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. 2014 Jun 13;289(24):17228-39.
doi: 10.1074/jbc.M113.522565. Epub 2014 Apr 24.

Dissection of the neonatal Fc receptor (FcRn)-albumin interface using mutagenesis and anti-FcRn albumin-blocking antibodies

Kine Marita Knudsen Sand  1 , Bjørn Dalhus  2 , Gregory J Christianson  3 , Malin Bern  1 , Stian Foss  1 , Jason Cameron  4 , Darrell Sleep  4 , Magnar Bjørås  2 , Derry C Roopenian  3 , Inger Sandlie  1 , Jan Terje Andersen  5
Affiliations

Dissection of the neonatal Fc receptor (FcRn)-albumin interface using mutagenesis and anti-FcRn albumin-blocking antibodies

Kine Marita Knudsen Sand et al. J Biol Chem. .

Abstract

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.

Keywords: Albumin; Antibody; Biodegradation; Bioengineering; Fc Receptor; FcRn; Half-life; Hydrophobic; pH Regulation.

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Figures

FIGURE 1.
FIGURE 1.
The 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) (25) 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) (26) 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.
FIGURE 2.
FIGURE 2.
Mutation of the hFcRn tryptophan residues eliminates binding to albumin. A, SDS-PAGE gel migration of recombinant GST-tagged hFcRn WT and variants showing the expected molecular sizes of the GST-fused HC and β2-microglobulin (β2m). Representative SPR sensorgrams showing binding of immobilized WT hFcRn (B), hFcRn-W51A (C), hFcRn-W53A (D), hFcRn-W59A (E), hFcRn-W61A (F), and hFcRn-H166A (G) to WT HSA at pH 6.0. ELISA binding of WT hFcRn and the tryptophan mutants to titrated amounts of HSA (H) and human IgG1 (I) at pH 6.0 (n = 3). All data are presented as mean ± S.D.
FIGURE 3.
FIGURE 3.
Identification of monoclonal Abs that block albumin binding to hFcRn. Competitive SPR analysis where monomeric hFcRn was injected alone or in the presence of WT HSA and HSA-K500A (A), or DVN1, ADM31, ADM32, and DVN24 (B) over immobilized WT HSA at pH 6.0. Relative binding was calculated by setting the maximum binding response of WT hFcRn toward WT HSA to 1.0. C, ELISA binding of hFcRn-E115A/E116A to titrated amounts of DVN24 and ADM31 coated in ELISA wells at pH 7.4 (n = 3). All data are presented as mean ± S.D. Competitive SPR analysis where monomeric hFcRn was injected alone or in the presence of DVN1, ADM31, ADM32, and DVN24 over immobilized ADM31 at pH 7.4 (D) and pH 6.0 (E). Representative sensorgrams showing binding of titrated amounts of monomeric hFcRn injected over immobilized DVN1 (F), ADM31 (G), ADM32 (H), and DVN24 (I) at pH 7.4. The binding kinetic constants are summarized in Table 1.
FIGURE 4.
FIGURE 4.
Trp-59 is required for binding to albumin-blocking anti-hFcRn Abs. Binding of WT hFcRn and the tryptophan mutants to titrated amounts of DVN1 (A), ADM31 (B), ADM32 (C), and DVN24 (D) coated in ELISA wells at pH 7.4 (n = 3). All data are presented as mean ± S.D.
FIGURE 5.
FIGURE 5.
The 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 Table 2.
FIGURE 6.
FIGURE 6.
Selective FcRn cross-species binding to the monoclonal Abs depends on His-161. A, close-up of the structural areas of hFcRn and rat FcRn encompassing the α1-domain loop with the tryptophan residues (red), hFcRn Val-52 and rat FcRn Ile-52 (cyan), and the α2-domain α-helix with hFcRn His-161 (blue), and rat FcRn Glu-163 (orange). The figures were made using the available crystal structures of hFcRn (25) and rat FcRn (24). B, an alignment of the stretch of amino acids of the α-helix containing His-166 in the α-2 domain of FcRn from 10 different species. A non-conserved amino acid variation is found at position 161 (human numbering). C, SDS-PAGE gel migration of recombinant GST-tagged hFcRn WT and mutants (V52I, V52M, H161E, and H161Q) showing the expected molecular sizes of the GST-fused HC and β2-microglobulin (β2m). Binding of WT hFcRn and mutants (V52I, V52M, W59A, H161A, H161E, and H161Q) to DVN1 (D), ADM31 (E), ADM32 (F), and DVN24 (G) at pH 7.4. Relative binding of the Abs to the hFcRn variants was calculated from an ELISA screen (data not shown), where binding of WT hFcRn to the Abs was set to 1.0 (n = 3). All data are presented as mean ± S.D.
FIGURE 7.
FIGURE 7.
Non-conserved FcRn residues modulate albumin binding. Representative SPR sensorgrams show binding of titrated amounts of WT HSA to immobilized WT hFcRn (A), hFcRn-V52I (B), hFcRn-V52M (C), hFcRn-Q56A (D), hFcRn-H161E (E), and hFcRn-H161Q (F) at pH 6.0. Representative SPR sensorgrams show binding of titrated amounts of WT MSA to immobilized WT hFcRn (G), hFcRn-V52I (H), hFcRn-V52M (I), hFcRn-Q56A (J), hFcRn-H161E (K), and hFcRn-H161Q (L) at pH 6.0. The binding kinetic constants are summarized in Table 1.
FIGURE 8.
FIGURE 8.
Hydrophobic interactions between hFcRn tryptophan residues and HSA DIII stabilize the complex. A, an overview of the crystal structure of the hFcRn-HSA13 complex showing the positions of residues Trp-53, Trp-59, His-161, and His-166 (yellow spheres) in the α1-α2 domain platform of hFcRn, and their positions relative to the three α-helical domains in HSA; DI (pink), DII (orange), and DIII (cyan/blue). The DIII is split into subdomains DIIIa (cyan) and DIIIb (blue). B, a close-up of the region around Trp-53 showing interactions with hydrophobic HSA residues Phe-507, Phe-509, and Phe-551. The hydrophobic part of Thr-527 and Glu-531 also defines the hydrophobic pocket into which Trp-53 is inserted. C, a close-up of the region around Trp-59 showing the hydrophobic surroundings, with HSA residues Met-418, Thr-422, Leu-460, and Leu-463 being closest to Trp-59. Also, His-464 and Val-426 contribute to the hydrophobic pocket. D, a close-up of His-161 in hFcRn that points toward a loop in the HSA DII domain, containing residues Glu-82 and Thr-83. β2m, β2-microglobulin.
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