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immunological screening of post-translationally modified peptides to characterize autoantibodies in patients’ sera
Cédric Rentier
To cite this version:
Cédric Rentier. Autoimmune diseases : design, synthesis and immunological screening of post- translationally modified peptides to characterize autoantibodies in patients’ sera. Organic chem- istry. Université de Cergy Pontoise; Università degli studi (Florence, Italie), 2015. English. �NNT : 2015CERG0746�. �tel-01214960�
AND OF
THE UNIVERSITY OF FIRENZE
Autoimmune diseases: design, synthesis and immunological screening of post-translationally modified peptides to characterize autoantibodies in
patients' sera
Presented by Cédric RENTIER
French-Italian Laboratory of Peptide and Protein Chemistry and Biology Laboratoire de Chimie Biologique (LCB EA 4505)
Platforme PeptLab@UCP
and Laboratorio di Chimica e Biologia di Peptidi e Proteine (PeptLab@UniFi) Thesis defended on July, 17th 2015
Evaluation Committee:
Prof. Anna Maria PAPINI PhD supervisor
Prof. Paolo ROVERO PhD co-supervisor
Prof. Nadège LUBIN-GERMAIN PhD co-supervisor Prof. Solange LAVIELLE Referee
Prof. Giancarlo MORELLI Referee
Prof. Claudio TONIOLO Examiner
Prof. Thierry BRIGAUD Examiner
Dr. Karen WRIGHT Examiner
Dr. Elisa PERONI Invited member
This PhD work was realized in the French-Italian Laboratory of Peptide and Protein Chemistry and Biology, a research unit joining the Laboratory of Chemical Biology and the platform PeptLab@UCP of the University of Cergy- Pontoise and the Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology (PeptLab@UniFi) of the University of Firenze.
As a project that occupied four years of my life, it would have happened much differently (or not happen at all) without a few people. In the next paragraphs, I will express my gratitude and thoughts to a few of these people, in the same way I worked these last few years: in three languages at once...
En premier lieu, je tiens à remercier ma directrice de thèse, la Prof. Anna Maria Papini. Elle m’aura fait découvrir le monde des peptides et des protéines en tant qu’étudiant, avant de m’offrir une place au sein de son équipe pour y effectuer ma thèse. Son énergie au travail et son exigence à me pousser toujours plus haut et plus loin dans mes limites auront été une formidable source d’inspiration. C’est également grâce à elle que j’aurai eu la chance de pouvoir effectuer une partie de mes travaux de recherche à l’Université de Florence, dans le contexte d’une cotutelle de thèse. Sans son soutien et sa ténacité, ce projet n’aurait probablement pas vu le jour. Qu’elle trouve ici l’expression de toute ma gratitude, mon estime et ma haute considération.
Mes deux co-directeurs de thèse, la Prof. Nadège Lubin-Germain de l’Université de Cergy-Pontoise et le Prof. Paolo Rovero de l’Université de Florence, sont également chaleureusement remerciés. L’un comme l’autre ont toujours été disponibles pour m’écouter dans les moments d’incertitude, fut- elle personnelle ou scientifique, pour me conseiller et me guider au mieux.
I would also like to thank the two referees of this PhD thesis: Prof. Solange Lavielle, from the Université Pierre et Marie Curie and the Prof. Giancarlo Morelli, from the University of Napoli “Federico II”. They kindly accepted to read my manuscript and evaluate this work.
The same goes for the examiners of my thesis: Prof. Claudio Toniolo from the University of Padova, Dr. Karen Wright from the Université de Versailles and Prof. Thierry Brigaud from the Université de Cergy-Pontoise. I am glad they accepted to be present for the discussion.
A Cergy-Pontoise, quelques collègues ont occupé une place particulière.
Chacun saura ce que je lui dois et ce qu’il m’aura apporté, et il n’est donc nul besoin d’exprimer par écrit une liste détaillée. Merci donc aux personnes suivantes: Dr. Olivier Monasson, Dr. Elisa Peroni, Mlle. Matthaia Ieronimaki, Mr. Nicolas Pasternak, Dr. Lotfi Tak-Tak, Dr. Andrea Bordessa, Mr. Simon Gonzalez, Mme Jocelyne Gougerot. Que ceux qui ne figurent pas dans la liste me pardonnent, et qu’ils soient assurés que j’aurai un mot pour eux.
A Firenze, vorrei anche ringraziare tutti coloro che ho potuto incontrare e frequentare, che mi hanno aiutato a imparare la lingua italiana, che mi hanno aiutato in laboratorio o che mi hanno aiutato per qualsiasi cosa. Lo sapete che senza tutti voi, non avrei mai potuto fare ciò che ho fatto a Firenze, quindi ecco i ringraziamenti per: Dr. Francesca Nuti, Dr. Giulia Pacini, Dr. Giada Rossi, Dr. Feliciana Real-Fernandez, Dr. Enrico Koenig, Dr. Mario Chelli, Dr.
Giuseppina Sabatino, Dr. Fabio Rizzolo, Dott. Francis Ciolli, Dr. Chiara Testa, Dr. Caterina Tiberi, Dr. Margherita Di Pisa, Dott.ssa Simona Pascarella, Dott.
Raffaelo Paolini, Dr. Alessandro Pratesi, Dott.ssa Martina Trabucco, Dott.ssa Martina Crulli. Grazie mille a tutti!
A word for the collaborators working with me on the various projects I have been involved on: Prof. Annunziata Lapolla (University of Padova), Prof. Piero Traldi (CNR Padova), Dott. Marco Roverso (CNR Padova), Prof. Carlo Selmi
(University of Milano), Prof. Alfonso Carotenuto (University of Napoli), Prof.
Paola Migliorini (University of Pisa).
This thesis would not have been possible without the financial support of several institutions that I would like to thank here: the French National Research Agency (ANR) for the Chaire d’Excellence 2009-2014 project
“Pepkit: development of peptide-based diagnostic kits for autoimmune diseases” (grant n° ANR-09-CEXC-013-01); the French-Italian University for their mobility program (Vinci Project grant n° C2-133) and the Ente Cassa di Risparmio di Firenze for the support to PeptLab@UniFi.
