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Dissection of the Neonatal Fc Receptor (FcRn)-Albumin Interface Using Mutagenesis and Anti-FcRn Albumin-blocking Antibodies*

  • Kine Marita Knudsen Sand
    Footnotes
    Affiliations
    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
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  • Bjørn Dalhus
    Footnotes
    Affiliations
    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
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  • Gregory J. Christianson
    Affiliations
    The Jackson Laboratory, Bar Harbor, Maine 04609
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  • Malin Bern
    Footnotes
    Affiliations
    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
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  • Stian Foss
    Footnotes
    Affiliations
    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
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  • Jason Cameron
    Affiliations
    Novozymes Biopharma UK, Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom
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  • Darrell Sleep
    Affiliations
    Novozymes Biopharma UK, Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom
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  • Magnar Bjørås
    Footnotes
    Affiliations
    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
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  • Derry C. Roopenian
    Affiliations
    The Jackson Laboratory, Bar Harbor, Maine 04609
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  • Inger Sandlie
    Affiliations
    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
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  • Jan Terje Andersen
    Correspondence
    Supported by the Research Council of Norway (Grants 179573/V40; 230526/F20, and 233710/O30) and the Southeastern Norway Regional Health Authority (Grant 39375). To whom correspondence should be addressed: Centre for Immune Regulation and Dept. of Immunology, Oslo University Hospital Rikshospitalet, P.O. Box 4956, Oslo N-0424, Norway
    Affiliations
    CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, N-0424 Oslo, Norway
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  • Author Footnotes
    * This work was supported in part by the Research Council of Norway through its Centres of Excellence funding scheme (Project 179573). I. S., J. T. A., B. D., J. C., and D. S. are co-inventors of pending patent applications related to the data described in this work.
    This article contains supplemental Figs. S1 and S2.
    1 Supported by the University of Oslo.
    2 Supported by the South-Eastern Norway Regional Health Authority to establish the Regional Technology Platform for Structural Biology and Bioinformatics (Grants 2009100, 2011040, and 2012085).
    3 Supported by the Research Council of Norway through its programme for Global Health and Vaccination Research (GLOBVAC) Grant 143822.
Open AccessPublished:April 24, 2014DOI:https://doi.org/10.1074/jbc.M113.522565
      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.

      Introduction

      Albumin is a product of hepatocytes and is the most abundant protein in blood (34–54 g/liter). It serves as versatile transporter of a wide range of endogenous and exogenous compounds such as metal ions, hormones, fatty acids, metabolites, toxins, and drugs (
      • T. P. Jr.,
      ). Similar to all serum proteins, its serum concentration is determined by its rate of synthesis and its size above the renal clearance threshold. However, a third feature of albumin that is only shared with IgG antibodies (Abs)
      The abbreviations used are:
      Ab
      antibody
      FcRn
      neonatal Fc receptor
      HC
      heavy chain
      DIII
      domain III
      HSA
      human serum albumin
      mFcRn
      mouse FcRn
      hFcRn
      human FcRn
      SPR
      surface plasmon resonance
      MSA
      mouse serum albumin.
      is a greatly extended persistence in the circulatory system, which in both cases is caused by their interaction with the neonatal Fc receptor (FcRn) (
      • T. P. Jr.,
      ,
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ,
      • Chaudhury C.
      • Brooks C.L.
      • Carter D.C.
      • Robinson J.M.
      • Anderson C.L.
      Albumin binding to FcRn: distinct from the FcRn-IgG interaction.
      ,
      • Chaudhury C.
      • Mehnaz S.
      • Robinson J.M.
      • Hayton W.L.
      • Pearl D.K.
      • Roopenian D.C.
      • Anderson C.L.
      The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.
      ).
      As implied by its name, FcRn was first recognized as the neonatal transporter of maternal IgG from mother's milk across the intestinal barrier to the blood of rats (
      • Simister N.E.
      • Mostov K.E.
      Cloning and expression of the neonatal rat intestinal Fc receptor, a major histocompatibility complex class I antigen homolog.
      ). It also proved to be the transporter of IgG across the maternofetal barrier in both humans and rodents (
      • Story C.M.
      • Mikulska J.E.
      • Simister N.E.
      A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus.
      ,
      • Firan M.
