N-linked glycosylation sites of the motor protein prestin

Effects on membrane targeting and electrophysiological function

Keiji Matsuda, Jing Zheng, Guo Guang Du, Nikolaj Klöcker, Laird D. Madison, Peter Dallos

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N-glycoprotein with three potential N-linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N-glycosylation were investigated by producing single-point (N163Q and N166Q) or double-point mutations (NN163/166QQ and NN163/166AA) at putative N-glycosylation sites. Further, treatment with tunicamycin or glycopeptidase-F was used to determine the consequences of removing N-linked glycosylation in wild-type prestin. We determined the effects of these manipulations on prestin's cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell-types. Data indicate that prestin is a glycoprotein with N-linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N-linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non-linear capacitance, a signature of prestin's motor function. Compared to glycosylated prestin, the fully de-glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.

Original languageEnglish (US)
Pages (from-to)928-938
Number of pages11
JournalJournal of Neurochemistry
Volume89
Issue number4
DOIs
StatePublished - May 1 2004

Fingerprint

Glycosylation
Membranes
Proteins
Outer Auditory Hair Cells
Glycoproteins
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Tunicamycin
Trimming
Audition
Molecular mass
Cell membranes
Point Mutation
Membrane Potentials
Hearing
Polysaccharides
Charge transfer
Capacitance
Cells
Cell Membrane

Keywords

  • Electromotility
  • Hair cells
  • N-linked glycosylation
  • Prestin
  • Solute Carrier Family 26

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Matsuda, Keiji ; Zheng, Jing ; Du, Guo Guang ; Klöcker, Nikolaj ; Madison, Laird D. ; Dallos, Peter. / N-linked glycosylation sites of the motor protein prestin : Effects on membrane targeting and electrophysiological function. In: Journal of Neurochemistry. 2004 ; Vol. 89, No. 4. pp. 928-938.
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N-linked glycosylation sites of the motor protein prestin : Effects on membrane targeting and electrophysiological function. / Matsuda, Keiji; Zheng, Jing; Du, Guo Guang; Klöcker, Nikolaj; Madison, Laird D.; Dallos, Peter.

In: Journal of Neurochemistry, Vol. 89, No. 4, 01.05.2004, p. 928-938.

Research output: Contribution to journalArticle

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T1 - N-linked glycosylation sites of the motor protein prestin

T2 - Effects on membrane targeting and electrophysiological function

AU - Matsuda, Keiji

AU - Zheng, Jing

AU - Du, Guo Guang

AU - Klöcker, Nikolaj

AU - Madison, Laird D.

AU - Dallos, Peter

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AB - Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N-glycoprotein with three potential N-linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N-glycosylation were investigated by producing single-point (N163Q and N166Q) or double-point mutations (NN163/166QQ and NN163/166AA) at putative N-glycosylation sites. Further, treatment with tunicamycin or glycopeptidase-F was used to determine the consequences of removing N-linked glycosylation in wild-type prestin. We determined the effects of these manipulations on prestin's cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell-types. Data indicate that prestin is a glycoprotein with N-linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N-linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non-linear capacitance, a signature of prestin's motor function. Compared to glycosylated prestin, the fully de-glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.

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KW - N-linked glycosylation

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