Special highlights to Sofia, my soon-to-be sister-in-law, who kindly accepted to proofread the manuscript and fix my English. You did an amazing job, and I will probably never be grateful enough for that. Thanks !
Je termine par un mot à ma famille: ma mère France, mon père Michel, mon frère Frédéric. Sans vous, rien n’aurait été possible, et vous savez pourquoi.
Merci. Cette thèse, si elle est pour moi, elle vous est aussi dédiée.
Et Marie. Inutile d’écrire quoique ce soit ici, les mots ne suffiraient pas.
Cette thèse est dédiée à mon grand-père, Maurice, parti trop tôt, avant d’avoir pu savourer ce manuscrit dans sa version finale. Sa discrète efficacité et son goût du travail bien fait auront été une grande source d’inspiration.
Abstract (English)
This research work aims to apply the novel concept of “Chemical Reverse Approach” to the design, the production, and the immunological screening of synthetic antigens able to specifically detect autoantibodies in sera of patients affected by immune-mediated diseases. Such specific autoantibodies are considered disease biomarkers and can be used to develop novel diagnostic/prognostic tools for the aforementioned pathologies.
In particular, three diseases have been investigated.
Primary biliary cirrhosis (PBC) is an autoimmune cholestatic disease of the liver, characterized by progressive destruction of intrahepatic bile ducts.
Specific antimitochondrial autoantibodies directed against a lipoylated epitope of the PDC-E2 protein, are considered relevant for the disease. The PDC-E2 protein is involved in the energetic cycle of mitochondria. Synthesis of lipoylated molecular probes based on PDC-E2(167-186) was carried out and optimized. These new synthetic antigens were tested on PBC patients’ sera.
The results showed that: 1) the sequence is fundamental for antibody recognition; 2) dithiolane lipoyl-lysine chirality does not seem to have any significant influence on antibody recognition; 3) the unlipoylated analogue of the native protein appears to detect a more relevant antibody titre than the lipoylated one.
Diabetes is a disease characterized by hyperglycaemia. This condition is caused by the reduced or inexistent action of insulin. Hyperglycaemia can cause various modifications on circulating proteins (such as glycation, O- glycosylation, N-glycosylation).
The synthesis of post-translationally modified peptides containing such structures was carried out. These new synthetic antigenic probes were tested on sera from patients suffering from diabetes. The results showed that three peptides among those tested can differentiate patients with type-1 diabetes
(autoimmune form) from those with type-2 (non-autoimmune form) and healthy patients.
Multiple sclerosis is a demyelinating disease of the central nervous system. The immune system destroys proteins components of myelin sheath. Kynurenine is a major metabolite of tryptophan, and it has been shown that in multiple sclerosis, the metabolic pathway of tryptophan may be deregulated.
Thus, the synthesis of modified peptides incorporating a kynurenine as an aberrant post-translational modification was carried out. The results show no specific antibody detection in multiple sclerosis sera.
Abstract (French)
Ces travaux de recherche visent à mettre au point par une nouvelle « Approche Chimique Inverse » des antigènes synthétiques pour le diagnostic de maladies auto-immunes; utilisant les autoanticorps spécifiques de ces pathologies en tant que biomarqueurs.
L’efficacité de ces peptides modifiés en tant qu’outils pour le diagnostic est évaluée par un screening au moyen de tests immunoenzymatiques (SP-ELISA), de sérums de patients atteints de pathologies auto-immunes sélectionnées.
Trois maladies ont été étudiées en particulier.
La cirrhose primitive biliaire est une maladie auto-immune cholestatique affectant le foie, caractérisée par une destruction progressive des canaux intra- hépatiques, pouvant mener à une cirrhose. Il existe des autoanticorps antimitochondriaux spécifiques à cette maladie qui reconnaissent un epitope lipoylé de la PDC-E2, protéine impliquée dans le cycle énergétique mitochondrial. La synthèse de sondes moléculaires lipoylées basées sur la PDC-E2(167-186) a été mise au point et optimisée. Les antigènes synthétiques ont ainsi été testés sur des sérums de patients de PBC. Les résultats montrent que: 1) la séquence a une importance pour la reconnaissance des anticorps; 2) la chiralité du dithiolane de la lipoyl-lysine ne semble pas avoir d’influence majeure sur la reconnaissance des autoanticorps; 3) l’absence de lipoylation sur le mime de la protéine native semble donner de meilleurs résultats que l’antigène synthétique lipoylé.
Le diabète est une maladie caractérisée par une hyperglycémie provoquée par l’action réduite ou inexistante de l’insuline. L’excès de sucre dans le sang peut provoquer des modifications de diverses natures sur les protéines circulantes (glycation, O-glycosylation, N-glycosylation).
La synthèse de sondes moléculaires portant ces structures en tant que modifications post-traductionnelles a été mise au point. Les antigènes
synthétiques ont ainsi été testés sur des sérums de patients atteints de diabète.
Les résultats montrent que trois des peptides testés permettent de différencier les patients atteints de diabète de type 1 (forme autoimmune) de ceux de type 2 (forme non autoimmune) ainsi que des controles sains.
La sclérose en plaques est une maladie impliquant une démyélinisation des fibres nerveuses du système nerveux central. Le système immunitaire détruit les protéines composante cette gaine de myéline. La kynurénine est un des métabolites principaux du tryptophane, et il a été montré que dans la sclérose en plaques, la voie métabolique du tryptophane pourrait être déréglée.
Ainsi, la synthèse de peptides modifiés portant une kynurénine comme modification post-traductionnelle aberrante a été menée à bien. Les résultats ne montrent pas de détection particulière d’anticorps dirigés contre cette modification dans le cas de la sclérose en plaques.
Abstract (Italian)
Questo progetto di ricerca è basato sull’ ”Approccio Chimico Inverso”. Questo approccio si basa sulla la progettazione, la sintesi e lo screening immunologico di sonde antigeniche sintetiche capaci di riconoscere anticorpi specifici nel siero di pazienti affetti da malattie autoimmuni. Gli anticorpi specifici sono utili come biomarker di malattia e possono quindi essere usati per sviluppare strumenti diagnostici e/o prognostici innovativi.
