Secretory Heavy Chain Research Articles

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein.

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.

Gene transfer of immunoglobulin light chain restores heavy chain secretion.

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.

Cell-type-specific and regulated expression of a human gamma 1 heavy-chain immunoglobulin gene in transgenic mice.

A functionally rearranged human gamma 1 heavy-chain immunoglobulin gene was cloned from a human plasma cell leukemia cell line, ARH-77, into the phage lambda Charon 4A. The recombinant phage DNA was introduced into fertilized mouse eggs (about 200 copies of the human gene per egg). A total of 30 mice were born and were screened for the presence of the human gamma 1 gene by dot hybridization. Two of these 30 mice had integrated one or two copies of the gene. The gamma 1 mRNAs were detected only in spleen. Levels of gamma 1 mRNA and the percentage of spleen cells producing human gamma chain increased up to 50-fold after treatment with bacterial lipopolysaccharide (a B-cell mitogen) but not with concanavalin A (a T-cell mitogen), suggesting B-cell-specific and regulated expression of the human gamma 1 heavy-chain gene. Human gamma chain-producing cells were found only in the periphery of the germinal center of the white pulp in histological sections of the spleen but not in sections of other tissues. Human gamma chains appeared to be coupled with mouse light chains to form a complete IgG molecule and were secreted into the cell supernatant. The production and secretion of endogenous immunoglobulin heavy and light chains in transgenic mice appeared to be the same as in normal mice. About one-seventh of the spleen cells that produced endogenous mouse heavy chains also produced human gamma chains, but no cells that produced only human gamma chain were observed.

Introduction of a Cloned Gene into Fertilized Mouse Eggs and its Expression*

Abstract In order to analyze the role of enhancer sequence for the tissue‐specific and stage specific expression of immunoglobulin gene in normal diploid cells we have introduced cloned human y1 immunoglobulin genes into fertilized mouse eggs. Human y1 genes lacking enhancer sequence were not expressed in any tissues of transgenic mice. On the other hand human y1 genes containing enhancer sequence were expressed only in B lymphocytes. Levels of HIGI mRNA and percentage of human y chain producing cells in spleen cells were increased up to 50 times after treatment with lipopolysaccharide but not with Concanavalin A, suggesting B cell specific and regulated expression of HIGI gene. Human y chain producing cells were only found in the periphery of germinal center of the white pulp in histological section of the spleen suggesting stage‐specific expression. Human y chains appeared to be coupled with mouse light chains to form a complete IgG molecule and are secreted into cell supernatant. The production and secretion of endogenous immunoglobulin heavy and light chains in transgenic mice appeared to be the same as in normal mice. About one‐seventh of spleen cells that are producing endogenous mouse heavy chain produced human y1 chain at the same time. But no cell which produce only human y chain was observed. These results suggest that human γ chain expression had no effect on mouse immunoglobulin expression.

The synthesis and in vivo assembly of functional antibodies in yeast.

The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here the synthesis, processing and secretion of light and heavy chains, the glycosylation of heavy chain, the intracellular localization of these foreign proteins by immunofluorescence, and the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo. Furthermore, only low-level assembly of these chains was found in vitro.

The ultrastructural localization of immunoglobulin in chronic lymphocytic lymphoma cells: changes in light and heavy chain distribution induced by mitogen stimulation

The distribution of light and heavy immunoglobulin chains in chronic lymphocytic leukemia (CLL) cells has been investigated at the ultrastructural level using an immunoperoxidase technique. Light chains were localized in the lumens of the perinuclear space and rough endoplasmic reticulum, while staining for heavy chains was weak or negative and generally confined to the membranes of the rough endoplasmic reticulum. This pattern is consistent with immunoglobulin biosynthetic studies on CLL cells in which light chains are synthesized in excess over heavy chains and secreted as the exclusive immunoglobulin product. The pokeweed mitogen stimulation of two populations of CLL cells for 6 days resulted in a balanced synthesis and secretion of light and heavy chains that was reflected in concomitant change in light and heavy chain distribution and intensity of staining using the immunoperoxidase technique.

