Recombinant anti-polyamine antibodies: identification of a conserved binding site motif.
Polyamines are small linear polycations found ubiquitously in eukaryotic cells. They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation. Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy. Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens. These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%). We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies. While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology. However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif. The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues. The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft. Aromatic residues contribute to polyamine binding interacting with the carbon backbone. The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.
Johnston
J S
JS
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK.
Athwal
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D000918:Antibody Specificity; D001665:Binding Sites; D003429:Cross Reactions; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000276:immunology; D011073:Polyamines / Q000737:chemistry / Q000276:immunology*; D011700:Putrescine / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013095:Spermidine / Q000276:immunology; D013096:Spermine / Q000276:immunology
D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011073:Polyamines; D011994:Recombinant Proteins; D013096:Spermine; D010047:Ovalbumin; D013095:Spermidine; D011700:Putrescine