A novel probe to assess cytosolic entry of exogenous proteins

Nat Commun. 2018 Aug 6;9(1):3104. doi: 10.1038/s41467-018-05556-z.

Abstract

Dendritic cells use a specialized pathway called cross-presentation to activate CD8+ T cells by presenting peptides from exogenous protein antigens on major histocompatibility complex class I molecules. Considerable evidence suggests that internalized antigens cross endocytic membranes to access cytosolic proteasomes for processing. The mechanism of protein dislocation represents a major unsolved problem. Here we describe the development of a sensitive reporter substrate, an N-glycosylated variant of Renilla luciferase fused to the Fc region of human IgG1. The luciferase variant is designed to be enzymatically inactive when glycosylated, but active after the asparagine to aspartic acid conversion that occurs upon deglycosylation by the cytosolic enzyme N-glycanase-1. The generation of cytosolic luminescence depends on internalization, deglycosylation, the cytosolic AAA-ATPase VCP/p97, and the cytosolic chaperone HSP90. By incorporating a T cell epitope into the fusion protein, we demonstrate that antigen dislocation into the cytosol is the rate limiting step in cross-presentation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antigen Presentation
  • Antigens / metabolism
  • CD8-Positive T-Lymphocytes / cytology*
  • Cross-Priming*
  • Cytosol / metabolism*
  • Dendritic Cells / immunology
  • Endocytosis
  • Epitopes / chemistry
  • Glycosylation
  • HEK293 Cells
  • HSP90 Heat-Shock Proteins / metabolism
  • Histocompatibility Antigens Class I / immunology
  • Humans
  • Immunoglobulin G / metabolism*
  • Luciferases / metabolism
  • Molecular Chaperones / metabolism
  • Protein Binding
  • Protein Transport
  • Renilla

Substances

  • Antigens
  • Epitopes
  • HSP90 Heat-Shock Proteins
  • Histocompatibility Antigens Class I
  • Immunoglobulin G
  • Molecular Chaperones
  • Luciferases