The Ultrastructural Localization of Immunoglobulin in Chronic Lymphocytic Lymphoma Cells: Changes in Light and Heavy Chain Distribution Induced by Mitogen Stimulation

The distribution of light and heavy immunoglobulin chains in chronic lymphocytic leukemia (CLL) cells has been investigated at the ultrastructural level using an immunoperoxidase technique. Light chains were localized in the lumens of the perinuclear space and rough endoplasmic reticulum, while staining for heavy chains was weak or negative and generally confined to the membranes of the rough endoplasmic reticulum. This pattern is consistent with immunoglobulin biosynthetic studies on CLL cells in which light chains are synthesized in excess over heavy chains and secreted as the exclusive immunoglobulin product. The pokeweed mitogen stimulation of two populations of CLL cells for 6 days resulted in a balanced synthesis and secretion of light and heavy chains that was reflected in concomitant change in light and heavy chain distribution and intensity of staining using the immunoperoxidase technique.

Asymmetrical surface IgG on MOPC-21 plasmacytoma cells contains membrane heavy chain and one secretory heavy chain.

Membrane proteins from the B lymphomas WEHI-231 and 2PK3 and from the plasmacytomas MPC-11 and MOPC-21 were radioiodinated in situ by the lactoperoxidase method and were subjected to two-dimensional (nonreduced, reduced) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Few heavily labeled membrane proteins were composed of disulfide-bonded subunits. One such protein (m.w. 200,000 intact and 116,000 reduced) shared some properties with the PC-1 alloantigen, although it was not conclusively identified. A second major disulfide-bonded protein (m.w. 200,000 intact and 95,000 reduced) has been identified previously as the receptor for transferrin. Membrane immunoglobulins of WEHI-231 (IgM) and 2PK3 (IgG2a) had the expected subunit structure, whereas membrane immunoglobulin was not detected on MPC-11. In contrast, surface IgG1 of MOPC-21 appeared to consist almost entirely of hybrid molecules containing one membrane gamma 1 chain and one secretory gamma 1 chain. This hybrid IgG molecule appeared to exist in both monomeric and dimeric forms. It is concluded that i) the synthetic and assembly mechanisms of secretory and membrane IgG1 are shared; ii) there are no special mechanisms to prevent pairing of membrane and secretory gamma 1 chains; iii) the presence of one hydrophobic tail is sufficient for membrane insertion of gamma 1 chains; and iv) the C-terminal extension cysteine residues of membrane gamma 1 chains in hybrid IgG molecules are either unpaired or may allow the formation of hybrid IgG dimers.

Synthesis and secretion of mouse immunoglobulin chains from Xenopus oocytes.

The secretion of immunoglobulins from mouse plasmacytoma cell lines is a valuable model for the study of mechanisms of protein secretion from eukaryotic cells. Some cells do not synthesize heavy chain immunoglobulin while retaining the ability to synthesize though not to secrete the complementary light chain1–3. In the MOPC 21 variant tine, NS1, it is absence of heavy chain that prevents secretion4–6. A reciprocal dependence, that is, a requirement of light chain for heavy chain secretion, has not been tested directly because no variants producing normal heavy chain alone have been detected4,7–9. In view of the known insolubility characteristics of isolated heavy chains in vitro10–12, this failure has led to the idea that free heavy chains would be toxic to the cell7,9. Recently, Xenopus laevis oocytes have been shown to provide a surrogate system for studying protein secretion after microinjection of the encoding mRNA13,14. Using this technique we demonstrate here that tetrameric mouse immunoglobulin is assembled and secreted by the oocyte but that free light chains are not secreted. Moreover, using purified heavy chain mRNA we show that heavy chain dimers accumulate in the oocyte and are not secreted unless light chain mRNA is also injected.

Control of J Chain Biosynthesis in Relation to Heavy and Light Chain Synthesis, Polymerization and Secretion

Mouse myeloma tumors and some variants derived from them were labeled in vitro with tritiated leucine and the radioactive J chain was assayed in cell lysates by precipitation with an antiserum specific for mouse J chain. The major findings were: 1) J chain can be found in an IgG2b-secreting cells (MPC-11). These data, together with previous findings suggest that cells secreting all classes of IgG synthesize J chain, even though there is no apparent requirement for J chain in assembly of the IgG molecule. Hence production of J chain does not depend upon secretion of a polymeric immunoglobulin. 2) Intracellular J chain can be found in myeloma variants that do not produce heavy chains showing that production of J chain may not coordinately be linked to the synthesis of heavy chain. 3) J chain was found in cells synthesizing, but not secreting, immunoglobulin. Thus production of J chain is not linked to secretion of immunoglobulin. 4) J chain could not be detected in plasma cells that do not produce immunoglobulins. It was also not found in mouse leukemic cells, suggesting that production of J chain is probably linked in some way to immunoglobulin production.