A novel probe to assess cytosolic entry of exogenous proteins

Qiao Lu, Jeff E. Grotzke and Peter Cresswell ()
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Qiao Lu: Yale University School of Medicine
Jeff E. Grotzke: Yale University School of Medicine
Peter Cresswell: Yale University School of Medicine

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-05556-z

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