Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

J Biol Chem. 2012 Apr 20;287(17):14040-51. doi: 10.1074/jbc.M111.322487. Epub 2012 Feb 20.

Abstract

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism
  • Circular Dichroism
  • Cloning, Molecular
  • Conserved Sequence
  • Crystallography, X-Ray / methods*
  • Disulfides / chemistry
  • Glycoproteins / chemistry
  • Glycosylation
  • Glypicans / chemistry*
  • HEK293 Cells
  • Heparitin Sulfate / chemistry
  • Humans
  • Models, Molecular
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proteoglycans / chemistry

Substances

  • Disulfides
  • Glycoproteins
  • Glypicans
  • Proteoglycans
  • Heparitin Sulfate

Associated data

  • PDB/4ACR
  • PDB/AD7