Engineering a Camelid Antibody Fragment That Binds to the Active Site of Human Lysozyme and Inhibits Its Conversion into Amyloid Fibrils

Abstract

A single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…65 It was recognized from early structural studies of anti-lysozyme V H Hs 6 and V NAR s 7 that these molecules interacted with the enzyme in unusual fashion, probing deeply into its active site using extended complementarity-determining region (CDR)3 loops. These results were later replicated independently using additional V H s, 51 V H Hs 11,47,48 and V NAR s 12 directed against the active site of lysozyme, as well as with active site-binding V H Hs against α-amylase, 31,32 carbonic anhydrase, 32 and urokinase. 62,63 Inhibition of α-amylase was achieved by one V H H through penetration of the active site cleft with its CDR2 loop, 31 demonstrating that CDR3-centric binding is not the only mechanism of competitive enzyme inhibition by sdAbs.…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…The average length of the CDR3 from human V H s is 12 residues while that from dromedary V H Hs contains most frequently~16e18 amino acids [34,43]. Several V H Hs with CDR3 longer than 25 residues have been reported [44,45]. The larger size of the CDRs is believed to compensate for the absence of the three CDRs from the V L s (the antigen is recognised by only three CDRs instead of six in conventional antibodies) and therefore provide a sufficiently large antigeninteracting surfaces of 600e800 Å 2 [46].…”

“…3D). In this species, the b-domain and the C-helix (referred to as the amylotope [44]) are cooperatively unfolded while the rest of the adomain remains native [81,82] (Fig. 3A).…”

“…Three nanobodies (named cAb-HuL5, cAb-HuL6 and cAb-HuL22) have been selected by phage display from the blood of a dromedary immunised with the monomeric Wt-HuL; this protein was also used for the panning [37,44,83]. These nanobodies bind to three different epitopes on the surface of lysozyme ( Fig.…”

“…However, several results such as (i) surface plasmon resonance (SPR) competition experiments with an analogue substrate, (ii) the lower affinity for D67H compared to that for Wt-HuL, and (iii) the comparison of NMR heteronuclear single quantum coherence (HSQC) spectra of 15 N-Wt-HuL free and in complex with cAb-HuL22 suggest that cAb-HuL22 binds into the active site of the protein through its long CDR3 loop (i.e. containing 29 residues) [44]. Thus, the epitope of cAb-HuL22 likely encompasses some residues from the amylotope (Fig.…”