triglycated lysozyme model compared to the native enzyme. The solventaccessible surface area of the active site region increased from 470 Å2 in native lysozyme to 550 Å2 in the triglycated model. This increase of 80 Å2 is significant, given that it represents ∼20% of the total net increase in solvent accessibility of lysozyme (407.27 Å2), when the contribution of the active site region to the total solvent accessible surface area of native lysozyme is only 7.2%. Figure 5 shows the solvent-accessible surface area of the triglycated lysozyme model (A) and of native lysozyme (B). The solvent-accessible surface area of the active site region is indicated in red for clarity. The reorientation of the side chains of the active site residues, coupled with the increase in the solvent-accessibility of the active site, is consistent with the increased susceptibility of the active site residues in the glycated protein to tryptic digestion. CONCLUSION
The findings of this study show that limited glycation of lysozyme resulted in conformational changes that increased the susceptibility of its active site region to the proteolytic activity of trypsin. The extent and nature of the conformational change depended on the extent of glycation, as well as the site(s) of glycation. The results also showed that conformational changes could occur at the glycation sites or regions remote from the site of glycation. Although the observed glycation sites of lysozyme in both sugar systems were similar, fructose showed a higher degree of specificity in glycating the -amino groups of the most reactive lysine residues (Lys1, -33, and -97) during the early stages of the glycation reaction, due to the lower reactivity of fructose. The results of this study also show that ESI-MS peptide mapping is a powerful technique for revealing specific and global changes in protein structure due to chemical modifications such as glycation. Glycationinduced changes in the peptide profile of the chromatographic fractions of the tryptic digest of lysozyme at different extends of glycation provided information about the dynamic nature of the changes in the structure and conformation the protein.
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BC034083V
