The primary structure and specificity determining residues displayed by recombinant salmon antibody domains.
Previously, single chain fragments of salmon (Salmo salar L.) immunoglobulin variable regions (scFv) were isolated by reactivity towards trinitrophenyl (TNP) or fluorescein (FITC) using phage display technology. The fine specificity of six scFv clones were analysed by ELISA, while the primary structure was determined by DNA sequencing. In addition, preliminary models of one anti-TNP and one anti-FITC clone were built. Here, a follow-up analysis of the primary and tertiary structure of all six clones is focused on the structural basis for hapten specificity. Tertiary structure was analysed by molecular modelling of the antigen combining site. The analysis shows that reactivity to each hapten is maintained by a number of different combinations of VH, D, JH and VL sequences. Accordingly, various sizes of CDR3 on both the heavy and light chain and CDR2 of IgH may support TNP binding. Due to variability of the antigen combining site each clone probably has a distinct binding affinity. However, a feature common among the four scFv antibodies that recognise TNP is a positively charged Arg in CDR2 of either the heavy or light chain. In the majority of the anti-TNP clones localisation of this side-chain is stabilised by a negatively charged Asp in LCDR1. In addition, a Trp in LCDR3 is conserved in all the anti-TNP clones. Also, the anti-FITC clones display a Trp in the LCDR3, suggesting its participation in binding of FITC as well. In combination with a large aromatic amino acid near the N-terminus of HCDR2 and a positively charged Arg in CDR1, these residues probably determine both specificity and affinity towards the FITC moiety.
Solem
Stein Tore
ST
Department of Marine Biotechnology, Norwegian College of Fishery Science, N-9037 Tromsø, Norway. steins@nfh.uit.no
Brandsdal
Bjørn O
BO
Smalås
Arne
A
Jørgensen
Trond Ø
TØ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003429:Cross Reactions; D016650:Fluorescein-5-isothiocyanate; D006241:Haptens / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D020317:Salmo salar / Q000276:immunology*; D016415:Sequence Alignment; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D014301:Trinitrobenzenes / Q000276:immunology
D000906:Antibodies; D006241:Haptens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014301:Trinitrobenzenes; D016650:Fluorescein-5-isothiocyanate