title: Characterization of fibrinogen-like protein 2 (FGL2): monomeric FGL2 has enhanced immunosuppressive activity in comparison to oligomeric FGL2.
abstract: 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.
authors.affiliation: Multi-Organ Transplant Program, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada M5G 2N2.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.forename: Peter S
authors.initials: PS
authors.lastname: Yang
authors.forename: Tina
authors.initials: T
authors.lastname: Zhu
authors.forename: Justin
authors.initials: J
authors.lastname: Manuel
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: He
authors.forename: Itay
authors.initials: I
authors.lastname: Shalev
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Myron I
authors.initials: MI
authors.lastname: Cybulsky
authors.forename: David R
authors.initials: DR
authors.lastname: Grant
authors.forename: M James
authors.initials: MJ
authors.lastname: Phillips
authors.forename: Gary A
authors.initials: GA
authors.lastname: Levy
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005260:Female; D005340:Fibrinogen / Q000737:chemistry / Q000494:pharmacology* / Q000502:physiology; D006031:Glycosylation; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology* / Q000502:physiology; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012995:Solubility; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000502:physiology
chemical_list: C490637:Fgl2 protein, mouse; D007166:Immunosuppressive Agents; D010446:Peptide Fragments; D005340:Fibrinogen; D003545:Cysteine