      • Bawdon R.
      • Radu C.
      • Ober R.J.
      • Eaken D.
      • Antohe F.
      • Ghetie V.
      • Ward E.S.
      The MHC class I-related receptor, FcRn, plays an essential role in the maternofetal transfer of γ-globulin in humans.
      ,
      • Medesan C.
      • Radu C.
      • Kim J.K.
      • Ghetie V.
      • Ward E.S.
      Localization of the site of the IgG molecule that regulates maternofetal transmission in mice.
      ). A large body of subsequent evidence has revealed that FcRn is expressed and functionally operative in a broad range of cells and tissues throughout life (
      • Roopenian D.C.
      • Akilesh S.
      FcRn: the neonatal Fc receptor comes of age.
      ,
      • Ward E.S.
      • Ober R.J.
      Chapter 4: Multitasking by exploitation of intracellular transport functions the many faces of FcRn.
      ), transporting IgG across epithelial and endothelial barriers, enhancing IgG-mediated antigen presentation by dendritic cells and phagocytosis by neutrophils (
      • Baker K.
      • Qiao S.W.
      • Kuo T.T.
      • Aveson V.G.
      • Platzer B.
      • Andersen J.T.
      • Sandlie I.
      • Chen Z.
      • de Haar C.
      • Lencer W.I.
      • Fiebiger E.
      • Blumberg R.S.
      Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8-CD11b+ dendritic cells.
      ,
      • Dickinson B.L.
      • Badizadegan K.
      • Wu Z.
      • Ahouse J.C.
      • Zhu X.
      • Simister N.E.
      • Blumberg R.S.
      • Lencer W.I.
      Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line.
      ,
      • Qiao S.W.
      • Kobayashi K.
      • Johansen F.E.
      • Sollid L.M.
      • Andersen J.T.
      • Milford E.
      • Roopenian D.C.
      • Lencer W.I.
      • Blumberg R.S.
      Dependence of antibody-mediated presentation of antigen on FcRn.
      ,
      • Spiekermann G.M.
      • Finn P.W.
      • Ward E.S.
      • Dumont J.
      • Dickinson B.L.
      • Blumberg R.S.
      • Lencer W.I.
      Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in the mammalian lung.
      ,
      • Vidarsson G.
      • Stemerding A.M.
      • Stapleton N.M.
      • Spliethoff S.E.
      • Janssen H.
      • Rebers F.E.
      • de Haas M.
      • van de Winkel J.G.
      FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis.
      ), and substantially extending the serum persistence of IgG (
      • Ghetie V.
      • Hubbard J.G.
      • Kim J.K.
      • Tsen M.F.
      • Lee Y.
      • Ward E.S.
      Abnormally short serum half-lives of IgG in β 2-microglobulin-deficient mice.
      ,
      • Ghetie V.
      • Popov S.
      • Borvak J.
      • Radu C.
      • Matesoi D.
      • Medesan C.
      • Ober R.J.
      • Ward E.S.
      Increasing the serum persistence of an IgG fragment by random mutagenesis.
      ,
      • Kim J.K.
      • Firan M.
      • Radu C.G.
      • Kim C.H.
      • Ghetie V.
      • Ward E.S.
      Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.
      ,
      • Montoyo H.P.
      • Vaccaro C.
      • Hafner M.
      • Ober R.J.
      • Mueller W.
      • Ward E.S.
      Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice.
      ).
      As FcRn has also been demonstrated to prolong the serum half-life of albumin (5, 20), it acts as a regulator of the circulatory half-life of two totally unrelated proteins. This is evident from the fact that mice lacking FcRn have serum levels of IgG and albumin 4–5 and 2–3-fold lower than normal mice, respectively, as do mice where FcRn is conditionally deleted in endothelial and hematopoietic cells (
      • Chaudhury C.
      • Mehnaz S.
      • Robinson J.M.
      • Hayton W.L.
      • Pearl D.K.
      • Roopenian D.C.
      • Anderson C.L.
      The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.
      ,
      • Montoyo H.P.
      • Vaccaro C.
      • Hafner M.
      • Ober R.J.
      • Mueller W.
      • Ward E.S.
      Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice.
      ,
      • Kobayashi K.
      • Qiao S.W.
      • Yoshida M.
      • Baker K.