In particolare sono state oggetto dei nostri studi: 1) la cirrosi biliare primitiva;
2) il diabete; 3) la sclerosi multipla.
La cirrosi biliare primitiva (CBP) è una malattia autoimmune che provoca un'infiammazione cronica delle vie biliari intraepatiche con conseguente distruzione di quest’ultime. È stato dimostrato che gli anticorpi antimitocondrio sono diretti contro un epitopo lipoilato della proteina PDC-E2. Questa proteina è coinvolta nel metabolismo energetico che si realizza nei mitocondri. In questo lavoro di ricerca è stata perciò effettuata e ottimizata la sintesi di sonde lipoilate derivate del frammento (167-186) della PDC-E2. Gli antigeni sintetici così ottenuti, sono stati testati sul siero di pazienti affetti da CBP. I risultati hanno mostrato che: 1) la sequenza peptidica è fondamentale per il riconoscimento anticorpale; 2) la chiralità del ditiolano sulla lisina lipoilata non sembra avere alcuna influenza sul riconoscimento anticorpale; 3) l’analogo non lipoilato della proteina nativa risulta la migliore sonda sintetica.
Il diabete è una malattia caratterizzata da una iperglicemia causata da una ridotta o assente attività dell’insulina. L’iperglicemia può indurre diverse modificazioni aberranti delle proteine (glicazione, O-glicosilazione, N- glicosilazione). In questo lavoro di ricerca è stata effettuata la sintesi di peptidi modificati con le strutture sopra riportate. Questi antigeni sintetici sono stati perciò testati su sieri di pazienti affetti da diabete. I risultati hanno dimostrato
che tre dei peptidi sintetizzati sono in grado di discriminare tra pazienti affetti da diabete di tipo 1, pazienti affetti da diabete di tipo 2 e donatori sani.
La sclerosi multipla (SM) è una patologia demielinizzante del sistema nervoso centrale nella quale il sistema immunitario distrugge alcune proteine presenti sulla guaina mielinica. La chinurenina è il principale metabolita del Triptofano il cui metabolismo può essere alterato nei pazienti affetti da SM. È stata perciò effettuata la sintesi di peptidi modificati con la chinurenina al fine di verificare la presenza di anticorpi specifici nel siero di pazienti affetti da SM. I risultati dei test biologici, tuttavia, non hanno mostrato alcuna interazione specifica tra i peptidi modificati e possibili anticorpi circolanti nel siero dei pazienti affetti da SM.
Abbreviations
AID Autoimmune Disease
AA Amino acid
Ab Antibody
Abs Absorbance
Ac Acetyl
ACP Acyl carrier protein
Ag Antigen
AMA Anti-Mitochondrial Antibodies amu Atomic mass unit
Auto-Ab Autoantibody Auto-Ag Autoantigen BB Building block Boc Tert-butoxycarbonyl
Bzl Benzyl
CoA Coenzyme A
CRA Chemical Reverse Approach DCC N,N-dicyclohexylcarbodiimide DCM Dichloromethane
DHLA Dihydrolipoic acid
DIC N,N-diisopropylcarbodiimide DIPEA Diisopropylethylamine DMF N,N-dimethylformamide DMSO Dimethylsulfoxide
DMTMM-BF4 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate
DTT Dithiothreitol E. Coli Escherichia Coli EDT Ethanedithiol
ELISA Enzyme-Linked Immunosorbent Assay
ESI-MS Electrospray ionization-mass spectrometry Fab Fragment antigen-binding
Fc Fragment crystallizable FBS Fetal Bovine Serum
FCC Flash column chromathography Fmoc 9-H-fluoren-9-ylmethoxycarbonyl
Glc Glucose
GlcNAc N-Acetyl-Glucosamine
HATU O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HFA Hexafluoroacetone HOBt Hydroxybenzotriazole
HPLC(-MS) High Performance Liquid Chromatography (Mass Spectrometry) IDO Indoleamine-2,3-dioxygenase
Ig Immunoglobulin
KP Kynurenine pathway
Kyn Kynurenine
KynA Kynurenic acid
LA Lipoic acid
LipA N-octanoyltransferase
LipB Lipoyl (octanoyl)-transferase LplA Lipoate-protein ligase
MHC Major Histocompatibility Complex MS Multiple Sclerosis
MW Microwaves
NBD Normal Blood Donors NHS N-hydroxysuccinimide NMM N-methylmorpholine NMR Nuclear magntic resonance
OAID Other non-hepatic autoimmune diseases OD Optical density
OSu O-succinimide OVA Ovalbumin
Oxyma(Pure) Ethyl-2-cyano-2-(hydroxyimino)acetate PBC Primary Biliary Cirrhosis
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PDC-E1 Thiamine diphosphate-dependent dicarboxylase PDC-E2 Dihydrolipoamide acetyltransferase
PDC-E3 Dihydrolipoyl dehydrogenase PDH Pyruvate dehydrogenase Pfp Pentafluorophenyl
PTM Post-Translational Modification QuinA Quinolinic acid
ROC Receiver operating characteristic
RP Reverse phase
SLCF Spot Liquid Chromatography Flash SP-ELISA Solid Phase ELISA
SPPS Solid-Phase Peptide Synthesis T1D Type-1 diabetes
T2D Type-2 diabetes
TBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
tBu Tert-butyl
TCEP Tris(2-carboxyethyl)phosphine TDO Tryptophan-2,3-dioxygenase TFA Trifluoroacetic acid
THF Tetrahydrofurane TIS Triisoprpylsilane
TLC Thin layer chromatography
Trt Trityl
UPLC Ultra High Performance Liquid Chromatography
UV Ultraviolet
TABLE OF CONTENTS
GENERAL INTRODUCTION ... 1
1.AUTOIMMUNITY: INTRODUCTION TO THE IMMUNE SYSTEM ... 1
1.1. Innate immune system and adaptive immune system ... 1
2.DISCRIMINATING SELF AND NON-SELF: ANTIGENS AND ANTIBODIES ... 2
2.1. Discrimination between “self” and “non-self” ... 2
2.2. Antigens and antibodies... 4
2.3. Antigen-antibody interactions: epitopes and paratopes ... 7
3.AUTOIMMUNE DISEASES: FROM SELF- TO NON-SELF RECOGNITION ... 8
