Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues.
The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding approximately 70 mg.L(-1) of fermentation of refolded soluble human FcRn. The structural and functional integrity was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response in goats immunized with the FcRn heavy chain alone was analyzed following affinity purification on heavy chain-coupled Sepharose. Importantly, purified antibodies blocked the binding of both ligands to soluble human FcRn and were thus directed to both binding sites. This implies that the FcRn heavy chain, without prior assembly with human beta2-microglobulin, contains the relevant epitopes found in soluble human FcRn, and is therefore sufficient to obtain binders to either ligand-binding site. This finding will greatly facilitate the selection and characterization of such binders.
Andersen
Jan T
JT
Department of Molecular Biosciences and Centre for Immune Regulation, University of Oslo, Norway. janta@imbv.uio.no <janta@imbv.uio.no>
Justesen
Sune
S
Fleckenstein
Burkhard
B
Michaelsen
Terje E
TE
Berntzen
Gøril
G
Kenanova
Vania E
VE
Daba
Muluneh B
MB
Lauvrak
Vigdis
V
Buus
Søren
S
Sandlie
Inger
I
D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011961:Receptors, Fc / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D004220:Disulfides; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D003545:Cysteine; C100688:Fc receptor, neonatal