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Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic.
Canassa-DeLeo, Thais; Campo, Vanessa Leiria; Rodrigues, Lílian Cataldi; Marchiori, Marcelo Fiori; Fuzo, Carlos; Brigido, Marcelo Macedo; Sandomenico, Annamaria; Ruvo, Menotti; Maranhão, Andrea Queiroz; Dias-Baruffi, Marcelo.
  • Canassa-DeLeo T; Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café S/N, CEP, Ribeirão Preto, SP, 14040-903, Brazil.
  • Campo VL; Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café S/N, CEP, Ribeirão Preto, SP, 14040-903, Brazil.
  • Rodrigues LC; Centro Universitário Barão de Mauá, Rua Ramos de Azevedo 423, Jardim Paulista, CEP, Ribeirão Preto, 14090-180, SP, Brazil.
  • Marchiori MF; Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café S/N, CEP, Ribeirão Preto, SP, 14040-903, Brazil.
  • Fuzo C; Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café S/N, CEP, Ribeirão Preto, SP, 14040-903, Brazil.
  • Brigido MM; Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café S/N, CEP, Ribeirão Preto, SP, 14040-903, Brazil.
  • Sandomenico A; Instituto de Ciências Biológicas, Universidade de Brasília, Asa Norte, Brasília, DF, CEP 70910-900, Brazil.
  • Ruvo M; Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16, 80134, Naples, Italy.
  • Maranhão AQ; Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16, 80134, Naples, Italy.
  • Dias-Baruffi M; Instituto de Ciências Biológicas, Universidade de Brasília, Asa Norte, Brasília, DF, CEP 70910-900, Brazil.
Glycoconj J ; 37(1): 77-93, 2020 02.
Article en En | MEDLINE | ID: mdl-31823246
ABSTRACT
Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.
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Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Glicoproteínas / Distroglicanos / Síndrome de Walker-Warburg / Anticuerpos Monoclonales / Mucinas Tipo de estudio: Diagnostic_studies / Prognostic_studies Límite: Humans Idioma: En Año: 2020 Tipo del documento: Article

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Glicoproteínas / Distroglicanos / Síndrome de Walker-Warburg / Anticuerpos Monoclonales / Mucinas Tipo de estudio: Diagnostic_studies / Prognostic_studies Límite: Humans Idioma: En Año: 2020 Tipo del documento: Article