N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins

Autor(es)

Ribela, Maria Teresa C. P. ; Damiani, Renata ; Silva, Felipe D. ; Lima, Eliana R. ; Oliveira, Joao E. ; Peroni, Cibele N. ; Torjesen, Peter A. ; Soares, Carlos R. ; Bartolini, Paolo

• Tipo de documento: Artigo Científico
• Fonte: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 18, n. 2 FEB 2017.
• Citações Web of Science: 2

Resumo

Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the -subunit and one in the -subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first time N-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%-87%) and carbohydrate mass (12%-19%) can be up to 34%-57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains similar to 3-fold fewer moles of galactose (p < 0.005) and sialic acid (p < 0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein. (AU)

• Processo FAPESP: 12/10779-2 - Clonagem, expressão e caracterização do hormônio folículo estimulante (FSH) e luteinizante (LH) de pirarucu (Arapaima gigas)
• Beneficiário: Paolo Bartolini
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 11/07289-0 - Síntese e caracterização de diferentes glicoformas de tireotrofina humana recombinante (rhTSH) farmacologicamente ativas
• Beneficiário: Maria Teresa de Carvalho Pinto Ribela
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/26058-0 - Estudo do perfil de N-glicosilação e dos sítios de ocupação em várias preparações de tireotropina humana (hTSH) hipofisária e recombinante
• Beneficiário: Carlos Roberto Jorge Soares
• Linha de fomento: Auxílio à Pesquisa - Regular