Investigating the Role of Artemin Glycosylation
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Investigating the Role of Artemin Glycosylation. / Danwen, Qiu; Code, Christian; Quan, Chao; Gong, Bang-Jin; Arndt, Joseph; Pepinsky, Blake; Rand, Kasper D; Houde, Damian.
In: Pharmaceutical Research, Vol. 33, No. 6, 06.2016, p. 1383-1398.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Investigating the Role of Artemin Glycosylation
AU - Danwen, Qiu
AU - Code, Christian
AU - Quan, Chao
AU - Gong, Bang-Jin
AU - Arndt, Joseph
AU - Pepinsky, Blake
AU - Rand, Kasper D
AU - Houde, Damian
PY - 2016/6
Y1 - 2016/6
N2 - PURPOSE: Oligosaccharides play diverse and unpredictable functional roles when attached to proteins and are a largely unexplored scaffold for deconstructing and attributing novel functions to proteins during drug development. Here, the glycoprotein Artemin (ART) was carefully assessed by multiple analytical methods that allow us to provide a comprehensive understanding of how N-linked glycosylation impact the structural and functional properties of ART.METHODS: Modification of the N-linked glycan of ART was performed by incubation with various enzymes. Biological assays and systems were used to examine the relative activity and pharmacokinetic properties of ART as a function of glycosylation. In order to reveal the conformational impact of glycosylation on ART, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed in addition to differential scanning calorimetry. The colloidal stability of ART glycovariants was assessed by dynamic light scattering, viscometry, and solubility assays.RESULTS: No difference in pharmacokinetics or relative potency was revealed between glycosylated and nonglycosylated ART. Surprisingly, the HDX-MS data indicated that the glycan does not greatly influence the conformation and dynamics of the protein. In contrast, differences in thermal and colloidal stability clearly revealed a role of glycosylation in increasing the solubility and stability of ART.CONCLUSIONS: Our findings demonstrate how careful analysis using multiple advanced techniques can be used to identify and dissect the multiple potential functions of protein glycosylation and form a prerequisite for glycoengineering and drug development of glycoproteins.
AB - PURPOSE: Oligosaccharides play diverse and unpredictable functional roles when attached to proteins and are a largely unexplored scaffold for deconstructing and attributing novel functions to proteins during drug development. Here, the glycoprotein Artemin (ART) was carefully assessed by multiple analytical methods that allow us to provide a comprehensive understanding of how N-linked glycosylation impact the structural and functional properties of ART.METHODS: Modification of the N-linked glycan of ART was performed by incubation with various enzymes. Biological assays and systems were used to examine the relative activity and pharmacokinetic properties of ART as a function of glycosylation. In order to reveal the conformational impact of glycosylation on ART, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed in addition to differential scanning calorimetry. The colloidal stability of ART glycovariants was assessed by dynamic light scattering, viscometry, and solubility assays.RESULTS: No difference in pharmacokinetics or relative potency was revealed between glycosylated and nonglycosylated ART. Surprisingly, the HDX-MS data indicated that the glycan does not greatly influence the conformation and dynamics of the protein. In contrast, differences in thermal and colloidal stability clearly revealed a role of glycosylation in increasing the solubility and stability of ART.CONCLUSIONS: Our findings demonstrate how careful analysis using multiple advanced techniques can be used to identify and dissect the multiple potential functions of protein glycosylation and form a prerequisite for glycoengineering and drug development of glycoproteins.
KW - Journal Article
U2 - 10.1007/s11095-016-1880-x
DO - 10.1007/s11095-016-1880-x
M3 - Journal article
C2 - 26908049
VL - 33
SP - 1383
EP - 1398
JO - Pharmaceutical Research
JF - Pharmaceutical Research
SN - 0724-8741
IS - 6
ER -
ID: 164347087