title: Development of monoclonal antibodies to interrogate functional domains and isoforms of FREM1 protein.
abstract: FREM1 was first identified as an extracellular matrix protein that is essential for the formation of the epithelial basement membrane during embryonic development. Recent studies have shown that FREM1 also modulates innate immunity through its isoform 2 splice variant protein, known as Toll-like/interleukin-1 receptor regulator (TILRR). TILRR is a co-receptor that enhances pro-inflammatory IL-1R1 signal transduction. Our previous study identified the minor allele of a SNP, rs1552896, in the intronic region of FREM1 gene to be associated with natural resistance to HIV-1 infection in a subgroup of Kenyan sex workers in the Pumwani cohort. To study the role of FREM1 and its variants in differential susceptibility to HIV-1 infection, we generated a panel of 17 monoclonal antibodies against two recombinant proteins of FREM1, rspD and rspF. Epitope mapping using overlapping pin peptides showed that the monoclonal antibody (MAb) panel interrogated seven unique regions across five different domains, including the C-type lectin domain disulfide bond and the TILRR GAG serine attachment site. Utility of three selected FREM1 MAbs were demonstrated by FACS and immunohistochemical detection of FREM1 in 293F kidney embryonic cells, HeLa 229 cervical cells, and Sup-T1 cells. Thus, these monoclonal antibodies could be used to study the functional domains of FREM1 and its isoforms.
authors.affiliation: 1 National Microbiology Laboratory , Public Health Agency of Canada.
authors.forename: Xin Yong
authors.initials: XY
authors.lastname: Yuan
authors.forename: Lewis Ruxi
authors.initials: LR
authors.lastname: Liu
authors.forename: Adriana
authors.initials: A
authors.lastname: Krawchenko
authors.forename: James
authors.initials: J
authors.lastname: Sainsbury
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Frank
authors.initials: F
authors.lastname: Plummer
authors.forename: Xi
authors.initials: X
authors.lastname: Yang
authors.forename: Ma
authors.initials: M
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis / Q000737:chemistry*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C548979:Frem1 protein, human; D020033:Protein Isoforms; D018123:Receptors, Interleukin