1092 Fig. 2 Analysis of glycosylation of VHHs a and VHH2s b by SDS- PAGE. Samples were either treated (+) or mock-treated (−) with endoglycosidase H (endoH). The molecular mass of the markers used is indicated single site. This clone was also found to neutralize LT most efficiently, suggesting that its N-glycosylation contributes to toxin neutralization. This is confirmed by the at least threefold-decreased neutralization efficiency of enzymatically deglycosylated LT109. About 98 % of the LT109 molecules are glycosylated. Such partial glycosylation of sites is more often observed (Kukuruzinska and Lennon 1998). The presence of some unglycosylated LT109 complicates deter- mination of the exact contribution of N-glycosylation to toxin neutralization since the glycosylated VHH could bind to LT with lower affinity. This is not unlikely since the glycosyl- ation site of LT109 is located within CDR2, a region which is often directly involved in antigen binding (Muyldermans et al. 2001). Most VHHs bound the B-subunit and their ability to neutralize LT in vitro seemed to correlate with their ability to inhibit toxin binding to the GM1 receptor, as measured by an inhibition ELISA. This is consistent with previous findings in the many studies on conventional monoclonal antibodies against LT or CT, and the view that toxin neutralization relies on inhibition of toxin receptor interac- Table 2 Effect of VHH2 glyco- sylation on FMDV neutralization + endoglycosidase H treatment, − mock incubation a No neutralization at the highest VHH concentration used VHH(2) M180shVI-12 M180shVI-12 M200shVI-12 M200shVI-12 M180 M200 VI-12 VI-12 Appl Microbiol Biotechnol (2009) 84:1087–1094 tion (Spangler 1992; Apter et al. 1993). Since LT contains five GM1 binding sites (Sixma et al. 1992), neutralizing VHHs must inhibit multiple receptor interactions. This can be accomplished either by binding of multiple VHHs to a single LT molecule or by blocking of multiple receptor sites by a single VHH. In the latter case, an increased size of the VHH due to N-glycosylation is likely to increase toxin neutralization. Possibly, the glycosylation occurring close to the antigen-binding site results in more efficient blocking of toxin receptor sites as compared to glycosylation occurring more distant from the antigen-binding site. It remains to be determined whether glycosylation of VHHs also increases inhibition of monovalent interactions. We also assessed the effect of VHH glycosylation on virus neutralization using two VHHs that are able to bind FMDV. These VHHs naturally lacked potential glycosyla- tion sites. They were indirectly glycosylated by genetic fusion to another VHH that was known to be glycosylated. This successful approach could generally be applied to obtain glycosylation of VHHs. In both cases, the N- glycosylation improved the FMDV neutralizing capacity at least fourfold. One of these VHHs, clone M200, binds to the GH-loop (Harmsen et al. 2007), which directly binds to the cellular receptor. As a result, it is unlikely that the increased neutralization relies on interference of the oligosaccharide with the GH-loop to which the VHH binds. However, each virion contains 60 GH-loop copies (Mateu and Verdaguer 2004). Possibly, the increased neutralization of the GH-loop-binding clone M200 due to N-glycosylation results from steric hindrance of other GH-loop-receptor interactions on the virion surface. Protein glycosylation can also contribute to protein stability after in vivo application (Sinclair and Elliott 2005). Since we studied the effect of neutralization in vitro and because non-glycosylated VHHs already have a high physicochemical stability (Van der Linden et al. 1999), it is unlikely that the decreased toxin or virus neutralization by deglycosylated VHHs results from reduced stability. Parenteral application to mammals of proteins containing terminally mannosylated carbohydrates, such as those occur- Endoglycosidase H treatment FMDV neutralization (mg/ml) − + − + − − − + 0.008 1 0.016 1 0.22 0.059 >10a >1