Structural differences between the heavy chain of membrane IgD (Bin) and the heavy chain of secreted IgD (8s) were investigated by using a human lymphoblastoid cell line that expresses idiotypically identical IgM and IgD. In a wheat germ cell-free system, mRNA from this cell line was shown to encode two distinct 8 Further analysis of these functional systems must eventually be accompanied by a detailed structural examination ofthe IgD heavy chain (8). Until recently, the low incidence of IgD myeloma and the extreme lability of IgD to proteolysis hampered the determination of the Fc amino acid sequence of the heavy chain of secretory IgD (8,) (7). The study of the heavy chain of membrane IgD (Sm) has been even more difficult. It is extremely susceptible to proteolysis and is present in low amounts, with a slow turnover rate on B cell membranes. Early structural studies on membrane IgD demonstrated that reduced 8 chains migrate as a broad band on NaDodSO4/polyacrylamide gel electrophoresis (8, 9). Subsequent analyses by two-dimensional gel electrophoresis indicated that the biosynthetic forms of 8 exist as a heterogeneous group, reflective of modifications of N-linked oligosaccharides (10). As shown by charge-shift electrophoresis, these forms probably have a hydrophobic COOH terminus (11, 12). Studies using one-dimensional NaDodSO4 gel electrophoresis systems have demonstrated that am exists as two molecular entities (8im and 8m2), distinguished from one another by a difference in molecular weight (ranging from 2000 to 7000) (13-16). Likewise, 8, has been resolved into two species (5s1 and 8s2) that differ in molecular weight (17)(18)(19). The relationship between these various forms ofthe 8 chain is obscure at present. As a result, the structural relationships between 5im and 8, as well as those between am and ILm have yet to be resolved.In this study, IgD biosynthesis was analyzed in a human B lymphoblastoid cell line (SeD) that coexpresses IgM. All Ig molecules synthesized by this cell line express the same idiotype. The 8 chains of this line were found, by cell-free translation ofextracted mRNA, to exist as two distinct primary translation products, most likely corresponding to 8m and 8s. When followed by pulse-chase experiments in vivo, these chains were found to be processed to four forms, differing in relative mobility as a result of differential N-glycosylation. 8m appears to be a higher molecular weight than 8, and does not have a large cytoplasmically exposed domain.
MATERIALS AND METHODSPreparation of RNA and Cell-Free Protein Synthesis. Total cellular RNA was extracted with NaDodSOjphenol/chloroform/isoamyl alcohol and proteinase K as described (20) and translated in a staphylococcal nuclease-treated wheat germ system (21).[35S]Methionine (New England Nuclear; 700 Ci/ mmol; 1 Ci = 3.7 x 10'°becquerels) was used at a final concentration of 1 mCi/ml, and salts were adjusted to final concentrations of 150 mM KOAc and 3.8 mM Mg(OAc)2. In indicated experiments, dog pancreatic microsomal membran...