RESUMEN
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 260 kDa, while under reducing conditions, FGL2 has a molecular weight of 65 kDa 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.