title: N-glycoprofiling analysis in a simple glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin.
abstract: Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A ∼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, 05508-000 São Paulo, Brazil.
authors.forename: Marcos V N
authors.initials: MV
authors.lastname: Capone
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, 05508-000 São Paulo, Brazil.
authors.forename: Miriam F
authors.initials: MF
authors.lastname: Suzuki
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, 05508-000 São Paulo, Brazil.
authors.forename: João E
authors.initials: JE
authors.lastname: Oliveira
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, 05508-000 São Paulo, Brazil.
authors.forename: Renata
authors.initials: R
authors.lastname: Damiani
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, 05508-000 São Paulo, Brazil. Electronic address: crsoares@ipen.br.
authors.forename: Carlos R J
authors.initials: CR
authors.lastname: Soares
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, 05508-000 São Paulo, Brazil.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bartolini
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003513:Cycloheximide / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry*; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D011388:Prolactin; D003513:Cycloheximide
keywords: Cycloheximide; G-hPRL; Glycosylated human prolactin; N-glycoprofiling; Prolactin