Generation of MHC Class I Peptide Antigens by Protein Processing in the Secretory Route by Furin

Abstract

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans-Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.

The Extra Domain A from Fibronectin Targets Antigens to TLR4-Expressing Cells and Induces Cytotoxic T Cell Responses In Vivo

“…This internalization seems to be conducted through a lipid raft-dependent pathway (35,36). It is tempting to speculate that this internalization process is used by the EDA-SIINFEKL protein to access the trans-Golgi compartment and be processed by endopeptidases such as furin (37,38) and by carboxypeptidases and to access the MHC class I presentation pathway in an TAP-independent and brefeldin A-sensitive process. However, this issue requires further investigation.…”

Furin-Processed Antigens Targeted to the Secretory Route Elicit Functional TAP1−/−CD8+ T Lymphocytes In Vivo

“…Furin is a rate-limiting enzyme in this pathway, because its inhibition fully prevents presentation from TAP-deficient cells to 9pp89-specific CTL (8), and its overexpression potentiates presentation of the 9pp89 epitope (9). Furin inhibition also blocked SIINFEKL presentation from TAP-deficient cells to specific CTL (data not shown).…”

“…Notably, when chimeric proteins containing a MHC class I epitope downstream of furin cleavage sites are directed to the secretory pathway in TAP-negative cells, so that there is no contribution from cytosolic epitopes, antigenic presentation is elicited in a strictly furindependent fashion (8,9). Furin is also essential for generating MHC class I ligands from some exogenous Ags (11,12).…”

“…1). Modified HBe was selected as carrier protein because it is a well-known furin natural target previously used to study furin-dependent Ag processing (8,9). Cytosolic HBc was used as control carrier protein.…”

“…Chimeras sC-Ova and sC-Ova⌬C were based on HBe and entered the secretory pathway. cC-Ova, sC-Ova, and sC-Ova⌬C DNA constructs were produced from plasmids encoding cC-A9A or sC-A9A (9). DNA sequence encoding RAAAAAYPHFMPTNLAAAAASRESQC was removed by AvaI and BstXI restriction enzymes (Roche) and replaced by sequences encoding RASIINFEKLSRESQC and RASIINFEKL, respectively.…”