Aberrant Glycosylation Augments the Immuno-Stimulatory Activities of Soluble Calreticulin

• Main Authors: Fang-Yuan Gong, Zheng Gong, Cui-Cui Duo, Jun Wang, Chao Hong, Xiao-Ming Gao
• Format: Article
• Language: English
• Published: MDPI AG 2018-02-01
• Series: Molecules
• Subjects: calreticulin eukaryotic; macrophage; glycosylation
• Online Access: http://www.mdpi.com/1420-3049/23/3/523

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. Since rCRT differs from nCRT in its lack of glycosylation, we wondered if aberrant glycosylation of eukaryotically expressed CRT (eCRT) would significantly enhance its immunological activity. In the present study, tunicamycin, an N-glycosyltransferase inhibitor, was employed to treat CHO cells (CHO-CRT) stably expressing full-length recombinant mouse CRT in secreted form for preparation of aberrantly glycosylated eCRT (tun-eCRT). Our biochemical and immunological analysis results indicate that eCRT produced by CHO-CRT cells is similar to nCRT in terms of glycosylation level, lack of self-oligomerization, relatively poor immunogenicity and weak macrophage-stimulatory activity, while tun-eCRT shows reduced glycosylation yet much enhanced ability to elicit specific humoral responses in mice and TNF-α and nitric oxide production by macrophages in vitro. Given that abberant glycosylation of proteins is a hallmark of cancer cells and also related to the development of autoimmune disorders in humans, our data may provide useful clues for better understanding of potentiating roles of dysregulated glycosylation of molecules such as CRT in tumorigenesis and autoimmunity.