3.1. Loss of T regulatory cells effect ... 9
3.2. Molecular mimicry ... 10
3.3. Post-translational modifications of proteins ... 11
3.4. Autoimmune diseases diagnosis and biomarkers ... 13
4.BIOMARKERS AND CHEMICAL REVERSE APPROACH ... 15
4.1. Synthetic peptides mimicking autoantigens ... 16
4.2. Synthetic peptides in SP-ELISA ... 18
5.CHEMICAL REVERSE APPROACH AND MULTIPLE SCLEROSIS ... 19
CHAPTER ONE: PRIMARY BILIARY CIRRHOSIS ... 21
1.INTRODUCTION ... 21
1.1. PBC, a cholestatic autoimmune disease of the liver ... 21
1.3. Identification of the immunogenic region of PDC-E2 ... 25
1.3.1. Immunogenicity of the sequence ... 27
1.3.2. Immunogenicity of the lipoylation ... 28
1.3.3. Immunogenicity of the conformation ... 29
1.4. General overview of lipoylation chemistry in vivo and in vitro ... 30
1.4.1. Lipoylation in vivo ... 33
1.4.2. Lipoylation in vitro ... 36
1.5. Objectives of the project ... 43
2.RESULTS AND DISCUSSION ... 46
2.1. Synthesis of the lipoylated lysines ... 46
2.1.1. Two-step reaction: the pentafluorophenylester route ... 47
2.1.2. Optimization of the synthesis of Fmoc-L-Lys(Nε-lipoyl) building block ... 50
2.1.3. Separation of the diastereomers ... 54
2.2. Peptide synthesis ... 57
2.2.1. Selection of the peptide sequences ... 57
2.2.2 Chemical modifications introduced in the peptide sequences ... 59
2.2.3. Preparation of a collection of modified peptides for PBC diagnosis ... 61
2.2.3.1. Lipoylated peptides cleavage: stability of the S-S bond ... 63
2.2.3.2. Management of lipoylated peptides S-S bond in ELISA ... 67
2.3. SP-ELISA ... 71
2.3.1. Importance of the lipoylation ... 71
2.3.2. Importance of the peptide sequence ... 73
2.3.3. N-glucosylation relevance in PBC ... 76
2.3.4. Use of modified peptides for PBC diagnosis ... 80
2.3.5. Tests of dihydrolipoylated peptides ... 85
2.4. Conformational studies ... 89
3.CONCLUSION ... 93
CHAPTER TWO: DIABETES ... 95
1.INTRODUCTION ... 95
1.1. Diabetes mellitus ... 95
1.1.1. Type-1 diabetes ... 97
1.1.2. Type-2 diabetes ... 99
1.2. Post-translational modifications possibly involved in diabetes ... 101
1.2.1. N-Glycosylation and glycation ... 102
1.2.2. O-Glycosylation and phosphorylation ... 105
1.3. Objectives of the project ... 109
2.RESULTS AND DISCUSSION ... 112
2.1. Sequence selection ... 112
2.2. Modifications introduced into peptides ... 112
2.3. Peptide synthesis ... 114
2.3.1. Peculiarities of GlcNAc(OAc)3 deprotection ... 116
2.3.2. Cleavage of 1-deoxyfructosyl-Lys moiety-containing peptides ... 117
2.3.3. Phosphorylated peptides ... 118
2.4. SP-ELISA ... 120
3.CONCLUSION ... 133
CHAPTER THREE: MULTIPLE SCLEROSIS ... 135
1.INTRODUCTION ... 135
1.1. Multiple Sclerosis ... 135
1.2. The kynurenine pathway ... 136
1.3. Kynurenine pathway involvement in Multiple Sclerosis ... 141
1.4. Introduction of Kynurenine in peptides ... 142
1.5. Objectives of the project ... 148
2.RESULTS AND DISCUSSION ... 149
2.1. Synthesis of Kyn-containing CSF114 analogues ... 149
2.2. SP-ELISA ... 151
3.CONCLUSION ... 155
CONCLUSIONS ... 157
EXPERIMENTAL PART ... 161
1.MATERIALS AND METHODS ... 161
2.GENERAL PROCEDURE FOR SOLID PHASE PEPTIDE SYNTHESIS ... 163
2.1. General procedure for in batch SPPS on the manual PLS 4x4 synthesizer ... 163
2.2. General procedure for in batch SPPS on an automatic peptide synthesizer... 164
2.3. Kaiser test procedure ... 165
2.4. General procedure for cleavages and micro-cleavages ... 166
2.5. General procedure for purification and characterization ... 167
3.GENERAL PROCEDURE FOR SP-ELISA ... 168
4.EXPERIMENTAL PART CHAPTER ONE ... 169
4.1. Synthesis of α-lipoic acid-OPfp esters (1, 2 and 3) ... 169
4.2. Synthesis of Fmoc-L-Lys(Nε-lipoyl)-OH building blocks 4, 5 and 6 ... 170
4.2.1. Using lipoyl pentafluorophenyl ester route ... 170
4.2.2. Synthesis of 4 using HATU/DIPEA ... 171
4.2.3. Synthesis of 4 using Oxyma/DIC ... 172
4.2.4. Synthesis of 4 using DMTMM-BF4 ... 173
4.3. Solid Phase Peptide Synthesis ... 173
4.3.1. Synthesis of CSF114 glycosylated analogues ... 173
4.3.2. Synthesis of CSF114 lipoylated analogues ... 174
4.3.3. Synthesis of PDC-E2(167-186) glycosylated analogues ... 175
4.3.4. Synthesis of PDC-E2(167-186) lipoylated analogues ... 176
4.3.5. Procedure for deprotection of Glc moieties... 177
4.3.6. Procedure for disulfide bond reduction ... 178
4.3.7. Procedure for disulfide bond oxidation ... 178
4.4. SP-ELISA protocol for dihydrolipoyl peptides ... 178
4.5. Circular dichroism ... 179
4.6. NMR spectroscopy for conformational studies ... 179
4.7. Structure calculation by molecular dynamics ... 180
5.EXPERIMENTAL PART CHAPTER TWO ... 182
5.1. Synthesis of glycated peptides ... 182
5.2. Synthesis of O-glycosylated and phosphorylated peptides ... 183
5.2.1. Deprotection of Glc moieties ... 184
6.EXPERIMENTAL PART CHAPTER THREE ... 185
6.1. Synthesis of CSF114 kynureninated analogues ... 185
LIST OF PUBLICATIONS AND COMMUNICATIONS OF C. RENTIER .... 187
SUPPLEMENTARY MATERIAL………...191
General introduction
1. Autoimmunity: introduction to the immune system
Immunity is one of the most wonderful examples of complex machineries merging together chemistry and biology to serve a very simple, yet complex purpose: protecting the organism against any form of external agent.
