Unusual amino acid usage in the variable regions of mercury-binding antibodies.
Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury-containing, hapten-carrier complex. The antibodies reacted by enzyme-linked immunosorbent assay with bovine serum albumin-glutathione-mercuric chloride (BSA-GSH-HgCl) but not with BSA-GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy-chain variable regions and seven light-chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region, which is unusual for murine antibodies. Mutagenesis of the cysteine to either tyrosine or serine in one of the Hg-binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza-specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal-protein interactions and for characterizing antibody responses to structurally simple haptens.
Westhoff
C M
CM
School of Biological Sciences, University of Nebraska, Lincoln.
Lopez
O
O
Goebel
P
P
Carlson
L
L
Carlson
R R
RR
Wagner
F W
FW
Schuster
S M
SM
Wylie
D E
DE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione; D006241:Haptens; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008628:Mercury / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012710:Serum Albumin, Bovine
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D012710:Serum Albumin, Bovine; D008628:Mercury; D005978:Glutathione; D003545:Cysteine