RESUMEN
Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined.
Asunto(s)
Antimaláricos , Malaria Falciparum , Humanos , Epítopos/metabolismo , Fosfatasa Alcalina/metabolismo , Plasmodium falciparum/metabolismo , Antimaláricos/farmacología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Anticuerpos/metabolismo , Sueros Inmunes , Resistencia a MedicamentosRESUMEN
Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD-TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS-TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD-TDP and ALS-TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-43S375) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD-TDP and ALS-TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD-TDP subtypes examined (A-C) and in ALS-TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43S375 antiserum among pathological subtypes. TDP-43 pathology of ALS-TDP, FTLD-TDP type B (including cases with the C9orf72 mutation), and FTLD-TDP type C all showed strong pTDP-43S375 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD-TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43S375 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers (i.e., TDP-43 strains) as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.