Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils

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 -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.

Jaar van publicatie:2008

Trefwoorden:nanobody, fibril