title: Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.
abstract: Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity.
authors.affiliation: Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands. michiel.harmsen@wur.nl
authors.forename: M M
authors.initials: MM
authors.lastname: Harmsen
authors.forename: C B
authors.initials: CB
authors.lastname: van Solt
authors.forename: H P D
authors.initials: HP
authors.lastname: Fijten
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D001427:Bacterial Toxins / Q000276:immunology*; D002161:Camelids, New World; D002522:Chlorocebus aethiops; D004768:Enterotoxins / Q000276:immunology*; D000939:Epitopes; D029968:Escherichia coli Proteins / Q000276:immunology*; D005536:Foot-and-Mouth Disease / Q000276:immunology / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000276:immunology*; D006031:Glycosylation; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014709:Vero Cells
chemical_list: D057134:Antibodies, Neutralizing; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C027538:heat-labile enterotoxin, E coli