In the following sections, this thesis will outline the basis of immunity, in order to give a comprehensive overview of notions such as autoimmunity, antigens and antibodies.
1.1. Innate immune system and adaptive immune system
Protection against external aggressions, i.e. pathogens, such as bacteria or viruses can be seen as a multilayer system.
The first layer is purely physical. In the case of human beings, the easiest example is the skin, but gastrointestinal system and respiratory airways are protected as well.
The second layer, which intervenes in the case of breaching of the first barrier, is the innate immune system, a general and immediate (but non persistent) response of the organism against pathogens. This system is present in almost all living organisms. Cells of the innate immune response are called leukocytes (or also white blood cells) and can be of a diverse nature: phagocytes (macrophages, neutrophils, monocytes, dendritic cells) absorb pathogens to destroy them, releasing inflammatory mediators, e.g. cytokines, which stimulate other responses from the organism against the potential threat; or natural killer cells, which induce apoptosis in infected cells. The complement system, which is a biochemical cascade of reactions between specific plasma
proteins that “complement” the immune system in cleaning, tagging or destroying pathogens, is also a part of the innate immune system.
The third layer, much more specific, is the adaptive immune system. Unlike the innate immune system, it is found exclusively in vertebrates and is highly specific to a given pathogen. Adaptive immunity can create immunological memory after an initial response to a specific pathogen, leading to an enhanced response to subsequent encounters with that pathogen.
The adaptive response mainly involves the clonal expansion of a subtype of leukocytes, namely B and T lymphocytes, which are produced by stem cells in the bone marrow.
T lymphocytes (also called T cells) are characterized by the presence at their surface of T cell receptors, while B cells express B cell receptors. The main difference between T cells and B cells lies in their recognition of foreign objects. B cells can recognize linear or conformational epitopes on their own, while T cells need the assistance of antigen presenting cells. Their mode of action is also different: T cells will either enhance the immune response by cytokines production (T helper cells, also known as CD4+ T cells), or destroy infected cells (cytotoxic T cells, also known as CD8+ T cells); while B cells either produce antibodies that will bind to antigens, or survive in the immune system for long periods of time (up to years) in order to quicken the reaction of the immune system in case of exposure to the same antigen.
2. Discriminating self and non-self: antigens and antibodies 2.1. Discrimination between “self” and “non-self”
Discrimination between self- and non-self recognition is absolutely fundamental for the adaptive immune system. This is partly accomplished by
the major histocompatibility complex (MHC), a molecule expressed at the surface of the cells, that can be either class I or class II.
MHC class I molecules are expressed by nearly all nucleated cells in the organism and comprise peptides of 8-9 amino acids in length presented to CD8+ T cells to alert possible intracellular infections (this is pictured in Figure 1 below). If an anomaly is detected in presentation by MHC, death of the defective cell is programmed by apoptosis.
Figure 1: Epitope presentation to the T cell receptor by a MHC molecule
MHC class II molecules, on the other hand, are expressed by antigen presenting cells (such as macrophages or dendritic cells) which are in charge of the presentation of the epitope (usually 15-30 amino acids), obtained by proteolytic cleavage of the antigens. The epitope-MHC complex is presented to CD4+ T cells that then activate the B cells that will then start to produce specific antibodies.
B cells can also recognize antigens by themselves, without the assistance of MHC molecules, as the B cell receptors expressed are antigen-specific.
Binding of a B cell with its cognate antigen, if a CD4+ T cell gives an additional signal, leads to the production of specific antibodies.
Thus, self- and non-self recognition is strongly dependent on presentation of epitopes to T lymphocytes by MHC molecules (either class I or class II, each
one eliciting a different mechanism), or direct recognition by B lymphocytes.
Fortunately, the immune system is able to distinguish between the self and therefore peptides presented in this way to MHC do not trigger any immune response under normal circumstances. This is called “immune tolerance”.
2.2. Antigens and antibodies
Strictly speaking, an antigen (Ag) can be defined as a substance that can bind to the receptors of the adaptive immune response (T and B cell receptors).
However, three different kinds of antigens are recognized:
- exogenous antigens: these Ags have entered the body from the outside, after breaching through physical barriers and innate immune system.
- endogenous antigens: these Ags are generated inside the cells, as by-products of normal cellular metabolism, or after a cell infection by viruses or bacteria.
- autoantigens: these Ags consist of normal peptides or proteins, components of the organism that are not recognized anymore as self by the immune system because of a malfunction of the “immune tolerance” system.
An immunogen is an Ag that elicits an immune response from the adaptive immune system. If an immunogen is always an Ag, the contrary is not true.
The best example that can be given are haptens, a family of low molecular weight antigenic compounds that are not immunogenic, unless they are bound to a carrier molecule.
Antibodies (Abs), also termed immunoglobulins (Igs), are molecules composed of two heavy (~55 kDa) identical polypeptides chains and two identical light (~25 kDa) polypeptide chains, held together by interchain disulfide bonds.
