Aberrant Glycosylation Augments the Immuno-Stimulatory Activities of Soluble Calreticulin.

Calreticulin (CRT), a luminal resident calcium-binding glycoprotein of the cell, is a tumor-associated antigen involved in tumorigenesis and also an autoantigen targeted by autoantibodies found in patients with various autoimmune diseases. We have previously shown that prokaryotically expressed recombinant murine CRT (rCRT) exhibits strong stimulatory activities against monocytes/macrophages in vitro and potent immunogenicity in vivo, which is partially attributable to self-oligomerization of soluble rCRT. However, even in oligomerized form native CRT (nCRT) isolated from mouse liver is much less active than rCRT, arguing against the possibility that self-oligomerization alone would license potent pro-inflammatory properties to nCRT.

Soluble calreticulin induces tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 production by macrophages through mitogen-activated protein kinase (MAPK) and NFκB signaling pathways.

We have recently reported that soluble calreticulin (CRT) accumulates in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus. Moreover, following self-oligomerization, soluble recombinant CRT (rCRT) polypeptides exhibit potent immunostimulatory activities including macrophage activation in vitro and antibody induction in vivo. This study was designed to further investigate the underlying molecular mechanisms for soluble CRT-induced macrophage activation.

Self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin.

We have recently reported that calreticulin (CRT), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant CRT (rCRT) exhibits extraordinarily strong immunological activities. We herein further demonstrate that rCRT fragments 18-412 (rCRT/18-412), rCRT/39-272, rCRT/120-308 and rCRT/120-250 can self-oligomerize in solution and are 50-100 fold more potent than native CRT (nCRT, isolated from mouse livers) in activating macrophages in vitro. We narrowed down the active site of CRT to residues 150-230, the activity of which also depends on dimerization.