Understanding the biosynthesis of human IgM SAM-6 through a combinatorial expression of mutant subunits that affect product assembly and secretion.

Autor: Hasegawa H; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America., Wang S; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America., Kast E; Molecular Analytics, Department of Biologic Therapeutic Discovery, Amgen Inc., South San Francisco, CA, United States of America., Chou HT; Structural Biology, Department of Small Molecule Therapeutic Discovery, Amgen Inc., South San Francisco, CA, United States of America., Kaur M; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America., Janlaor T; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America., Mostafavi M; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America., Wang YL; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America., Li P; Discovery Protein Science, Department of Large Molecule Discovery and Research Data Science, Amgen Inc., South San Francisco, CA, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2024 Jun 07; Vol. 19 (6), pp. e0291568. Date of Electronic Publication: 2024 Jun 07 (Print Publication: 2024).
DOI: 10.1371/journal.pone.0291568
Abstrakt: Polymeric IgMs are secreted from plasma cells abundantly despite their structural complexity and intricate multimerization steps. To gain insights into IgM's assembly mechanics that underwrite such high-level secretion, we characterized the biosynthetic process of a natural human IgM, SAM-6, using a heterologous HEK293(6E) cell platform that allowed the production of IgMs both in hexameric and pentameric forms in a controlled fashion. By creating a series of mutant subunits that differentially disrupt secretion, folding, and specific inter-chain disulfide bond formation, we assessed their effects on various aspects of IgM biosynthesis in 57 different subunit chain combinations, both in hexameric and pentameric formats. The mutations caused a spectrum of changes in steady-state subcellular subunit distribution, ER-associated inclusion body formation, intracellular subunit detergent solubility, covalent assembly, secreted IgM product quality, and secretion output. Some mutations produced differential effects on product quality depending on whether the mutation was introduced to hexameric IgM or pentameric IgM. Through this systematic combinatorial approach, we consolidate diverse overlapping knowledge on IgM biosynthesis for both hexamers and pentamers, while unexpectedly revealing that the loss of certain inter-chain disulfide bonds, including the one between μHC and λLC, is tolerated in polymeric IgM assembly and secretion. The findings highlight the differential roles of underlying non-covalent protein-protein interactions in hexamers and pentamers when orchestrating the initial subunit interactions and maintaining the polymeric IgM product integrity during ER quality control steps, secretory pathway trafficking, and secretion.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Hasegawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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