RESUMEN
Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing â¼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.
Asunto(s)
Actinina/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Esclerosis Múltiple/inmunología , Proteínas del Tejido Nervioso/inmunología , Péptidos/inmunología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/sangre , 2',3'-Nucleótido Cíclico Fosfodiesterasas/inmunología , Actinina/sangre , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/sangre , Autoantígenos/sangre , Encéfalo/inmunología , Encéfalo/metabolismo , Proteínas Portadoras/sangre , Proteínas Portadoras/inmunología , Forma BB de la Creatina-Quinasa/sangre , Forma BB de la Creatina-Quinasa/inmunología , Epítopos/sangre , Epítopos/inmunología , Glicosilación , Humanos , Proteínas de Microfilamentos/sangre , Proteínas de Microfilamentos/inmunología , Datos de Secuencia Molecular , Esclerosis Múltiple/sangre , Esclerosis Múltiple/patología , Proteínas del Tejido Nervioso/sangre , Péptidos/sangre , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). OBJECTIVE: The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. METHODS: Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. RESULTS: RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. CONCLUSIONS: Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Autoantígenos/metabolismo , Citrulina/inmunología , Histonas/metabolismo , Neutrófilos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Autoantígenos/inmunología , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Histonas/inmunología , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Péptidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
Among the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH3 features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde could be employed as well without a substantial increase in terms of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless, only a single paper reports about a diethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation.