Molecular profile of mannan-binding lectin in hepatitis C patients with MBL gene polymorphisms by a modified mannan-coated nitrocellulose assay.

Diego A P Albuquerque, Igor T Cavalcanti, Luydson R S Vasconcelos, Francisco Montenegro, Leila M M B Pereira, Maria S M Cavalcanti, Patrícia Moura, Luiz B C Júnior, Sinara Mônica Vitalino de Almeida, Eduardo I C Beltrão

Journal: Journal of immunological methods 2019;460():101-106

PMID: 30056939

Full Text on Elsevier Science View on PubMed

Abstract

The aim of this study was to develop an assay to analyze the serum profile of Mannose-binding lectin (MBL) through a simple and "in-house" method (called "dot-N-man"). Furthermore, the study attempted to associate molecular masses of MBL to the profile of MBL gene polymorphisms in patients with hepatitis C. Heterogeneity in molecular masses of MBL is due to the impairment of oligomers formation, which is linked to genetic polymorphisms in the MBL gene. Individuals with AA genotype (wild-type) produce high-molecular-mass proteins, whereas AO and OO individuals produce intermediate and low-molecular-mass proteins, respectively. Sera of thirty patients carrying the hepatitis C virus (HCV) were investigated using MBL binding assay with mannan-coated nitrocellulose (dot-N-man). Purified MBL was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Dot-N-Man assay yielded MBL with molecular masses ranging between 55 and 320 kDa, comparable to low and high molecular mass forms of MBL. Nonreducing SDS-PAGE showed high molecular mass bands in all AA individuals while bands of 270 and 205 kDa were observed in sera for a number of patients with AO and OO genotypes, respectively. Immunoblotting confirmed the MBL samples obtained from the dot-N-man. These results provide new insights to understand the MBL molecular forms profile in patients infected with HCV- which could be useful in future investigations on the influence of the MBL structure/genotype on both the progression of infection and the response to hepatitis C therapy.

Address: Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife, PE, Brazil.; Instituto do Fígado e Transplantes de Pernambuco - IFP, Recife, PE, Brazil; Faculdade de Ciências Médicas, Universidade de Pernambuco (UPE), Recife, PE, Brazil; Instituto de Pesquisas Aggeu Magalhães - FIOCRUZ, Recife, PE, Brazil.; Laboratório de Biologia Molecular de Vírus, Instituto de Ciências Biológicas, Universidade de Pernambuco, Recife, PE, Brazil.; Instituto do Fígado e Transplantes de Pernambuco - IFP, Recife, PE, Brazil; Faculdade de Ciências Médicas, Universidade de Pernambuco (UPE), Recife, PE, Brazil.; Laboratório de Biologia Molecular de Vírus, Instituto de Ciências Biológicas, Universidade de Pernambuco, Recife, PE, Brazil; Faculdade de Ciências Médicas, Universidade de Pernambuco (UPE), Recife, PE, Brazil.; Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife, PE, Brazil; Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife, PE, Brazil.; Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife, PE, Brazil; Universidade de Pernambuco (UPE), Faculdade de Ciências, Educação e Tecnologia de Garanhuns (FACETEG), Garanhuns, PE, Brazil. Electronic address: [email protected].

DOI: 10.1016/j.jim.2018.06.015