Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

Abstract

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...

Pathogenic Mutations Located in the Hydrophobic Core of the Prion Protein Interfere with Folding and Attachment of the Glycosylphosphatidylinositol Anchor

“…Mutant PrP might fold into an alternate tertiary structure, burying the GPI anchor signal sequence. An impact of protein folding on a different posttranslational modification has been shown in previous studies, indicating that the structure of the nascent chain determines the efficiency of N-linked glycosylation (40,41).…”

The export of immunoglobulin D by human neoplastic B lymphocytes.

“…A similar procedure appears to operate for synthesis of membrane and secreted IgD (13). It is of interest that investigation of a human lymphoblastoid cell line expressing surface IgM and IgD of g light chain type, but secreting only IgM, revealed the presence of two mRNA molecules encoding 6 chains (14). One appeared to encode surface 6, the other a ~ chain of lower molecular weight consistent with that of secreted IgD.…”

Complete amino acid sequence of the delta heavy chain of human immunoglobulin D.

“…Althdugh the entire Fc sequence is identical in the two human IgD proteins for which it has been reported-i.e., WAH (2) and NIG-65 (7)-the length of the tailpiece in these 8 chains is only 6 amino acid residues compared to 20 in the mouse 8 tailpiece. Multiple forms ofthe tailpieces ofthe human 8 chain have been reported (16); some though not all of the differences are attributed to the degree of glycosylation, but neither amino acid nor DNA sequence data are available. Several distally coded exonic segments that are potential alternate carboxyl termini for the mouse 8 chain have been identified, but their function is unclear (12,13).…”

“…Like IgM, IgD exists in two forms: sIgD, which is secreted into the serum, and mIgD, which is bound to the B-cell membrane. Cloning studies (12)(13)(14)(15)(16) have shown that the A& and 8 structural genes may both be expressed in a single primary transcript that can be processed to yield either IgM or IgD having the same VH region. Although the recent findings on the cellular role and biosynthesis of tL and 8 chains are similar for mice and humans (9-16), and although human and mouse IgM are similar in structure (17, 18), the mouse 8 chain is unusual in that it lacks the C82 domain present in the human 8 chain (1-7).…”

“…Cloning studies (12)(13)(14)(15)(16) have shown that the A& and 8 structural genes may both be expressed in a single primary transcript that can be processed to yield either IgM or IgD having the same VH region. Although the recent findings on the cellular role and biosynthesis of tL and 8 chains are similar for mice and humans (9)(10)(11)(12)(13)(14)(15)(16), and although human and mouse IgM are similar in structure (17,18), the mouse 8 chain is unusual in that it lacks the C82 domain present in the human 8 chain (1-7). The hinge structures of the 8 chains ofthe two species also differ, and there is unexpectedly low homology in the C81 domains (3).…”