      • Lencer W.I.
      • Blumberg R.S.
      An FcRn-dependent role for anti-flagellin immunoglobulin G in pathogenesis of colitis in mice.
      ,
      • Roopenian D.C.
      • Christianson G.J.
      • Sproule T.J.
      • Brown A.C.
      • Akilesh S.
      • Jung N.
      • Petkova S.
      • Avanessian L.
      • Choi E.Y.
      • Shaffer D.J.
      • Eden P.A.
      • Anderson C.L.
      The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs.
      ). Genetic linkage in humans is also found by the rare human disease, familial hypercatabolic hypoproteinemia, which is characterized by abnormally low levels of both ligands that correlates with FcRn expression deficiency (
      • Wani M.A.
      • Haynes L.D.
      • Kim J.
      • Bronson C.L.
      • Chaudhury C.
      • Mohanty S.
      • Waldmann T.A.
      • Robinson J.M.
      • Anderson C.L.
      Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant β2-microglobulin gene.
      ).
      FcRn is a major histocompatibility class I-related molecule consisting of a unique transmembrane heavy chain (HC) with three extracellular domains (α1, α2, and α3) that are non-covalently bound to the common soluble β2-microglobulin. Crystal structures of the extracellular part of FcRn show that the amino-terminal α1-α2 platform is made up of eight antiparallel β-pleated strands topped by two long α-helices followed by the α3-domain (
      • Burmeister W.P.
      • Huber A.H.
      • Bjorkman P.J.
      Crystal structure of the complex of rat neonatal Fc receptor with Fc.
      ,
      • Mezo A.R.
      • Sridhar V.
      • Badger J.
      • Sakorafas P.
      • Nienaber V.
      X-ray crystal structures of monomeric and dimeric peptide inhibitors in complex with the human neonatal Fc receptor, FcRn.
      ,
      • West Jr., A.P.
      • Bjorkman P.J.
      Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,).
      ). The soluble β2-microglobulin is tightly bound to the α1-α2 platform and the α3-domain is proximal to the membrane (Fig. 1A). Although major histocompatibility class I molecules bind peptides in their peptide-binding groove that is located between the two α-helices on the α1-α2 platform, the corresponding groove on FcRn is closed (
      • Burmeister W.P.
      • Huber A.H.
      • Bjorkman P.J.
      Crystal structure of the complex of rat neonatal Fc receptor with Fc.
      ,
      • Mezo A.R.
      • Sridhar V.
      • Badger J.
      • Sakorafas P.
      • Nienaber V.
      X-ray crystal structures of monomeric and dimeric peptide inhibitors in complex with the human neonatal Fc receptor, FcRn.
      ,
      • West Jr., A.P.
      • Bjorkman P.J.
      Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,).
      ). Instead, FcRn has evolved to bind IgG and albumin.
      Figure thumbnail gr5
      FIGURE 5The monoclonal Abs bind in a species-dependent manner to FcRn. A, SDS-PAGE gel migration of recombinant GST-tagged human, macaque, pig, dog, mouse, and rat FcRn showing the expected molecular sizes of the GST-fused HCs and β2-microglobulin (β2m). ELISA binding of human, macaque, pig, dog, mouse, and rat FcRn toward DVN1 (B), ADM31 (C), ADM32 (D), DVN24 (E), ADM11 (F), ADM12 (G), DVN21 (H), DVN22 (I), and DVN23 (J) at pH 7.4 (n = 3). All data are presented as mean ± S.D. An overview of relative binding of the Abs to the FcRn species is given in .
      Figure thumbnail gr1
      FIGURE 1The structure of hFcRn and location of the pH-dependent flexible loop containing a cluster of tryptophans. A, overall structure of the extracellular part of hFcRn. The His-166 residue (blue ball-and-stick) within the α2-domain regulates a pH-dependent flexible loop (residues 51–61) within the α1-domain, which contains four tryptophan residues Trp-51, Trp-53, Trp-59, and Trp-61 (red ball-and-stick). The binding site for albumin is indicated relative to the IgG binding site that involves Glu-115 and Glu-116 (yellow ball-and-stick). The hFcRn HC is shown in green, and the β2-microglobulin (β2m) subunit is shown in gray. B, close up view of the FcRn HC loop at low pH (4.2) (
      • Mezo A.R.
