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Post-translational fate of variant MOPC 315 lambda chains in Xenopus oocytes and mouse myeloma cells
Abstract:
The post-translational fates of three immunoglobulin lambda chain variants of MOPC 315 were investigated in mouse plasmacytoma cell lines and in mRNA-microinjected Xenopus oocytes. Quite unexpectedly we found that one non-secretory variant chain (lambda-43) underwent extensive post-translational N-glycosylation: however the presence of the oligosaccharide moiety did not account for the nonsecretory phenotype nor did it affect the rate of degradation of this lambda chain. Another variant chain (lambda-47) at first believed to be non-secretory, was found to be secreted from oocytes at a very low level, but mostly as a lambda-lambda dimer. In myeloma cells a low level of lambda-47 chain was secreted and again lambda-lambda dimers were the favoured secretory form. The secretory lambda-48 chain also formed lambda-lambda dimers, whereas lambda-43, which was never secreted, was only found as a monomeric lambda chain in both oocytes and myeloma cells. A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (gamma 1) chain mRNA and MOPC 315 lambda chain mRNAs. The wild type lambda chain (lambda-48) was able to assemble with the gamma chain in a covalently bound tetramer (gamma gamma lambda lambda). The variant lambda-47 chain was also able to form gamma gamma lambda lambda tetramers, whereas the lambda-43 was not, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted. These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell-type specific.
Insights
Investigating immunoglobulin lambda chain variants revealed that protein conformation, not glycosylation, dictates secretion. Misfolded variants fail to assemble into functional tetramers, preventing their secretion from cells.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Immunoglobulin lambda light chains are essential components of antibodies.
- Understanding protein secretion pathways is crucial for antibody engineering and treating related diseases.
- Post-translational modifications and protein assembly significantly influence protein fate.
Purpose of the Study:
- To investigate the post-translational modifications and secretion mechanisms of three MOPC 315 immunoglobulin lambda chain variants.
- To determine the role of N-glycosylation and protein assembly in the non-secretory phenotype of variant lambda chains.
- To explore the cell-type specificity of protein secretion pathways using mouse plasmacytoma cells and Xenopus oocytes.
Main Methods:
- Investigated three MOPC 315 immunoglobulin lambda chain variants (lambda-43, lambda-47, lambda-48).
- Utilized mouse plasmacytoma cell lines and mRNA-microinjected Xenopus oocytes for studying protein fate.
- Analyzed protein secretion, glycosylation, monomer/dimer formation, and tetramer assembly (with MOPC 21 heavy chain).
- Administered tunicamycin to assess the impact of glycosylation inhibition on secretion.
Main Results:
- The non-secretory lambda-43 variant underwent N-glycosylation, but this modification did not cause its non-secretory phenotype or affect degradation.
- Variant lambda-47 was secreted at low levels, primarily as lambda-lambda dimers, and could form gamma gamma lambda lambda tetramers.
- Secretory lambda-48 and variant lambda-47 formed lambda-lambda dimers and gamma gamma lambda lambda tetramers, respectively, which were secreted; lambda-43 remained monomeric and was not secreted.
- Lambda-43 failed to assemble into gamma gamma lambda lambda tetramers, even when glycosylation was blocked.
Conclusions:
- Protein conformational changes, rather than N-glycosylation, are the primary determinants of immunoglobulin lambda chain secretion.
- The ability to form specific oligomeric structures (dimers and tetramers) is critical for the secretion of immunoglobulin chains.
- The cellular mechanisms recognizing conformational changes for protein secretion are conserved across different cell types, suggesting a universal pathway.