RESUMEN
Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients' sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.
RESUMEN
The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the ß-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Transportadora de Acilo/química , Ácido Graso Sintasas/química , Ácido Graso Sintasas/genética , Complejos Multienzimáticos/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Antibodies to MOG in serum have a dubious prognostic value in multiple sclerosis. The MOG recombinant protein conformational properties relevant to the antigenic activity are unknown. We employed a solid-phase ELISA based on a product (rMOG(ED)(His)(6)) expressed in E. coli after subcloning the cDNA of the extracellular domain of rat MOG, performing a refolding procedure on column and affinity purification. The far-UV Circular Dichroism (CD) spectra of rMOG(ED)(His)(6) showed a ß-sheet, a characteristic feature of the Ig-fold. However, in MS sera and controls we failed to detected IgM or IgG antibodies.
Asunto(s)
Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Glicoproteína Asociada a Mielina/inmunología , Pliegue de Proteína , Adulto , Animales , Espacio Extracelular/química , Espacio Extracelular/inmunología , Femenino , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/diagnóstico , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/química , Glicoproteína Mielina-Oligodendrócito , Estructura Terciaria de Proteína/fisiología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
The fungal type I fatty acid synthase (FAS) is a 2.6 MDa multienzyme complex, catalyzing all necessary steps for the synthesis of long acyl chains. To be catalytically competent, the FAS must be activated by a posttranslational modification of the central acyl carrier domain (ACP) by an intrinsic phosphopantetheine transferase (PPT). However, recent X-ray structures of the fungal FAS revealed a barrel-shaped architecture, with PPT located at the outside of the barrel wall, spatially separated from the ACP caged in the inner volume. This separation indicated that the activation has to proceed before the assembly to the mature complex, in a conformation where the ACP and PPT domains can meet. To gain insight into the auto-activation reaction and also into the fungal FAS assembly pathway, we structurally and functionally characterized the Saccharomyces cerevisiae FAS type I PPT as part of the multienzyme protein and as an isolated domain.
Asunto(s)
Ácido Graso Sintasas/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Proteína Transportadora de Acilo/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Coenzima A/química , Coenzima A/metabolismo , Activación Enzimática , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Magnesio/química , Magnesio/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoAsunto(s)
Anticuerpos/inmunología , Biomarcadores/análisis , Esclerosis Múltiple/inmunología , Glicoproteína Asociada a Mielina/síntesis química , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Humanos , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/química , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Asociada a Mielina/metabolismo , Glicoproteína Mielina-OligodendrócitoRESUMEN
Sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. Identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. However, posttranslational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. In line with these observations, we described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. We describe here the conformation-activity relationship of a focused library of glycopeptides based on structural diversity, with the aim of defining the structural requirements for the interaction of these glycopeptide antigens with specific autoantibodies. The final goal is the optimization of an antigenic probe for multiple sclerosis, to be used in the development of a simple diagnostic test based on an immunoenzymatic assay. The reported results clearly indicate that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type I' beta-turn around the minimal epitope Asn(Glc), which allows an efficient exposure of this moiety to antibodies interactions, in the context of a solid-phase immunoenzymatic assay.
Asunto(s)
Autoanticuerpos/sangre , Diseño de Fármacos , Glicopéptidos/inmunología , Sondas Moleculares/inmunología , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/inmunología , Reacciones Antígeno-Anticuerpo , Biomarcadores/sangre , Glicopéptidos/química , Sondas Moleculares/química , Esclerosis Múltiple/sangre , Biblioteca de Péptidos , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Multiple sclerosis (MS) is a complex disease that seems to depend on several pathophysiological processes. Because of its varied clinical presentation, natural history, and response to therapeutic interventions, MS can be considered to be a group of diseases that have not been yet characterized, thus resulting in difficult evaluation of prognosis. In the last few years, the role of autoAbs in MS has been reevaluated, and, therefore, their identification as specific biomarkers became a relevant target. In this paper, we demonstrate that an aberrant N-glucosylation is a fundamental determinant of autoAb recognition in MS. Thus, we developed CSF114(Glc), an antigenic probe accurately measuring IgM autoAbs in the sera of a patient population, as disease biomarker. The relevance of CSF114(Glc) is demonstrated by its clinical application and correlation with disease activity and prognosis. In fact, CSF114(Glc), a structure-based designed glycopeptide, is able to recognize, by ELISA, the presence of specific IgM autoAbs in the sera of a MS patient population but not in blood donors and other autoimmune conditions. AutoAbs specific for CSF114(Glc) isolated from MS patients recognized myelin and oligodendrocyte antigens by immunohistochemistry but not other nonrelevant tissues. We demonstrate that CSF114(Glc) is a reliable, specific probe in a longitudinal study of untreated MS patients. Development of IgG/IgM anti-CSF114(Glc) Abs paralleled clinical activity and brain lesions positive to MRI. Therefore, a CSF114(Glc)-based immunoassay on sera may have important prognostic value in monitoring MS disease progression guiding optimal therapeutic treatment.