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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

N-glycoprofiling analysis in a simple glycoprotein model: A comparison between recombinant and pituitary glycosylated human prolactin

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Author(s):
Capone, Marcos V. N. [1] ; Suzuki, Miriam F. [1] ; Oliveira, Joao E. [1] ; Damiani, Renata [1] ; Soares, Carlos R. J. [1] ; Bartolini, Paolo [1]
Total Authors: 6
Affiliation:
[1] IPEN CNEN SP, Ctr Biotechnol, BR-05580000 Sao Paulo, SP - Brazil
Total Affiliations: 1
Document type: Journal article
Source: Journal of Biotechnology; v. 202, p. 78-87, MAY 20 2015.
Web of Science Citations: 4
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 25 kDa, 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-GhPRL) 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. NGlycan 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. (C) 2014 Elsevier B.V. All rights reserved. (AU)

FAPESP's process: 11/22985-3 - Characterization of the oligosaccharide structure of native and recombinant human prolactinobtained from CHO cells treated or not with cycloheximide
Grantee:Carlos Roberto Jorge Soares
Support Opportunities: Regular Research Grants