Site-Specific Albumin-Selective Ligation to Human Serum Albumin under Physiological Conditions

Abstract

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It plays a critical role in the native transportation of numerous drugs, metabolites, nutrients, and small molecules. HSA has been successfully used clinically as a noncovalent carrier for insulin (e.g., Levemir), GLP-1 (e.g., Liraglutide), and paclitaxel (e.g., Abraxane). Site-specific bioconjugation strategies for HSA only would greatly expand its role as the biocompatible, non-toxic platform for theranostics purposes. Using the enabling one-bead one-compound (OBOC) technology, we generated combinatorial peptide libraries containing myristic acid, a well-known binder to HSA at Sudlow I and II binding pockets, and an acrylamide. We then used HSA as a probe to screen the OBOC myristylated peptide libraries for reactive affinity elements (RAEs) that can specifically and covalently ligate to the lysine residue at the proximity of these pockets. Several RAEs have been identified and confirmed to be able to conjugate to HSA covalently. The conjugation can occur at physiological pH and proceed with a high yield within 1 h at room temperature. Tryptic peptide profiling of derivatized HSA has revealed two lysine residues (K225 and K414) as the conjugation sites, which is much more specific than the conventional lysine labeling strategy with N-hydroxysuccinimide ester. The RAE-driven site-specific ligation to HSA was found to occur even in the presence of other prevalent blood proteins such as immunoglobulin or whole serum. Furthermore, these RAEs are orthogonal to the maleimide-based conjugation strategy for Cys34 of HSA. Together, these attributes make the RAEs the promising leads to further develop in vitro and in vivo HSA bioconjugation strategies for numerous biomedical applications.

Figure 1

(a) α,β-unsaturated sulfonamides specifically modified K64 of HSA; (b) sulfonyl acrylate specifically modified K573 of HSA; (c) this work: site-specific HSA lysine modification via unactivated acrylamide in the reactive ligand driven by the proximity effect in a complex protein mixture such as human serum.

Figure 2

(a) Published X-ray diffraction studies reveal 5 hydrophobic pockets where myristic acid can bind (gray balls). Myristic acid can bind to domains I and III (red circle) more strongly; (b) design of OBOC libraries. The library compounds comprised 3 parts: myristylated N-terminus (red), random peptides made by 36 different amino acids (blue), and unactivated acrylamide branched from the side chain of a lysine residue (green); (c) sequence of discovered RAEs (LYL1–4) that are reactive toward HSA.

Figure 3

(a–e) Chemical structure of biotin-tagged RAEs LYL1–4 and non-selective 4-nitrophenyl biotin ester (NBE) as positive control used to chemically biotinylate HSA; (f) western blots to detect HSA biotinylated by peptidomimetic RAEs. The conjugation was performed in PBS buffer (pH = 7.2) with 20 μM HSA and 100 μM biotinylation reagents for 1 h at room temperature; (g) bio-layer interferometry assay measured the intrinsic binding affinity using non-covalent biotin-LYLs; (h) MALDI-TOF intact MS for HSA-LYL1BT conjugates prepared at various pH values in PBS. The conjugates were prepared by mixing 20 μM HSA and 200 μM B-LYL1 for 16 h; (i) the intensity of corresponding western blots is proportional to signal intensity from biotinylated HSA in MS and increases as pH elevates.

Figure 4

(a–c) Chemical structure of biotin-tagged maleimide (B-Mal), FITC-tagged maleimide (FC-Mal), and FITC-tagged LYL1 (FC-LYL1) used for selective HSA conjugation in a complicated protein matrix; (d, e) comparison between FC-LYL1 and FC-Mal in modifying proteins in serum. Protein SDS-PAGE gels were exposed to a green channel (Ex = 490 nm, Em = 525 nm) and then stained with Coomassie Blue. Electrophoresis showed that FC-LYL1 can selectively label albumin content (d) while FC-Mal labels multiple proteins (e).

Figure 5

(a) Ability of conjugation to specific lysine allows B-LYL1 to modify HSA concurrently with other conjugation strategies in one pot to afford dual-modified HSA conjugates; (b) western blots for dual-labeled HSA conjugates. B-LYL1 can biotinylate HSA on lysine in the presence of fluorescein-Mal (F-Mal) that modifies cysteine residues. HSA biotinylation was detected by streptavidin Alexa 647 conjugates at the red channel (Ex = 594 nm, Em = 633 nm), while the fluorescein tag was detected at the green channel (Ex = 490 nm, Em = 525 nm). (c) Western blots for dual-labeled HSA conjugates using FC-LYL1 and Sulfo-NHS-Biotin. Dual-conjugation status was examined by similar approaches to (b). (d). Flow cytometry of the LLP2A-HSA-B-LYL1 (20 μM) complex that binds to Jurkat cells. Biotinylated LLP2A ligands (B-LLP2A, 20 μM) were used as the positive control.