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

Author(s)

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

• Document type: Journal article
• Source: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 18, n. 2 FEB 2017.
• Web of Science Citations: 2

Abstract

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)

• FAPESP's process: 12/10779-2 - Cloning, expression and characterization of Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of pirarucu (Arapaima gigas)
• Grantee: Paolo Bartolini
• Support type: Regular Research Grants
• FAPESP's process: 11/07289-0 - Synthesis and characterization of different glycoforms of recombinant human thyrotropin (rhTSH) pharmacologically actives
• Grantee: Maria Teresa de Carvalho Pinto Ribela
• Support type: Regular Research Grants
• FAPESP's process: 15/26058-0 - Glycoprofiling and N-glycosylation site occupancy of different human thyrotropin (hTSH) preparations of pituitary or recombinant origin
• Grantee: Carlos Roberto Jorge Soares
• Support type: Regular Research Grants