      • Sridhar V.
      • Badger J.
      • Sakorafas P.
      • Nienaber V.
      X-ray crystal structures of monomeric and dimeric peptide inhibitors in complex with the human neonatal Fc receptor, FcRn.
      ) where the positively charged His-166 makes charge-stabilized hydrogen bonds with Glu-54 and Tyr-60, structuring the loop of surrounding tryptophan residues. C, close up view of the FcRn HC loop at high pH (8.2) (
      • West Jr., A.P.
      • Bjorkman P.J.
      Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,).
      ) where the uncharged His-166 loses the interactions with Glu-54 and Tyr-60, and the loop becomes flexible and structurally disordered (shown as a dashed line). D, alignment of a stretch of amino acids (residues 50–61) of the α1-domain of FcRn from 10 species. Asterisks indicate fully conserved amino acid residues. The four tryptophan residues are fully conserved, whereas a non-conserved amino acid is found in position 52. E, Trp-53 and Trp-59 are fully exposed at the surface of hFcRn, whereas Trp-61 is partially exposed, and Trp-51 is buried in the hydrophobic core of the molecule.
      A hallmark of the interaction is that both IgG and albumin bind FcRn in a strictly pH-dependent manner, with binding at acidic pH (6.5–5.5) and no binding or release at neutral pH (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ,
      • Chaudhury C.
      • Brooks C.L.
      • Carter D.C.
      • Robinson J.M.
      • Anderson C.L.
      Albumin binding to FcRn: distinct from the FcRn-IgG interaction.
      ,
      • Chaudhury C.
      • Mehnaz S.
      • Robinson J.M.
      • Hayton W.L.
      • Pearl D.K.
      • Roopenian D.C.
      • Anderson C.L.
      The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.
      ,
      • Kim J.K.
      • Firan M.
      • Radu C.G.
      • Kim C.H.
      • Ghetie V.
      • Ward E.S.
      Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.
      ,
      • Burmeister W.P.
      • Huber A.H.
      • Bjorkman P.J.
      Crystal structure of the complex of rat neonatal Fc receptor with Fc.
      ). This is fundamental for FcRn rescue of both ligands from lysosomal degradation. The underlying cellular mechanism has been deduced from live-cell imaging studies of the FcRn-IgG complex (
      • Ward E.S.
      • Ober R.J.
      Chapter 4: Multitasking by exploitation of intracellular transport functions the many faces of FcRn.
      ,
      • Ober R.J.
      • Martinez C.
      • Lai X.
      • Zhou J.
      • Ward E.S.
      Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level.
      ,
      • Ober R.J.
      • Martinez C.
      • Vaccaro C.
      • Zhou J.
      • Ward E.S.
      Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn.
      ,
      • Prabhat P.
      • Gan Z.
      • Chao J.
      • Ram S.
      • Vaccaro C.
      • Gibbons S.
      • Ober R.J.
      • Ward E.S.
      Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy.
      ,
      • Ward E.S.
      • Martinez C.
      • Vaccaro C.
      • Zhou J.
      • Tang Q.
      • Ober R.J.
      From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling.
      ), which demonstrate that FcRn resides predominantly within acidic endosomes where it encounters IgG taken up by fluid-phase endocytosis. The acidic milieu triggers binding of IgG via its Fc fragment to FcRn followed by exocytosis at the cell surface, where exposure to the near neutral pH of the blood results in release of IgG. Albumin is likely to follow the same rescue pathway, while proteins that do not bind the receptor are eliminated by lysosomal degradation.
      Site-directed mutagenesis and inspection of the atomic resolution structure of rat FcRn in complex with rat IgG2a Fc have revealed that conserved histidine residues located at the IgG Fc elbow region (His-310 and His-435) regulate the strict pH dependence of the interaction (
      • Ghetie V.
      • Popov S.
      • Borvak J.
      • Radu C.
      • Matesoi D.
      • Medesan C.
      • Ober R.J.
      • Ward E.S.
      Increasing the serum persistence of an IgG fragment by random mutagenesis.
      ,
      • Kim J.K.
      • Firan M.
      • Radu C.G.
      • Kim C.H.
      • Ghetie V.
      • Ward E.S.
      Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.
