Membrane Orientation and Location of Multiple and Distinct Allotypic Determinants of Mouse Lymphocyte IgD

Steven W. Kessler; Virgil L. Woods; Fred D. Finkelman; Irwin Scher

Abstract

The general structure, surface orientation, and location of allotypic determinants of mouse lymphocyte IgD have been deduced by enzymatic and immunochemical dissection of this molecule on the cell membrane. After limited trypsin digestion of IgD of the a (and cross-reacting c) and b allotypes, IgD was no longer detectable on cell surfaces by immunofluorescent staining with an antiserum against light chain determinants, an allotype specific heteroantiserum against IgD^a, and a hybridoma antibody specific for the IgD^b allotype. Immunoprecipitations performed with these reagents and m.w. analyses by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, after lactoperoxidase-catalyzed cell radioiodination and trypsin digestion, indicated that the respective cleavage fragments were contained in the incubation medium and corresponded to the Fab portion of IgD. On the other hand, trypsin digestion reduced, but did not eliminate immunofluorescent staining with a heteroantiserum that bound IgD determinants common to all allotypes, and straining with two other hybridoma antibodies specific for the a and b allotypes of IgD was only minimally affected. Immunoprecipitations with these reagents indicated that the respective antigenic fragments, corresponding to the Fc portion of IgD, were retained on the cell membrane. Therefore, mouse lymphocyte IgD contains distinct allotypic determinants on both the Fab and Fc portions. Furthermore, it was determined that both fragments bore carbohydrate moieties by lentil lectin chromatography, and that cleavage took place NH₂-terminal to a disulfide bond joining the two heavy chains in the Fc region. These findings point to a high degree of structural homology between mouse membrane IgD and human IgD myeloma proteins.

Footnotes

↵² Please address correspondence to Dr. Steven Kessler, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20214.
↵¹ This work was supported by Uniformed Services University of the Health Sciences Grants CO8310 and RO8308 and Naval Medical Research and Development Command Research Task No. M0095-PN.001.1030. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council.