Tyrosinase degradation is prevented when EDEM1 lacks the intrinsically disordered region

PLoS One. 2012;7(8):e42998. doi: 10.1371/journal.pone.0042998. Epub 2012 Aug 8.

Abstract

EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies / chemistry
  • Crystallography, X-Ray / methods
  • Endoplasmic Reticulum / metabolism
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Melanoma / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / physiology*
  • Molecular Sequence Data
  • Monophenol Monooxygenase / genetics
  • Monophenol Monooxygenase / metabolism*
  • Mutation
  • Polysaccharides / chemistry
  • Protein Binding
  • Protein Folding
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Antibodies
  • EDEM1 protein, human
  • Membrane Proteins
  • Polysaccharides
  • Monophenol Monooxygenase

Grant support

This work was supported by the Romanian Academy projects 1 and 3 of the Institute of Biochemistry(http://www.acad.ro) and CNCSIS Grants ID 1171 - 1018/2008 and ID 3-0342 - 181/2011 (http://www.uefiscdi.gov.ro). SG & LNS also acknowledges the postdoctoral program POSDRU/89/1.5/S/60746, from European Social Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.