      ,
      • Burmeister W.P.
      • Huber A.H.
      • Bjorkman P.J.
      Crystal structure of the complex of rat neonatal Fc receptor with Fc.
      ), as histidines, positively charged at acidic pH, interact with negatively charged residues on FcRn. The more recently identified albumin interaction has not been mapped in the same detail. However, we and others have shown that the principal site for FcRn is located to the C-terminal domain III (DIII) of albumin, whereas domain I (DI) seems to modulate the interaction (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ,
      • Chaudhury C.
      • Brooks C.L.
      • Carter D.C.
      • Robinson J.M.
      • Anderson C.L.
      Albumin binding to FcRn: distinct from the FcRn-IgG interaction.
      ,
      • Andersen J.T.
      • Daba M.B.
      • Sandlie I.
      FcRn binding properties of an abnormal truncated analbuminemic albumin variant.
      ). The pH dependence of the interaction is regulated by three conserved histidine residues, two were found within subdomains DIIIa (His-464) and DIIIb (His-535), and the last was in a loop connecting the two (His-510). These residues are involved in pH-sensitive interactions at the interfaces and internally in each protein (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ). Furthermore, His-166 in the α2-domain plays a regulatory role by stabilizing a flexible loop within the α1-domain. The loop is disordered at basic pH but forms an ordered structure at acidic pH (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ).
      Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to characterize the binding sites for human serum albumin (HSA) and antibodies blocking HSA binding. Our studies reveal the hFcRn epitopes that are crucial for binding of the antibodies, which overlap with amino acid residues that are required for binding to HSA. Furthermore, we show that the FcRn-albumin interaction is not only pH-dependent but also hydrophobic in nature. Binding is explained by a cluster of tryptophans within a pH-sensitive loop of the α1-domain of FcRn that is structurally ordered at acidic pH, and not at neutral pH, whereas divergent cross-species binding properties of albumin and the blocking antibodies are determined by structural differences in proximity to these hot spot residues. We structurally provide explanations for why the anti-hFcRn antibodies block HSA binding and show selective cross-species FcRn binding. The studies increase our understanding of the FcRn-albumin interaction at the atomic level that will facilitate the design of albumin variants with extended serum half-life for therapeutic applications.

      DISCUSSION

      FcRn is a versatile cellular receptor for the two most abundant proteins in blood, IgG and albumin. Their biodistribution, high concentration, and long serum half-life depend on FcRn-mediated transcytosis or rescue from intracellular degradation. Therefore, it is essential to unravel how FcRn binds both ligands at the atomic level. In addition, as IgG and albumin variants and fusions are increasingly utilized as diagnostics and therapeutics (
      • Andersen J.T.
      • Sandlie I.
      The versatile MHC class I-related FcRn protects IgG and albumin from degradation: implications for development of new diagnostics and therapeutics.
      ,
      • Sleep D.
      • Cameron J.
      • Evans L.R.
      Albumin as a versatile platform for drug half-life extension.
      ), such knowledge will guide design of novel bio-pharmaceuticals. Although the FcRn-IgG interaction has been studied for more than two decades, the more recently discovered interaction with albumin is less well understood. Thus, we aimed for a mechanistic understanding of how hFcRn binds HSA and anti-hFcRn antibodies that block HSA binding. In this report, we identify key amino acid residues on hFcRn that are required for binding to HSA and the blocking antibodies.
      Previously, we demonstrated that mutation of His-166 within the α2-domain of hFcRn eliminated binding to HSA without affecting IgG binding (
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ). His-166 is only partly exposed on the surface, as it is engaged in an intramolecular network of interactions that stabilize an exposed loop within the α1-domain (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ). This happens in a pH-dependent manner, where an ordered structure of the loop is obtained at acidic pH only. In line with this, targeting of charged residues within this loop (E54 and Q56) by mutagenesis eliminates or reduces binding to albumin (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ).
      Moreover, inspection of available crystal structures revealed that His-166 of hFcRn (His-168 in rat FcRn) is surrounded by a cluster of four tryptophan residues that are located within the flexible loop. Two of these tryptophans are fully exposed (Trp-53 and Trp-59), one is partly exposed (Trp-61) and one is buried (Trp-51). As all four tryptophans are conserved among species, we hypothesized that they are important for binding, and performed alanine scanning in hFcRn. Binding studies then revealed that each of them attenuated binding to HSA.
