Man Of Iga Fc Receptor Fc¦Ári (cd89) The Structural And Functional Analysis

FcαRI (also called CD89) is the Fc receptor for Inununoglobulin A (IgA). It connects IgA and effector cells that are mainly from the myeloid lineage including neutrophils, eosinophils, monocytes/macrophages, etc. When clustered by IgA immune complexes, CD89 can trigger a broad range of immune responses including phagocytosis, antigen presentation, ADCC, superoxide generation and release of various cytokines and inflammatory mediators. Structurally, CD89 is a type I glycoprotein containing two extracellular Ig-like domains and a short cytoplasmic tail. Although CD89 is an Fc receptor, its sequence is less homology to other Fc receptors. In addition, it has different chromosome location, ligand binding and signal transduction pathways, indicating that CD89 is a distinct Fc receptor.In order to understand interactions of CD89 with IgA, we tried to resolve crystal structure of CD89. Therefore, cDNA of the extracellular part of CD89 (sCD89) was subcloned into various vectors and expressed in bacteria and insect cells. In E. coli, sCD89 with or without expression tags such as GST·tag, His₆·tag and pelB leader peptide formed inclusion bodies. In insect cells, sCD89 cDNA was cloned in the reformed transfer vector of pAcHLT-Nat and expressed in soluble form. Western blotting showed sCD89 expressed in insect cells had similar molecular weight with that expressed in bacteria. Two consecutive AGA codons corresponding to Arg50 and Arg51 of the mature CD89 peptide were found to be harmful to the expression of CD89 in E. coli. Synonymous mutations of AGA to CGC or CGT significantly increased expression of sCD89 in E. coli.Arginine salt, urea and GuHCl were used as reversible detergents for sCD89 refolding. The refolding of sCD89 peptide was achieved in renatured buffer containing 2.40-2.70 M urea or 1.8 M GuHCl but failed in 0.5 M arginine solution. The renatured sCD89 could recognize native IgA, indicating it recovered its biological functions. In addition, sCD89 could be recognized by a set of anti-native CD89 antibodies, indicating it held a right conformation as native CD89.Refolded sCD89 and its selenomethionine derivative were successfully crystallized. The crystal structure of sCD89 was solved by multiwavelength anomalous diffraction using selenomethionine derivative. The overall structure of sCD89 takes a characteristic shape of elbow or heart with an angle of 85°. The ectodomain of CD89 is consisting of two Ig-like domains (EC1, residues 2-101; EC2, residues 105-200) with a three-residue linker (102-104) that holds a hydrophobic core with several amino acid residues from interface of the two Ig folds. Each domain is composed of two anti-parallels βsheets connected by an S-S bond formed by two conservative cysteine residues located in B strand and F strand...