Characterization of fibrinogen-like protein 2 (FGL2): Monomeric FGL2 has enhanced immunosuppressive activity in comparison to oligomeric FGL2

• Published in: The international journal of biochemistry & cell biology 2013-02, Vol.45 (2), p.408-418
• Main Authors: Liu, Hao, Yang, Peter S., Zhu, Tina, Manuel, Justin, Zhang, Jianhua, He, Wei, Shalev, Itay, Zhang, Li, Cybulsky, Myron I., Grant, David R., Phillips, M. James, Levy, Gary A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; antigen-presenting cells; binding capacity; Cell Proliferation; Cercopithecus aethiops; COS Cells; cysteine; Cysteine - chemistry; Cysteine - genetics; Deglycosylation; Disulfide bond; Female; fibrinogen; Fibrinogen - chemistry; Fibrinogen - pharmacology; Fibrinogen - physiology; Fibrinogen like protein 2; Functional domain; Glycosylation; Immunosuppressive Agents - chemistry; Immunosuppressive Agents - pharmacology; innate immunity; lectins; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Sequence Data; molecular weight; Mutagenesis, Site-Directed; mutation; Oligomerization; pathogenesis; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Peptide Fragments - physiology; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Processing, Post-Translational; Protein Stability; Protein Structure, Quaternary; Protein Structure, Secondary; receptors; rheumatoid arthritis; Solubility; structure-activity relationships; sugars; T-lymphocytes; T-Lymphocytes - drug effects; T-Lymphocytes - immunology; T-Lymphocytes - physiology; therapeutics; viral hepatitis; Western blotting
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/j.biocel.2012.10.014

Fibrinogen-like protein 2 (FGL2), a novel effector molecule of CD4+CD25+Foxp3+ regulatory T cells (Treg), mediates its suppressive activity through binding to low affinity Fcγ receptors expressed on antigen presenting cells (APCs). FGL2 has been implicated in the pathogenesis of viral hepatitis, xeno- and allotransplant rejection, and rheumatoid arthritis. Here we fully analyzed the structure–function relationships of recombinant murine FGL2 generated in COS-7 cells and identified the receptor binding domains. Native FGL2 exists as an oligomer with a molecular weight of approximately 260kDa, while under reducing conditions, FGL2 has a molecular weight of 65kDa suggesting that native FGL2 is composed of four monomers. By site-directed mutation, cysteines at positions 94, 97, 184 and 187, found in the coiled-coil domain were shown to be crucial for FGL2 oligomerization. Monomeric FGL2 had a lower affinity binding to APCs, but increased immunosuppressive activity compared to oligomeric FGL2. Deglycosylation demonstrated that sugar moieties are critical for maintaining solubility of FGL2. SWISS-MODEL analysis suggested that FGL2 has a similar tertiary structure with other members of the fibrinogen family such as fibrinogen and tachylectin. Mutational analysis of cysteine residues and Western blots suggested an asymmetric bouquet-shaped quaternary structure for oligomeric FGL2, resembling many pattern-recognition molecules in the lectin pathway of innate immunity. The functional motifs of FGL2 were mapped to the C terminal globular domain, using a peptide blockade assay. These results collectively define the biochemical and immunological determinants of FGL2, an important immunosuppressive molecule of Treg providing important insights for designing FGL2-related therapeutics.