Abstract
Albumin has a serum half-life of three weeks in humans and is utilized to extend the serum persistence of drugs that are genetically fused or conjugated directly to albumin or albumin-binding molecules. Responsible for the long half-life is FcRn that protects albumin from intracellular degradation. An in-depth understanding of how FcRn binds albumin across species is of importance for design and evaluation of albumin-based therapeutics. Albumin consists of three homologous domains where domain I and domain III of human albumin are crucial for binding to human FcRn. Here, we show that swapping of two loops in domain I or the whole domain with the corresponding sequence in mouse albumin results in reduced binding to human FcRn. In contrast, humanizing domain I of mouse albumin improves binding. We reveal that domain I of mouse albumin plays a minor role in the interaction with the mouse and human receptors, as domain III on its own binds with similar affinity as full-length mouse albumin. Further, we show that P573 in domain III of mouse albumin is required for strong receptor binding. Our study highlights distinct differences in structural requirements for the interactions between mouse and human albumin with their respective receptor, which should be taken into consideration in design of albumin-based drugs and evaluation in mouse models.
Highlights
Albumin consists of 67% α-helices connected via flexible loops that make up three homologous domains (DI, DII and DIII)[1]
We have demonstrated that sequence variation in DIII is largely responsible for the cross-species differences, as mouse serum albumin (MSA) with human DIII, instead of mouse DIII, showed comparable binding as WT human serum albumin (HSA) to the mouse and human forms of FcRn, while binding of HSA with mouse DIII to both receptors improved, but showed even stronger binding than WT MSA towards hFcRn34
From the multiple albumin sequence alignment, we found that HSA has four amino acids that differ from MSA in the two DI loops, namely T83N and M87L in loops in DI (loop I) and N111S and R114P in loop II (Fig. 1b)
Summary
Albumin consists of 67% α-helices connected via flexible loops that make up three homologous domains (DI, DII and DIII)[1]. The binding sites for albumin and IgG on FcRn are distinct and non-overlapping, and binding of both ligands is pH-dependent, occurring only at acidic pH below 6.5 and not at neutral pH11,15–18 This enables FcRn to rescue albumin and IgG from lysosomal degradation, as binding readily occurs in acidified endosomes and cease at the cell surface when exposed to the physiological pH of the extracellular surroundings. Chain stabilizes a loop in the α1-domain and conserved tryptophan residues (W51, W53, W59 and W61) within the loop[16,18,26,27] This allows binding of W53 and W59 to hydrophobic pockets in DIII of human serum albumin (HSA) that are sustained in open conformations by H464, H510 and H53518,26. We have demonstrated that sequence variation in DIII is largely responsible for the cross-species differences, as MSA with human DIII, instead of mouse DIII, showed comparable binding as WT HSA to the mouse and human forms of FcRn, while binding of HSA with mouse DIII to both receptors improved, but showed even stronger binding than WT MSA towards hFcRn34
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