Structure-function studies of anti-3-fucosyllactosamine (Le(x)) and galactosylgloboside antibodies.
We are studying murine mAbs against two carbohydrate epitopes, 3-fucosyllactosamine (Le(x), CD15) and galactosylgloboside. The VH domains of both panels of Ab are encoded by VH441, a member of the X24 family of Ig genes. To evaluate the contribution of the heavy chain CDR3 to the affinity of the anti-3-fucosyllactosamine Ab, CDR3-H of PMN6, a low affinity Ab, was replaced by the CDR3 of PM81, a higher affinity Ab. The affinity of the chimeric 6/81 Ab was increased when the heavy chain was paired with the PM81 light chain, but not when paired with another light chain (M5), which differs from PM81 light chain by three amino acids. To evaluate the contribution of somatic mutations to the binding of GalGb4, the 3A9 VH sequence, which contains three amino acid substitutions, was replaced by a germ-line sequence encoded by either VH441 or VHX24. The chimeric Ab, 441/3A9 and X24/3A9, bound Ag as well as the wild-type 3A9 Ab. Computer models of the Fv fragments of PM81 and 3A9 were compared with the crystal structure of the Fv fragment of J539, a galactan-binding myeloma protein that is encoded by the same VH and VK genes as 3A9. The surfaces of 3A9 and J539 have shallow pockets that are potential Ag-binding sites. Replacement of CDR3-H Tyr99, which is a prominent component of the pocket, by Ala abolished the binding of Ag. In contrast, the Fv surface of PM81 contains a large cleft rather than a pocket. These models indicate how the same VH gene segment can be used to encode Abs that exhibit different specificities.
Snyder
J G
JG
Dept. of Medicine, Baylor College of Medicine, Houston, TX 77030.
Dinh
Q
Q
Morrison
S L
SL
Padlan
E A
EA
Mitchell
M
M
Yu-Lee
L Y
LY
Marcus
D M
DM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D015295:Antigens, Tumor-Associated, Carbohydrate; D001483:Base Sequence; D002240:Carbohydrate Sequence; D017931:DNA Primers / Q000737:chemistry; D006028:Glycosphingolipids / Q000276:immunology*; D016256:Lewis X Antigen / Q000276:immunology*; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055746:Stage-Specific Embryonic Antigens; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D017931:DNA Primers; D006028:Glycosphingolipids; D016256:Lewis X Antigen; D046911:Macromolecular Substances; D055746:Stage-Specific Embryonic Antigens; C052924:stage-specific embryonic antigen-3