Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features

In addition to conventional antibodies (Abs), camelids possess Abs consisti ng of only heavy chains. The variable domain of such a heavy-chain Ab (VHH) is fully capable of antigen (Ag) binding. Earlier analysis of 47 VHHs show ed sequence features unique to VHH domains. These include the presence of c haracteristic amino acid substitutions in positions which, in conventional VH domains are involved in interdomain interactions, and the presence of a long third complementarity-determining region (CDR3) which is frequently co nstrained by an interloop disulphide bond. Here, we describe a large (152) set of Lama glama VHH cDNAs. Based on amino acid sequence similarity, these and other published camelid VHHs were classified into four subfamilies. Th ree subfamilies are absent in dromedaries, which have been the primary sour ce of VHHs thus far. Comparison of these subfamilies to conventional VH reg ions reveals new features characteristic of VHHs and shows that many featur es earlier regarded as characteristic of VHHs in general are actually subfa mily specific. A long CDR3 with a concomitant putative additional disulphid e bond is only observed in two VHH subfamilies. Furthermore, we identified new VHH-characteristic residues at positions forming interdomain sites in c onventional VH domains. The VHH subfamilies also differ from each other and conventional VH domains in the canonical structure of CDR1 and CDR2, mean CDR3 length, and amino acid residue variability. Since different VHH-charac teristic residues are observed in all four subfamilies, these subfamilies m ust have evolved independently from classical VH domains. (C) 2001 Elsevier Science Ltd. All rights reserved.