Dissection of the neonatal Fc receptor (FcRn)-albumin interface using mutagenesis and anti-FcRn albumin-blocking antibodies.
Albumin is the most abundant protein in blood and plays a pivotal role as a multitransporter of a wide range of molecules such as fatty acids, metabolites, hormones, and toxins. In addition, it binds a variety of drugs. Its role as distributor is supported by its extraordinary serum half-life of 3 weeks. This is related to its size and binding to the cellular receptor FcRn, which rescues albumin from intracellular degradation. Furthermore, the long half-life has fostered a great and increasing interest in utilization of albumin as a carrier of protein therapeutics and chemical drugs. However, to fully understand how FcRn acts as a regulator of albumin homeostasis and to take advantage of the FcRn-albumin interaction in drug design, the interaction interface needs to be dissected. Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to unmask the binding sites for albumin blocking antibodies and albumin on the receptor, which revealed that the interaction is not only strictly pH-dependent, but predominantly hydrophobic in nature. Specifically, we provide mechanistic evidence for a crucial role of a cluster of conserved tryptophan residues that expose a pH-sensitive loop of FcRn, and identify structural differences in proximity to these hot spot residues that explain divergent cross-species binding properties of FcRn. Our findings expand our knowledge of how FcRn is controlling albumin homeostasis at a molecular level, which will guide design and engineering of novel albumin variants with altered transport properties.
From the Centre for Immune Regulation (CIR) and Department of Biosciences, University of Oslo, N-0316 Oslo, Norway, CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, N-0424 Oslo, Norway.
Kine Marita Knudsen
KM
Sand
the Department for Microbiology, Oslo University Hospital Rikshospitalet and University of Oslo, Nydalen, N-0424 Oslo, Norway, the Department of Medical Biochemistry, Oslo University Hospital Rikshospitalet and University of Oslo, Nydalen, N-0424 Oslo, Norway.
Bjørn
B
Dalhus
The Jackson Laboratory, Bar Harbor, Maine 04609, and.
Gregory J
GJ
Christianson
From the Centre for Immune Regulation (CIR) and Department of Biosciences, University of Oslo, N-0316 Oslo, Norway, CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, N-0424 Oslo, Norway.
Malin
M
Bern
From the Centre for Immune Regulation (CIR) and Department of Biosciences, University of Oslo, N-0316 Oslo, Norway, CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, N-0424 Oslo, Norway.
Stian
S
Foss
Novozymes Biopharma UK, Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom.
Jason
J
Cameron
Novozymes Biopharma UK, Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom.
Darrell
D
Sleep
the Department for Microbiology, Oslo University Hospital Rikshospitalet and University of Oslo, Nydalen, N-0424 Oslo, Norway, the Department of Medical Biochemistry, Oslo University Hospital Rikshospitalet and University of Oslo, Nydalen, N-0424 Oslo, Norway.
Magnar
M
Bjørås
The Jackson Laboratory, Bar Harbor, Maine 04609, and.
Derry C
DC
Roopenian
From the Centre for Immune Regulation (CIR) and Department of Biosciences, University of Oslo, N-0316 Oslo, Norway, CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, N-0424 Oslo, Norway.
Inger
I
Sandlie
CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, N-0424 Oslo, Norway, j.t.andersen@ibv.uio.no.
Jan Terje
JT
Andersen
D000418:Albumins / Q000378:metabolism*; D000595:Amino Acid Sequence; D019138:Antibodies, Blocking / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011961:Receptors, Fc / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000418:Albumins; D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D011961:Receptors, Fc; C100688:Fc receptor, neonatal
Albumin; Antibody; Biodegradation; Bioengineering; Fc Receptor; FcRn; Half-life; Hydrophobic; pH Regulation