1088 cellular receptor. Alternatively, binding of a bulky antibody close to a site involved with receptor interaction may also prevent receptor interaction, a process called steric hin- drance (Mateu and Verdaguer 2004). Due to their small size, VHHs are probably less well suited for steric hindrance. Increasing the size of VHHs by N-glycosylation could increase their neutralizing capacity. N- glycosylation occurs at Asn-X-Ser/Thr sequons, where X represents any amino acid except Pro. About 90% of such sites become N-glycosylated (Gavel and von Heijne 1990). About 10% of the VHHs have such potential N-glycosyla- tion sites (Harmsen et al. 2000). N-glycosylation is a complex sequential process involving the transfer of a core- oligosaccharide to the protein within the endoplasmic reticulum and the addition of an outer-chain oligosaccharide within the Golgi apparatus. In mammals, the type of outer- chain glycosylation is highly variable between different proteins and sites and can be classified into high-mannose, hybrid, and complex-type glycosylation. Baker’s yeast (S. cerevisiae) only adds outer chains consisting of α1–3- and α1–6-linked mannose residues. Depending on the particular glycosylation site, such yeast outer-chain glycosylation can result either in relatively short chains of about 10 kDa (core- like N-glycosylation) or very long chains with different lengths (high-mannose-type N-glycosylation) that have a molecular mass of 50–100 kDa (Kukuruzinska and Lennon 1998). As a result, yeast N-glycosylation of small proteins such as VHHs that have an average size of 15 kDa, can result in a large increase in molecular mass. We therefore analyzed the contribution of N-glycosyla- tion of VHHs to their neutralizing capacity. We isolated several LT-binding VHHs and produced them by secretory yeast expression. A potent LT-neutralizing VHH was found to be N-glycosylated. We have further shown that its glycosylation contributes to its neutralization. We also showed that we could increase the FMDV-neutralizing capacity of two non-glycosylated VHHs by genetic fusion to another VHH that is glycosylated. The possible mechanism by which N-glycosylation improves neutraliza- tion capacity of VHHs is discussed. Materials and methods Strains and genetic constructs Appl Microbiol Biotechnol (2009) 84:1087–1094 Phage display selection of LT-binding VHHs The basic procedure for selection of antigen specific VHHs from immunized llamas by phage display using vector pUR4676 has been described before (Frenken et al. 2000; Harmsen et al. 2002). We immunized a single llama with 100 µg LT (Sigma, Saint Louis, MO) per immunization. Phage display selections of clones producing LT-binding VHHs were performed by biopanning (McCafferty and Johnson 1996), using polystyrene 96-well plates coated with 0.1 or 0.01 µg/ml LT (Sigma). The VHH encoding regions of clones binding LT in ELISA were subjected to automated DNA sequencing as described previously (Harmsen et al. 2000). The deduced VHH amino acid sequences were aligned according to the IMGT system ( L e f r a n c 2 0 0 4 ) f o r a l i g n m e n t , n u m b e r i n g , a n d complementarity-determining region (CDR) definition of immunoglobulins. Additional gaps had to be introduced at the end of CDR2 to accommodate VHHs with unusually long CDR2s. Yeast production of VHHs LT-binding VHHs containing a C-terminal hexahistidine tag were produced by secretory yeast expression using plasmid pUR4584 and yeast strain SU51 (Harmsen et al. 2005b). For this purpose, VHH clones were transferred from pUR4676 to pUR4584 using the PstI and BstEII restriction sites. Single FMDV-binding VHHs M180 and M200 and the porcine immunoglobulin G binding VHH VI-12 were produced as described previously using vector pUR4585 (Harmsen et al. 2007) with C-terminal c-myc and hexahis- tidine tags. The VHH2s M180shVI-12 and M200shVI-12 were produced as described previously (Harmsen et al. 2005a), but using the llama short-hinge sequence without cysteines (amino acid sequence AHHSEDPSSKSP) instead of the synthetic Gly–Gly–Ser linker for fusion of the two VHH domains. Secreted VHHs or VHH2s were purified by immobilized-metal affinity chromatography as described previously (Harmsen et al. 2005b). VHHs were deglycosy- lated by 16 h incubation of 5 mg/ml VHH with 2 U/ml endoglycosidase H (Roche Applied Science, Mannheim, Germany) at 37 °C in 50 mM sodium citrate, pH 5.5. Vero cell assay E. coli XL1-Blue (Stratagene, La Jolla, CA) was used for routine recombinant DNA manipulation. E. coli TG1 (Stratagene) was used for phage display purposes. S. cerevisiae SU51 (YT6-2-1L) MATa; can1; his4-519; leu2- 3, −112; cir+, which is a cir+ derivative of AH22 (ATCC 38626), was used for VHH production as described previously (Van der Vaart 2002). The cytopathogenic effect (CPE) of LT on African green monkey kidney (Vero) cells was used to measure LT toxicity (Speirs et al. 1977). Monolayers of Vero cells were grown in Minimal Essential Medium Eagles with Earle’s salts and L-glutamine (EMEM; Invitrogen, San Diego, CA) and containing 10% normal calf serum in 96-well plates at 37 °C in a 5% CO2 atmosphere. The ability of VHHs to