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Glycoprofiling and N-glycosylation site occupancy of different human thyrotropin (hTSH) preparations of pituitary or recombinant origin


The great majority of the recombinant proteins for farmaceutical use is glycosylated and it has, therefore, one more potential glycosylation sites, not always occupied. The importance of the carbohydrate moiety of a glycoprotein is well known and it is directly related to solubility, stability, folding, assembling, circulatory half-life and in-vivo biological activity. Only recently, however, the importance of the macro- and micro-structures of carbohydrate, especially identified with the glycoprofiling and its variations and with occupancy, i.e. the percentage of glycosylation sites effectively occupied, has been considered. The above mentioned parameters , rarely determined, have been in fact directly related with intracellular trafficking, inflammation, defense mechanisms and also with various disease states such as different congenital disorders of glycosylation (CDG). For these reasons, also considering the experience of our research group in the study of glycoprotein hormones in general and of prolactin and thyrotropin in particular, we decided to set up a methodology that allows the simultaneous determination of the following two parameters: N-glycan microstructure and percentage of occupancy. This can be done, in our opinion, with basis on N-glycoprofiling and glycoprotein molecular mass determination, both carried out via MALDI-TOF. The present project, therefore, will carry out these determinations in 6 hTSH preparations of different origins, two pituitariy and four recombinant ones. The results will be quite interesting either for validating the methodology in itself or for studying the differences existing between the native product, secreted by the pituitary, and the CHO- or human HEK 293 cell-derived recombinant products. We emphasize that CHO-derived hTSH is a well known pharmaceutical applied to the diagnosis or therapy of thyroid cancer. A specific identification will be, moreover, carried out concerning the occupancy and type of N-glycan that is present in each one of the three glycosylation sites. This will be done in one pituitary and two recombinant preparations and will possibly open new perspectives in the study of these structures. (AU)

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Scientific publications (6)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
SUZUKI, MIRIAM F.; ALMEIDA, LARISSA A.; POMIN, STEPHANIE A.; SILVA, FELIPE D.; FREIRE, RENAN P.; OLIVEIRA, JOAO E.; AFFONSO, REGINA; SOARES, CARLOS R. J.; BARTOLINI, PAOLO. Periplasmic synthesis and purification of the human prolactin antagonist Delta(1-11)-G129R-hPRL. AMB EXPRESS, v. 11, n. 1, . (12/24345-4, 17/50332-0, 15/26058-0)
DE AZEVEDO, MARIANGELA DE BURGOS M.; MELO, VITOR H. S.; SOARES, CARLOS R. J.; GAMARRA, LIONEL F.; BARROS, CAIO H. N.; TASIC, LJUBICA. Poly(3-hydroxi-butyrate-co-3-hydroxy-valerate) (PHB-HV) microparticles loaded with holmium acetylacetonate as potential contrast agents for magnetic resonance images. INTERNATIONAL JOURNAL OF NANOMEDICINE, v. 14, p. 6869-6889, . (15/26058-0)
DIAS, PAULO V. S.; ARTHUSO, FERNANDA S.; OLIVEIRA, JOAO E.; SUZUKI, MIRIAM F.; SOUSA, JOSE M.; RIBELA, MARIA TERESA C. P.; BARTOLINI, PAOLO; SOARES, CARLOS R. J.. Determination of recombinant Interferon-alpha 2 in E. coil periplasmic extracts by reversed-phase high-performance liquid chromatography. JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, v. 1072, p. 193-198, . (12/24345-4, 15/10928-6, 15/26058-0)
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. N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, v. 18, n. 2, . (15/26058-0, 11/07289-0, 12/10779-2)
SILVA, FELIPE D.; OLIVEIRA, JOAO E.; FREIRE, RENAN P.; SUZUKI, MIRIAM F.; SOARES, CARLOS R.; BARTOLINI, PAOLO. Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis. AMB EXPRESS, v. 9, n. 1, . (15/26058-0, 17/50332-0)
SANT'ANA, P. M.; OLIVEIRA, J. E.; LIMA, E. R.; SOARES, C. R. J.; PERONI, C. N.; BARTOLINI, P.; RIBELA, MARIA TERESA C. P.. Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination. Applied Microbiology and Biotechnology, v. 102, n. 3, p. 1215-1228, . (15/26058-0)

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