title: 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.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Pak-Ho
authors.initials: PH
authors.lastname: Chan
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Linda
authors.initials: L
authors.lastname: Menzer
authors.forename: Erwin
authors.initials: E
authors.lastname: De Genst
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
authors.forename: John
authors.initials: J
authors.lastname: Christodoulou
authors.forename: Dirk
authors.initials: D
authors.lastname: Saerens
authors.forename: Alain
authors.initials: A
authors.lastname: Brans
authors.forename: Fabrice
authors.initials: F
authors.lastname: Bouillenne
authors.forename: David B
authors.initials: DB
authors.lastname: Archer
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.forename: Christina
authors.initials: C
authors.lastname: Redfield
authors.forename: Lode
authors.initials: L
authors.lastname: Wyns
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: Mireille
authors.initials: M
authors.lastname: Dumoulin
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000915:Antibody Affinity; D002162:Camelus / Q000235:genetics / Q000276:immunology*; D020134:Catalytic Domain / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000682:Amyloid; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D009113:Muramidase