      Furthermore, we took advantages of a panel of nine monoclonal Abs directed toward hFcRn, which were made by immunizing mice lacking expression of FcRn with spleen cells from mice that transgenically express hFcRn (
      • Christianson G.J.
      • Sun V.Z.
      • Akilesh S.
      • Pesavento E.
      • Proetzel G.
      • Roopenian D.C.
      Monoclonal antibodies directed against human FcRn and their applications.
      ). Two of the generated Abs (ADM31 and ADM32) were shown to block HSA binding to recombinant hFcRn at both pH 6.0 and 7.4, whereas one (DVN1) bound to an overlapping binding site at pH 7.4 only. Furthermore, at pH 7.4, all three Abs competed for binding. The binding behavior of the Abs is in agreement with previous cellular studies (
      • Christianson G.J.
      • Sun V.Z.
      • Akilesh S.
      • Pesavento E.
      • Proetzel G.
      • Roopenian D.C.
      Monoclonal antibodies directed against human FcRn and their applications.
      ).
      Next, we aimed to identify their binding epitopes, and found them to have slightly different binding profiles. None bound hFcRn-W59A, all were affected by the mutation of Trp-61, whereas only DVN1 was dependent on the presence of Trp-51. None of these required the presence of Trp-53, which was crucial for HSA binding. Thus, the binding sites for the antibodies overlap with that of HSA, without being identical.
      His-161, which is exposed on the surface of an α-helix on the α2-domain, is in close structural proximity to Trp-59 and was shown to be important for binding to all three Abs, and crucial for binding of DVN1. Substitution of His-161 with alanine, glutamine, or glutamic acid eliminated binding to DVN1. The charge of His-161 changes with pH and thus may explain why DVN1 binds pH-dependent. In addition, Trp-61 and Trp-51 may also be less exposed when the α1-domain loop is stabilized by His-166 at acidic pH and contribute to this pH dependence. As His-161 is in contact with DI of HSA (
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ), the albumin-blocking antibodies mask this interaction.
      To gain structural insights, we inspected the crystal structure of the hFcRn-HSA13 complex, which revealed that the surface-exposed Trp-53 and Trp-59 make direct contact with HSA13 at acidic pH, whereas Trp-51 and Trp-61 stabilize the ends of the hFcRn loop comprising these residues. Thus, the co-crystal structure confirms our findings and together show that the FcRn-albumin interaction is not only pH-dependent but also hydrophobic in nature. This is supported by isothermal titration calorimetry analysis showing that the interaction has hydrophobic features as evidenced by a large positive change in entropy (
      • Chaudhury C.
      • Brooks C.L.
      • Carter D.C.
      • Robinson J.M.
      • Anderson C.L.
      Albumin binding to FcRn: distinct from the FcRn-IgG interaction.
      ). Furthermore, the co-crystal structure shows that the exposed Trp-53 in hFcRn is contacting the HSA partner on a hydrophobic platform in DIIIb of HSA. The platform is comprised of phenylalanine residues in the loop that correspond to residues 500–510, which connects the subdomains DIIIa and DIIIb of HSA as well as the next to last C-terminal α-helix. We recently showed that both these structural areas are of importance for binding of HSA to hFcRn (
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ). The platform is formed by the loop residues Phe-502, Phe-507, and Phe-551. Trp-53 makes direct contact with the platform by hydrophobic stacking with the three fully conserved phenylalanines.
      Furthermore, Trp-59 is interacting with the hydrophobic residues Met-418, Leu-460, and Leu-463 in DIIIa. The hydrophobic interface involving Trp-53 and Trp-59 is surrounded by the conserved histidines that mediate the strict pH dependence of the interaction. Notably, a binding pocket is located near the hydrophobic platform, and binding of compounds to this pocket may well affect FcRn binding. Indeed, a recent study shows that bound long fatty acids negatively modulate binding to hFcRn (
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ).
      In addition, a crystal structure of the hFcRn in complex with WT HSA was very recently made available (
      • Oganesyan V.
      • Damschroder M.M.
      • Cook K.E.
      • Li Q.
      • Gao C.
      • Wu H.
      • Dall'Acqua W.F.