These large proteins (~150 kDa), produced by plasma cells (differentiated B
lymphocytes) are Y-shaped and are used by the organism to selectively identify and neutralize Ags1,2.
The general structure of an Ab is represented below (Figure 2). Sugars can be linked to some of their amino acid residues, thus Igs are glycoproteins. The
“arms” of the antibody are termed the Fab region (for Fragment antigen- binding), whilst the “base” is called Fc region (Fragment crystallizable). The Fab region is involved in Ag binding (and thus important for specific Ag recognition), while the Fc region is more involved in interactions with surface- cell receptors of the immune system: for example, they allow recognition and ingestion of pathogens by phagocytic cells; or they can interact with proteins activating the complement system.
The connection between Fab and Fc regions is rich in Proline, Threonine, and Serine and is known as the hinge.
Figure 2: Y-Shaped antibody structure (including Fc and Fab regions), and typical Fc-linked N-glycans.
In mammals, there are five different types of Abs, termed isotypes: IgG, IgM, IgA, IgE, and IgD. They differ mainly in their biological properties, and each isotype contains a different heavy chain. This is summarized in the Table 1.
1 Poljak, R.J. Mol. Immun. 28, 1341-1345 (1991).
2 Davies, D.R.; Cohen, G.H. Proc. Natl. Acad. Sci. USA, 93, 7-12 (1996).
Table 1: The five isotypes of Ig in mammals.
IgG is the most important isotype among the five, providing the majority of antibody-based immunity against exogenous Ags. Found in blood and extracellular fluids in monomeric form, it is the most abundant (~75%) of serum Igs in humans. It is involved in the secondary immune response, produced in the later stages of the immune process, but lasting longer in the organism. Characterized by its ability to easily cross cell membranes, it is the only Ab able to cross the placenta to passively immunize the fetus.
IgM is the largest isotype, usually found in a pentameric form (~975 kDa), forming a “star” of Y-shaped Abs. They are found in blood and form the very first response to a pathogenic invasion, being the primary isotype produced by B cells. Although they are effective at binding Ags because of their higher valency, they cannot cross cell membranes.
IgA isotype is principally found in mucosal areas as a dimer (gastrointestinal, genitourinary, and respiratory tracts), but also in tears, saliva and mucus. It plays a critical role by eliminating pathogens in these zones (mainly by agglutination), thus avoiding pathogenic colonization.
IgE antibodies are present in mucous membranes, lungs, and skin. They have a function in immune response towards allergens and are also believed to protect
against certain parasitic worms. It is the least common isotype of Ab in the organism and comes as a monomer.
IgD is another monomeric antibody (with IgG and IgE) found in very small amounts in blood serum. They are mainly expressed on B cell surfaces as antigen receptors and may play a role in eliminating the B-lymphocytes, which generate self-reactive autoantibodies.
2.3. Antigen-antibody interactions: epitopes and paratopes
Antigen-antibody interaction is performed through an epitope-paratope interaction, which can be seen as a key-lock system. The epitope is the part recognized by the Ab and usually comprises 6-7 amino acids; the paratope is the binding part of the Ab that selectively recognizes the epitope. The paratope is located at the end of the Fab region, and usually is the same for both arms of an antibody.
These interactions are weak, non-specific and of a complex nature3,4. This means binding between an antibody and an antigen is reversible and for this reason interactions between antibodies and antigens are to be considered as relative rather than absolute. The formation and dissociation of antigen- antibody complexes follows the basic thermodynamic principles of any reversible biomolecular interaction.
Epitopes can be divided in two categories:
- linear epitopes: these epitopes are composed of a given amino acid sequence.
Also termed continuous epitopes, they are accessible for interaction with antibodies if they are exposed, e.g. at the surface of an antigen or in a conformationally extended region of a folded protein antigen.
3 Van Oss, C.J. Molecular Immunology 32, 199-211 (1995).
4 Reverberi, R., Reverberi, L. Blood Transfus. 5, 227-240 (2007).
- conformational epitopes: also called discontinuous epitopes, these need a particular conformation for the formation of the epitope. It often comes from the protein folding, which allows distant amino acids in the sequence to be spatially close one to each other.
Because most antigens are highly complex, they present numerous epitopes that are recognized by a large number of lymphocytes. Each lymphocyte is activated to proliferate and differentiate into plasma cells, and the resulting antibody response is polyclonal. In contrast, monoclonal antibodies are antibodies produced by a single B lymphocyte clone.
3. Autoimmune diseases: from self- to non-self recognition
Under normal circumstances, the self- and non-self are well differentiated by the organism and the innate and adaptive immune systems. A balance between immune response and regulatory mechanisms of the immune system allows the whole mechanism to be kept under control.
An autoimmune disease (AID) is characterized by loss of the tolerance to the self and an autoreactivity against self-Ags. A crucial feature of an autoimmune disease is that tissue injury is caused by the immunologic reaction of the organism to its own tissues, and the production of antibodies against the organisms own tissues (autoantibodies, autoAbs). It may lead to destruction of one or more types of body tissues, abnormal growth of an organ, and changes in organ functions. These attacks of the immune system can be directed against a particular type of cells, or instead can be more systemic and widespread.
A disease is considered as an AID if it is in line with Witebsky’s postulates5,6: direct evidence from transfer of pathogenic antibodies or pathogenic T cells;
indirect evidence based on reproduction of the autoimmune disease in experimental animals; and circumstantial evidence from clinical clues.
They affect about 8% of the total world population7, with 3 patients out of 4 being women8, which generally have a higher risk of AIDs development (although exceptions exist)9. Direct consequence of these diseases is the high social impact they have on the patients.
AIDs are always complex diseases, with mechanisms that might not always be completely understood. Currently, it is accepted that AIDs are multifactorial diseases: genetics, xenobiotic contaminants, bacterial or viral infections have been proved to be important aetiological agents.
