TAILIEUCHUNG - Báo cáo khoa học: Characterization of N-glycosylation consensus sequences in the Kv3.1 channel

N-Glycosylation is a cotranslational and post-translational process of pro-teins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K + channel (). Glycosylation of protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. | ềFEBS Journal Characterization of N-glycosylation consensus sequences in the channel Natasha L. Brooks Melissa J. Corey and Ruth A. Schwalbe Department of Biochemistry and Molecular Biology Brody Schoolof Medicine East Carolina University Greenville NC USA Keywords brain glycosylation K channel topology trafficking Correspondence R. A. Schwalbe Department of Biochemistry and Molecular Biology Brody Schoolof Medicine at East Carolina University 600 Moye Boulevard Greenville NC 27834 USA Fax 1 252 744 3383 Tel 1 252 744 2034 E-mail schwalber@ Received 25 August 2005 revised 18 May 2006 accepted 23 May 2006 doi N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding maturation stability trafficking and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltagegated K channel . Glycosylation of protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F PNGase F produced a much faster-migrating immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type in Sf9 cells cells were treated with tunicamycin. Also partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single N229Q and N220Q and double N220Q N229Q mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated were transported to the plasma membrane. Unitary conductance of N220Q N229Q was similar to that of the wild-type . However whole cell currents of N220Q N229Q channels had slower activation rates and a slight positive shift in voltage dependence compared to wild-type .

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