1090 Appl Microbiol Biotechnol (2009) 84:1087–1094 Fig. 1 Multiple amino acid sequence alignment of E. coli LT-binding VHHs. Dashes indicate gaps introduced for sequence alignment. Dots indicate amino acids identical to the consensus sequence. Residue numbering and CDR definitions are according to the IMGT numbering system (Lefranc 2004). Residues 1–5 (amino acid sequence QVQLQ) and 143–145 (amino acid sequence VSS) are not shown since they are vector-encoded. The VHH clones are ranked according to CDR3-group and clone number. VHHs of the same CDR3 group are boxed. Potential N- glycosylation sites are indicated in boldface Effect of VHH N-glycosylation on LT neutralization The VHHs LT109 and LT156 have potential N-glycosylation sites at IMGT positions 61 and 62, respectively, which are both located in CDR2 (Fig. 1). SDS-PAGE analysis showed that LT109 indeed consisted of a heterogeneous high- molecular-weight species, indicative of high-mannose type N-glycosylation, in addition to a minor amount of a discrete