PROTEIN CROSSLINKING BY THE MAILLARD REACTION WITH ASCORBIC ACID AND GLUCOSE by ZHENYU DAI Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Dissertation Advisor: Vincent M. Monnier, M. D. Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables……………………………………………………………………………2 List of Figures…………………………………………………………………………..3 Acknowledgements……………………………………………………………………..6 List of Abbreviations…………………………………………………………………....7 Abstract………………………………………………………………………………...10 Chapter 1. Introduction……………………………………………………………….. 12 Chapter 2. Histidino-threosidine: A novel crosslink from ascorbic acid degradation products Introduction…………………………………………………………………..49 Experimental Procedures…………………………………………………….50 Results………………………………………………………………………..55 Discussion……………………………………………………………………72 Chapter 3. Glucosepane: Site-specific crosslinking of ribonuclease A Introduction………………………………………………………………….76 Experimental Procedures……………………………………………………83 Results……………………………………………………………………….86 Discussion…………………………………………………………………..103 Conclusions and future studies…………………………………...………………….110 Bibliography…………………………………………………………………………113 1 List of Tables Table 1.1 The pKa comparison of several ε-lysines and one α-lysine in hemoglobin……………………………………………………………….31 Table 1.2 Distribution of glycated peptides in RNase A............................................34 Table 1.3 Distribution of glucose adducts among lysine residues on α1(I)CB3 and α2CB3-5 fragments of collagen prepared from rats of different ages…... 37 Table 2.1 Relative yield of glycation agents for histidino-threosidine…………….. 69 Table 3.1 Comparative levels of selected Maillard Reaction adducts and cross-links in normal human skin collagen in the eighth decade of life……………. 79 Table 3.2 Observed double peaks or triple peaks separated by Δm/z=6 in the tryptic map of glycated ribonuclease A…………………………………………..88 Table 3.3 Peptide maps from other enzymatic digestion. a) Asp-N digestion. b) chymotryptic digestion …………………………………………………..101 Table 3.4 The relative surface accessibility (RSA) values of lysine and arginine residues in ribonuclease A …………………………………..………. 106 2 List of Figures Fig 1.1 Proposed mechanism of pentosidine formation from D-ribose, D-fructose, and ascorbic acid via the common intermediate…………………………...17 Fig 1.2 Proposed mechanism of pentosidine formation by Lederer et al.…………18 Fig 1.3 Structure of formulas of important α-dicarbonyl intermediates…………... 23 Fig 1.4 Mechanism of glucosepane and crossline formation from glucose via N6-(2,3- dihydroxy-5,6-dioxohexyl)-L-lysinate intermediate compound…………..24 Fig 1.5 Structure of DODIC, MODIC, and GODIC crosslinks………………..…..25 Fig 1.6 Specific α-helix peptides models with catalytic residues positioned at specific Sites………………………………………………………………………...30 Fig 1.7 Pathway of glycation of protein and trapping methods to discriminate the Schiff base and the Amadori products..........................................................33 Fig 1.8 Catalytic mechanism of phosphate bound in a basic microenvironment on the Amadori rearrangement…………………………………………………….35 Fig 1.9 Diagrammatic representation of the aggregated forms of the collagen superfamily of proteins…………………………………………………… 39 Fig 1.10 Location of lysyl oxidase derived crosslinks in different types of collagen..41 Fig 1.11 Location of the divalent immature and the trivalent mature lysyl oxidase crosslinks derived from immature crosslinks……………………..……… 43 Fig 2.1 Reversed-phase HPLC UV and fluorescence profiles for the purification of Z-histidino-threosidine………………………………………………………56 Fig 2.2 Absorption spectra of BOC-histidino-threosidine…………………………..58 3 Fig 2.3 Electron spray ionization-MS/MS spectra of histidino-threosidine and histidino-threosidine analog………..…………………………………….….59 Fig 2.4 1H-NMR spectra of Z-histidino-threosidine and histidino-threosidine analog.61 Fig 2.5 1H – 1H correlation spectroscopy (COSY) of histidino-threosidine analog…..63 Fig 2.6 Proposed chemical structure of histidino-threosidine analog and histidino-threosidine………………………………………………………….65 Fig 2.7 Heteronuclear multiple bond correlation (HMBC) spectroscopy of imidazole- Threosidine…………………………………………………………………....66 Fig 2.8 13C NMR spectrum of histidino-threosidine analog……………….…………68 Fig 2.9 Effect of time, incubation ratios pH on Z-histidino-threosidine formation from Z-lysine, Z-histidine and threose………………………………………...