Characterization of biosynthetic IgG oligomers resulting from light chain variable domain duplication.
We have characterized a human IgG1 monoclonal antibody composed of altered light chains. Each light chain consists of two identical variable domains and a kappa constant domain, in association with a normal gamma chain. This antibody assembled biosynthetically into a mixture of stable oligomers and monomers. Employing gel filtration, PAGE, and electron microscopy, we examined the antibody and the nature of the associations involved in oligomer formation. By engineering a protease factor Xa site between the duplicated light chain variable domains and examining the fragments produced following factor Xa cleavage, we demonstrated the association of the IgG monomers occurred through their duplicated VL domains. Electron microscopy showed the oligomeric antibody to be predominantly dimers and trimers in which the monomeric units were associated through the tips of the Fab portion of the antibody, presumably through the protruding N-terminal VL domains. Similar examination of monomers demonstrated several molecular forms, including individual molecules with self-crosslinked Fab arms and others displaying the open Y and T shapes typically observed for IgG antibodies. The monomers also displayed distally protruding domain-like structures. The oligomers produced by this cell line therefore occurred through the noncovalent interaction between the extra light chain variable domains.
Roux
K H
KH
Department of Biological Science, Florida State University, Tallahassee.
Shuford
W W
WW
Finley
J W
JW
Esselstyn
J
J
Pankey
S
S
Raff
H V
HV
Harris
L J
LJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001704:Biopolymers; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family
D000911:Antibodies, Monoclonal; D001704:Biopolymers; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D004247:DNA; D015951:Factor Xa