Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease.
Although calreticulin (CRT) is a major Ca(2+)-binding luminal resident protein, it can also appear on the surface of various types of cells and it functions as an immunopotentiating molecule. However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still unclear. In the present study, a recombinant fragment (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed in Escherichia coli. The affinity-purified rCRT/39-272 assembles into homodimers and oligomers in solution and exhibits high binding affinity to various glycans, including carrageenan, alginic acids, and hyaluronic acids. Functionally, rCRT/39-272 is capable of driving the activation and maturation of B cells and cytokine production by macrophages in a TLR-4-dependent manner in vitro. It specifically binds recombinant mouse CD14, but not BAFFR and CD40. It is also able to trigger Ig class switching by B cells in the absence of T cell help both in vitro and in vivo. Furthermore, this fragment of CRT exhibits strong adjuvanticity when conjugated to polysaccharides or expressed as part of a fusion protein. Soluble CRT can be detected in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus, but not in healthy subjects. We argue that CRT, either on the membrane surface of cells or in soluble form, is a potent stimulatory molecule to B cells and macrophages via the TLR-4/CD14 pathway and plays important roles in the pathogenesis of autoimmune diseases.
Hong
Chao
C
Department of Immunology, Peking University Health Science Center, Beijing, China.
Qiu
Xiang
X
Li
Yue
Y
Huang
Qianrong
Q
Zhong
Zhaoyan
Z
Zhang
Yan
Y
Liu
Xiangyuan
X
Sun
Lin
L
Lv
Ping
P
Gao
Xiao-Ming
XM
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology; D015551:Autoimmunity / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D037282:Calreticulin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D008262:Macrophage Activation / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism; D055815:Young Adult
D037282:Calreticulin; D018950:Lipopolysaccharide Receptors; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4