……70 Fig 2.10 Detection of histidino-threosidine by LC-ESI/MS/MS analysis in bovine lens protein incubated with threose………………………………………………..71 Fig 2.11 Proposed mechanism of formation of histidino-threosidine and dideoxysone formation with histidine and threose……………………………….………….75 Fig 3.1 Structures of crosslinks of the Maillard reaction categorized by their precursors …...………………………………………………………………...77 Fig 3.2 Major glycation pathways expected in nonoxidative glucose-protein incubation……………………………………………………………………….81 Fig 3.3 Sequencing of glycated ribonuclease A tryptic peptide 40CKPVNTFVHESL ADVQAVCSQK61 with modification of K41 by Δm/z=160 (oxidized Amadori). ………………………………………………………………………90 Fig 3.4 Sequencing of glycated ribonuclease A tryptic peptide 36DRCKPVNTFVHESL 4 ADVQAVCSQK61…………………………………………………………….92 Fig 3.5a Sequencing of glycated ribonuclease A tryptic peptide 36DRCKPVNTFVHES LADVQAVCSQK61 with a possible intramolecular modification by DODIC at residues K41 and R39……………………………………………………..94 Fig 3.5b the co-elution of peptide with m/z of 2896.4 and the second peptide with m/z of 2914.4 suggest that the peptides with m/z value of 2914.4 may have different origins. …………………………………………………………… .95 Fig 3.6 Both tryptic and chymotryptic peptides indicate presence of intra-molecular glucosepane between K98 and R85……………………...………………….97 Fig 3.7a Sequencing assignment of the peptide with m/z value of 4064.0…………...99 Fig 3.7b In tryptic digestion of glycated ribonuclease A, two sets of peaks point to the inter-crosslinking between K1 and R39…………………………………….100 Fig 3.8 Distance of all nearby lysine and arginine pairs in ribonuclease A…………105 Fig 3.9 Schematic representation of crosslink formation in RNase A…….………...112 5 Acknowledgements My most sincere appreciation goes to: My dissertation advisor and mentor, Dr. Vincent M. Monnier, for accepting me as a lab member, constant scientific guidance and pressure. My wife, Xiaochen Hu and my family, for their support and love. My advisory committee, Drs. Vernon E. Anderson, Lawrence M. Sayre, Pieter deHaseth, for their interest, guidance and intriguing discussion. My defense committee member, Dr. Steven L. Sanders and Dr. Masaru Miyagi for advice on this dissertation. My collaborator, Ms. Ina Nemet, Dr. Wei Shen, Dr. Benlian Wang, and Dr. Gang Sun for scientific and technical assistance. Members of Dr. Monnier’s laboratory, for all the help on science, technology. 6 List of Abbreviations 3-DG 3-deoxyglucosone AG aminoguanidine AGE(s) advanced glycation end product(s) AGOEs advanced glycoxidation end products BOC- t-butyl oxy carbonyl CEL Nε-(1-carboxyethyl)lysine CML Nε-carboxymethyl-lysine CNBr or CB cyanogen bromide COSY correlation spectroscopy CTGF connective tissue growth factor DEPT distortionless enhancement by polarization transfer DHA dehydroascorbic acid DODIC 3-deoxyglucosone-derived imidazolium cross-link DTPA diethylenetriamine pentaacetic Acid GBM glomerular basement membrane GO glyoxal GODIC glyoxal-derived imidazolium cross-link GOLD glyoxal-lysine dimer GSH glutathione Hb Ao unmodified hemoglobin Hb A1c glycated hemoglobin 7 HHLNL histidino-hydroxylysinonorleucine HHMD histidino-hydroxymerodesmosine deH-LNL dehydro-lysinonorleucine HLKNL hydroxylysinoketonorleucine HL-pyr hydroxylysyl-pyridinoline HMBC heteronuclear multiple bond coherence HMQC heteronuclear multiple quantum coherence K2P 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl- amino)-3-hydroxy-2, 3-dihydropyridinium LC-MS liquid chromatography-mass spectrometry L-pyr lysyl-pyridinoline MFP-1 Maillard fluorescent product-1 MGO methylglyoxal MODIC methylglyoxal-derived imidazolium cross-link MOLD methylglyoxal-lysine dimer MALDI matrix-assisted laser desorption and ionization MRM multiple reaction monitoring NMR nuclear magnetic resonance PM pyridoxamine PTB N-phenacylthiazolium bromide RAGE receptor for advanced glycation end products RNase A ribonuclease A ROS reactive oxygen species 8 RP-HPLC reversed phase-high performance liquid chromatography SSAO semicarbazide-sensitive amine oxidase TFA trifluoroacetic acid Z or CBZ benzyloxycarbonyl 9 Protein Crosslinking by the Maillard Reaction with Ascorbic Acid and Glucose Abstract by ZHENYU DAI Nonenzymatic glycation has been implicated in diabetes, aging, and aging related disease such as Alzheimer’s disease. Nonenzymatic glycation has been implicated in the pathology of normal aging and diabetes. Glycation derived AGEs, especially