title: Self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin.
abstract: 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. By contrast, rCRT/18-197 is almost completely inactive. When rCRT/18-412 is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. Additionally, rCRT/18-412 oligomers are much better than monomers in binding to, and uptake by, macrophages. Inhibition of macrophage endocytosis partially blocks the stimulatory effect of rCRT/18-412. We conclude that the immunologically active site of CRT maps between residues 198-230 and that soluble CRT could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization.
authors.affiliation: Institute of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province, China.
authors.forename: Shang-Hui
authors.initials: SH
authors.lastname: Huang
authors.forename: Li-Xiang
authors.initials: LX
authors.lastname: Zhao
authors.forename: Chao
authors.initials: C
authors.lastname: Hong
authors.forename: Cui-Cui
authors.initials: CC
authors.lastname: Duo
authors.forename: Bing-Nan
authors.initials: BN
authors.lastname: Guo
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Gong
authors.forename: Si-Dong
authors.initials: SD
authors.lastname: Xiong
authors.forename: Fang-Yuan
authors.initials: FY
authors.lastname: Gong
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Gao
mesh_terms: D000818:Animals; D015153:Blotting, Western; D037282:Calreticulin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D055503:Protein Multimerization / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
chemical_list: D037282:Calreticulin; D008070:Lipopolysaccharides; D011994:Recombinant Proteins