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Core 2 GlcNAc modification and megalin ligand-binding activity.

Heng Zhang1, Shigemi Yoshioka, Masao Miyazaki

  • 1Sphingolipid Expression Laboratory, RIKEN Frontier Research System, Wako, Saitama, Japan.

Biochimica Et Biophysica Acta
|November 17, 2007
PubMed
Summary

Mouse megalins with different core 2 beta6GlcNAc transferase modifications exhibit distinct kinetic properties. These glycan chain variations on megalin impact its binding affinity and capacity for retinol-binding protein (RBP).

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Area of Science:

  • Biochemistry
  • Glycobiology
  • Molecular Biology

Background:

  • Megalin is a crucial glycoprotein transporter in kidney proximal tubules, responsible for reabsorbing small proteins.
  • Core 2 beta6GlcNAc transferase modifies megalin, influencing its function.
  • Variations in core 2 beta6GlcNAc transferase expression lead to distinct megalin glycoforms, as seen in BALB/c (wild-type) and DBA/2 (mutant) mice.

Purpose of the Study:

  • To investigate the impact of core 2 beta6GlcNAc transferase-mediated glycan modifications on megalin's ligand-binding kinetics.
  • To compare the kinetic properties of megalin from BALB/c mice (expressing core 2 extended Le(x) epitope) and DBA/2 mice (lacking the epitope).

Main Methods:

  • Purification of megalin from BALB/c and DBA/2 mice using lentil lectin chromatography.
  • Measurement of ligand-binding activity using Cy5-labeled retinol-binding protein (RBP).
  • Analysis techniques included gel permeation chromatography (GPC) and fluorescence correlation spectroscopy (FCS).
  • Scatchard analysis was employed to characterize binding sites.

Main Results:

  • GPC analysis revealed different apparent V(max) and K(m) values for BALB/c and DBA/2 megalins interacting with RBP.
  • Scatchard analysis indicated two binding sites for RBP on both megalin types, with varying affinities.
  • FCS analysis showed distinct K(m) and V(max) values compared to GPC, but similar K(m) values between the two mouse strains, with DBA/2 megalin having a lower V(max).

Conclusions:

  • Core 2 GlcNAc extended glycan chains on megalin significantly influence its ligand-binding affinity and capacity.
  • Glycosylation patterns of megalin play a critical role in its functional properties as a transporter.