Three pathways for immune tolerance breakdown are outlined in the following sections: by loss of the immune system downregulation (T-reg cells), by cross- reactivity of a self protein with microbial specific Abs (molecular mimicry), and by chemical modification of self proteins (aberrant post-translational modifications).
3.1. Loss of T regulatory cells effect
T regulatory cells (formerly known as T suppressor cells) play a crucial role in the control of this balance, by being responsible for the elimination of
5 Witebsky, E., Rose, N.R., Terplan, K., Paine, J.R., Egan, R.W. J. Am. Med. Assoc. 164, 1439- 1447 (1957).
6 Rose, N.R., Bona, C. Immunol. Today 14, 426-430 (1993).
7 Cooper, G.S., Bynum, M.L.K., Somers, E.C. J. Autoimmun. 33, 197-207 (2009).
8 Jacobson, D.L., Gange, S.J., Rose, N.R., Graham, N.M. Clin. Immunol. Immunopathol. 84, 223-243 (1997).
9 Beeson, P.B. Am. J. Med., 96, 457-462 (1994).
autoreactive T cells. They downregulate or suppress the reactivity of the CD4+ and CD8+ cells towards self- components of the organism.
If, for any reason, the balance between effector T cells (helpers and cytotoxics) and regulatory T cells is disrupted, then the immune system downregulation is lost and an immune process can be initiated.
This is a possible cause for Systemic Lupus Erythematosus and rheumatoid arthritis, as reviewed by Chavele et al.10 and pictured below (Figure 3).
Figure 3: Loss of the balance between T effectors and T regulatory cells (by Chavele et al.10)
3.2. Molecular mimicry
Due to the wide diversity of molecular structures, it is possible to have exogenous pathogens (in particular microbial molecules) that have similar structure and/or conformation to mammalian surface glycoproteins and/or glycolipids, as shown in the Figure 4.
10 Chavele, K.M., Ehrenstein, M.R. FEBS Lett. 585, 3603-3610 (2011).
A possible outcome is the cross-reactivity of the self molecule and the exogenous Ag, leading to activation of autoreactive lymphocytes. This is termed molecular mimicry.
Molecular mimicry has been reported in type I diabetes mellitus (T1DM), RA, and multiple sclerosis (MS)11. The most commonly identified triggering agents are pathogens having carbohydrate sequences (considered the antigens) in common with the peripheral nervous tissue.
Figure 4: Illustration of the molecular mimicry concept (by Oldstone12)
3.3. Post-translational modifications of proteins
Post-translational modifications (PTMs) are chemical modifications of proteins, after their synthesis or during their life cycle. It is estimated that between 50 and 90% of proteins are post-translationally modified in vivo13. PTMs are diverse and abundant, and most proteins undergo proteolytic processing and/or the attachment (or removal) of functional groups to control protein folding, localization, activity, lifespan, and interactions with other molecules. Common examples of PTMs include glycosylation,
11Oldstone, M.B.A. Cell 50, 819-820 (1987).
12 Oldstone, M.B.A. The FASEB J. 12, 1255-1265 (1998).
13 Doyle, H.A., Mamula, M.J. Trends Immunol. 22, 443–9 (2001).
phosphorylation, ubiquitinylation, acylation, methylation, and amino acid conversions (Figure 5).
Most PTMs are the result of controlled enzymatic action, but modifications may also be introduced as a consequence of aging or exposure of proteins to chemically reactive environments. When PTMs are introduced in a non- contolled manner, they are referred as aberrant PTMs.
Figure 5: Common PTMs, including associated chemical structures and most frequently modified amino acids (by Petersen et al.14)
Such alterations in amino acid structures or sequences can generate neo- epitopes from self-proteins, and thus result in the loss of immune recognition15, initiating immune system self-reactivity, ultimately leading to AIDs16,17.
14 Petersen, J., Purcell A.W., Rossjohn J. J. Mol. Med. 87, 1045-1051 (2009).
15 Anderton, S.M. Curr. Opin. Immunol. 16, 753-758 (2004).
16 Doyle, H.A., Mamula, M.J. Ann. N. Y. Acad. Sci. 1050, 1-9 (2005).
3.4. Autoimmune diseases diagnosis and biomarkers
AIDs are a particular class of diseases, with high social impact on patients’
lives, and complex mechanisms. Diagnostic methods exist, but since every AID is different, there is not one single diagnostic method for every AID.
Moreover, cures for immune diseases, when they are available, are extremely expensive and have huge secondary effects on the patients (e.g.
immunosuppressant treatments).
Thus, it appears crucial to have at disposal methods that allow an early diagnosis of AIDs. Most of these methods are based on the detection either of the inflammatory markers that are usually associated with the immune response; or on auto-Abs produced by the immune system.
There are several methods for the detection of Ag-Ab complexes (Table 2).
Table 2: Sensitivity of various immunoassays
17 Doyle, H.A., Mamula, M.J. Curr. Opin. Immunol. 24, 112-118 (2012).
Each method has different sensitivities and ideally a test should be characterized by a good sensitivity, low-invasivity, low-cost, and ease of execution. Unfortunately, to date, in most cases the diagnostic and prognostic methods used for AID do not meet these criteria.
Some of these are based on the tendency of antibody-antigen complexes to come out of solution in what is called a “precipitation reaction”. Some are based on the ability of antibodies to stick cells together, called an
“agglutination reaction”. The third category comprises the immunoassays, including:
- Radio immunoassay: this uses radio-labeled Ags (e.g. 125I attached to Tyr) to measure Ag-Ab interactions18. Sensitive and precise, this technique however requires specialized equipment, precautions and licensing, since radioactive substances are used.
- Immunofluorescence: this technique uses Abs labeled with fluorescent dyes that will emit light at a particular wavelength when excited. It can be direct immunofluorescence, if the label is attached directly to the Ab of interest, but these are costly and the number of fluorophores that can be linked is limited.
Indirect immunofluorescence uses a labeled secondary Ab that can recognize the primary Ab (more precisely, they are specific to the heavy chain of Igs considered). It is currently the golden standard for AIDs diagnostics, although it can suffer from cross-reactivity or photobleaching (loss of fluorescence during excitation). In addition, this procedure is invasive as it requires tissue sections or isolated cells.
