The intrinsic cysteine and histidine residues of the anti-Salmonella antibody Se155-4: a model for the introduction of new functions into antibody-binding sites.
New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a 'reagentless' fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity.
Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6 Martin.Young@nrc-cnrc.gc.ca Roger.MacKenzie@nrc-cnrc.gc.ca.
N Martin
NM
Young
Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6.
David C
DC
Watson
Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6.
Anna M
AM
Cunningham
Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6 Martin.Young@nrc-cnrc.gc.ca Roger.MacKenzie@nrc-cnrc.gc.ca.
C Roger
CR
MacKenzie
D000907:Antibodies, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005456:Fluorescent Dyes; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012475:Salmonella / Q000276:immunology*
D000907:Antibodies, Bacterial; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper; D003545:Cysteine
CDR4 loops; binding-site modification; histidine and cysteine residues; metal affinity