      Structural insights into neonatal Fc receptor-based recycling mechanisms.
      ). The HSA model was divided into separate parts to find a solution during the molecular replacement procedure. Overall, this model resembles that of the hFcRn-HSA13 complex, with the exception of the local conformation changes induced by the four amino acids of the HSA13 variant.
      Large differences exist across species in the FcRn-albumin interaction, which compromise preclinical in vivo evaluations (
      • Andersen J.T.
      • Daba M.B.
      • Berntzen G.
      • Michaelsen T.E.
      • Sandlie I.
      Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding.
      ). Specifically, mFcRn binds only weakly to HSA, whereas hFcRn binds more strongly to MSA than to HSA. Here, we demonstrate that binding of albumin blocking Abs require the presence of His-161. Neither mouse nor rat FcRn bind the Abs, and both have a glutamic acid at the corresponding position (Glu-163), whereas the surrounding residues are otherwise very similar in human, mouse and rat FcRn. Also, dog FcRn, with a glutamine at position 161, showed reduced Ab binding, and in line with this, hFcRn-H161Q bound the Abs with reduced affinity. Moreover, we previously showed that amino acid differences in the vicinity of His-166 of hFcRn (His-168 of mFcRn) modulate binding to albumin (
      • Andersen J.T.
      • Daba M.B.
      • Berntzen G.
      • Michaelsen T.E.
      • Sandlie I.
      Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding.
      ). Notably, although Oganesyan and colleagues (
      • Oganesyan V.
      • Damschroder M.M.
      • Cook K.E.
      • Li Q.
      • Gao C.
      • Wu H.
      • Dall'Acqua W.F.
      Structural insights into neonatal Fc receptor-based recycling mechanisms.
      ) suggest from the hFcRn-HSA WT co-crystal structure that His-161 is the only histidine that mediates strict pH-dependent binding to HSA, we find this unlikely as this position is not conserved across species. We have also previously shown that mutation of His-161 (H161A) reduced affinity toward HSA, but it still bound pH dependently (
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ).
      Although the tryptophans within the α1-domain loop are fully conserved, position 52 varies among species; human has a valine, rat has an isoleucine, and mouse has a methionine in this position. Despite the fact that these amino acids are similar in size and chemical properties, replacing Val-52 of hFcRn with an isoleucine or a methionine-reduced binding to both HSA and MSA. This shows that only minor changes in the composition of the loop affect albumin binding, which further emphasizes the importance of this pH-stabilized loop in albumin binding.
      In this study, all recombinant soluble hFcRn variants were secreted in equal amounts from HEK293E cells, and SDS-PAGE analysis demonstrated that the mutants migrated as distinct bands similar to that of the WT. Furthermore, measurements of their stability by differential scanning fluorimetry gave rise to melting temperatures ranging from 53.9–55.7 °C (data not shown). In addition, the amino acid substitutions that were shown to decrease binding to HSA did not disrupt binding to IgG. Thus, the results strongly indicate that the single point substitutions of the hFcRn heavy chain do not have a large impact on the global stability of the proteins. However, we cannot exclude the possibility that the mutations affect the local structural stability.
      The long serum half-life of albumin makes albumin ideal as a fusion partner or carrier for drugs (
      • Andersen J.T.
      • Sandlie I.
      The versatile MHC class I-related FcRn protects IgG and albumin from degradation: implications for development of new diagnostics and therapeutics.
      ,
      • Sleep D.
      • Cameron J.
      • Evans L.R.
      Albumin as a versatile platform for drug half-life extension.
      ). This is an attractive approach as the therapeutic efficacy of many small drugs is hampered by their very short in vivo half-life. This study provides an increased understanding of the FcRn-HSA interaction at the atomic level that may pave the way for design of albumin variants with extended serum half-life.
      At last, there is an urgent need for anti-hFcRn antibodies for cellular and in vivo studies. However, there are no such well characterized and high affinity monoclonal antibodies commercially available. Thus, the panel of anti-hFcRn antibodies described in this work should be of great interest and, in particular, the antibodies that bind hFcRn pH independently and block ligand binding at acidic pH.

      Acknowledgments

      We are grateful to Sathiaruby Sivaganesh and Kristin Støen Gunnarsen for excellent technical assistance.

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