- Flow cytometry: this is a laser-based, biophysical technology that allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second by suspending cells in a stream of fluid and passing them through an electronic detection apparatus.
18 Yalow, R.S., Berson, S.A. J. Clin. Invest. 39, 1157-1175 (1960).
- Enzyme-Linked ImmunoSorbent Assay (ELISA): Described for the first time in 1971 by Engvall et al.19 ELISA is based on the primary immunologcal concept of Ag binding to specific Ab. The greatest advantage of this technique lies in the immobilization of one of the reactants to a solid surface: this feature makes ELISA easy to design and to perform, and enables the separation of bound from non-bound material during the assay, facilitating the measuring of specific analytes within a crude preparation. Similar to immunofluorescence, ELISA can be direct or indirect.
4. Biomarkers and chemical reverse approach
A biomarker is defined20 as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”
Specific autoAbs produced during the course of AIDs can serve as biomarkers.
This makes auto-Abs a valuable tool in the diagnosis of AIDs21,22, especially when auto-Abs are produced long before the apparition of first symptoms of the disease23,24,25; or when seric concentration of auto-Abs can be correlated with the course of the disease (emission/remission, efficiency of a treatment, etc.).
Unfortunately, in AIDs only very low specific Abs were demonstrated to be detectable in serum, possibly because proteins used in the assays cannot
19 Engvall, E., Perlmann P. Immunochemistry 8, 871-874 (1971).
20 Biomarkers Definition Working Group, Clin Pharmacol Ther. 69, 89-95 (2001).
21 Leslie, D., Lipsky, P., Notkins, A.L. J. Clin. Invest. 108, 1417-1422 (2001).
22Alcaro, M.C., Lolli, F., Migliorini, P., Chelli, M., Rovero, P., Papini, A.M. Chem. Today 25, 14-16 (2007).
23 Bizzaro, N. Autoimmun. Rev. 6, 325-333 (2007).
24 Bizzaro, N. Clin. Rev. Allergy Immunol. 34, 326-331 (2008).
25 Hu, Z.D., Deng, A.M. Clin. Chim. Acta. 437, 14-18 (2014).
reproduce the real Ag. Moreover, an entire protein can contain more than one epitope, thus leading to reactivity towards multiple Abs.
These observations account, at least in part, for the limited success attained in the discovery of biomarkers for AIDs using proteomic analysis and/or protein microarrays.
The diagnostics and prognostics of AIDs can be done using specific auto-Abs as biomarkers. It was observed that native or recombinant proteins are difficult to obtain (isolation, synthesis) with the correct sequences or the correct post- or co-translational modifications; and the tridimensional structure (folding). This makes such proteins inconvenient tools for simple and reproducible AIDs diagnostics.
4.1. Synthetic peptides mimicking autoantigens
Therefore, only the relevant part of the Ag is synthesized by classical means (solid- and liquid-phase synthesis), derived from native protein sequences and reproducing specific epitopes, or mimicking immunogenic modifications. This approach could be even more effective and specific than native proteins in antibody detection.
Its principal advantage is the complete control of the synthesized molecule:
either on the sequence or on the insertion of the correct PTM at the relevant position26. The folding issue can also be partly avoided: synthetic peptides, through sequence optimization, can adopt specific conformations (α-helical, β- turns, etc.). The most difficult aspect remains the identification of the immunogenic part, i.e. to decide which sequence should be synthesized, which modification should be introduced. Usually, the construction of the correct
26 Papini, A.M. “Peptide-based Immunoassays for Biomarkers Detection: a Challenge for Translational Research” Zervas Award Lecture 30th European Peptide Symposium, August 31- September 5 2008, Helsinki, Finland.
peptide is based on the molecular mechanism of and the analysis of circulating Abs for each disease.
This novel approach was developed a few years ago at the Laboratory of Peptide and Protein Chemistry and Biology of the University of Florence (PeptLab@UniFi) and called the “Chemical Reverse Approach” (CRA)27.
“Reverse” because the screening of the synthetic antigenic probe is guided by autoantibodies circulating in autoimmune disease patients’ sera. “Chemical”
because autoantibody recognition drives selection and optimisation of the
“chemical” structure from defined peptide collections. The whole process is represented in Figure 6.
Figure 6: Overall representation of the CRA.
The upper part of the figure (blue and purple) represents the immune-mediated process in AIDs: an aberrant PTM together with a loss of the immune tolerance triggers the immune response. In particular the creation of auto-Abs directed against the neo-Ag. The lower part illustrates the CRA. Synthetic peptides are designed, trying to reproduce immunogenic parts of neo-Ags (continuous/discontinuous epitopes, PTM involved). These synthetic peptides are used to detect possible specific auto-Abs, biomarkers of the AID.
27 Papini, A.M. J. Pept. Sci. 15, 621-628 (2009).
4.2. Synthetic peptides in SP-ELISA
The most simple way to evaluate synthetic peptides as molecular probes for AIDs auto-Abs is by ELISA.
The general ELISA procedure (indirect ELISA, shown in Figure 7) involves the immobilization of the Ag on a solid phase (coating), usually on a 96-well microtiter plate, and the consequent blocking of any un-coated areas of the well with a solution of non-reactive protein (e.g. fetal bovine serum, FBS). Then the given biological sample is applied, allowing the binding of the coated Ag to a specific Ab, which is subsequently detected bound by a secondary enzyme- coupled Ab. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.
Secondary Abs are commonly labeled with alkaline phosphatase or horseradish peroxidase, and a large selection of substrates is available. The choice depends upon the required assay sensitivity and the instrumentation available for signal- detection.
Figure 7: Indirect ELISA procedure. The blocking step is not shown, and should be between the first and the second step.
Peptide-based ELISA diagnostics have several advantages over other current diagnostic techniques such as indirect immunofluorescence or radio immunoassay:
- easy to set up and can be routinely performed
- a large number of sera can be tested in a short period of time - do not need any special instrumentation or reagents
