title: Apple four in human blood coagulation factor XI mediates dimer formation.
abstract: Human blood coagulation factor XI is a dimer composed of two identical subunits. Each subunit contains four apple domains as tandem repeats followed by a serine protease region. A disulfide bridge between Cys321 of each fourth apple domain links the subunits together. The role of Cys321 in the dimerization of factor XI was examined by mutagenesis followed by expression of its cDNA in baby hamster kidney cells. The recombinant proteins were then purified from the tissue culture medium and shown to have full biological activity. Normal recombinant factor XI was secreted as a dimer as determined by SDS-PAGE, while recombinant factor XI-Cys321 Ser migrated as a monomer under these conditions. Gel filtration studies, however, revealed that each protein existed as a dimer under native conditions, indicating that the disulfide bond between Cys321 of each factor XI monomer was not necessary for dimer formation. The fourth apple domain (apple4) of factor XI was then introduced into tissue plasminogen activator (tPA) to investigate its role in the dimerization of other polypeptide chains. The fusion protein, containing apple4 (apple4-tPA), formed dimers as detected by SDS-PAGE and gel filtration. Furthermore, dimerization was specific to apple4, while apple3 had no effect on dimerization. These data further indicated that the apple4 domain of factor XI mediates dimerization of the two subunits and the interchain disulfide bond involving Cys321 was not essential for dimer formation.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195.
authors.forename: J C
authors.initials: JC
authors.lastname: Meijers
authors.forename: E R
authors.initials: ER
authors.lastname: Mulvihill
authors.forename: E W
authors.initials: EW
authors.lastname: Davie
authors.forename: D W
authors.initials: DW
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D005172:Factor XI / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D005172:Factor XI