UBR E3 ligases and the PDIA3 protease control degradation of unfolded antibody heavy chain by ERAD

Danming Tang; Wendy Sandoval; Cynthia Lam; Benjamin Haley; Peter Liu; Di Xue; Deepankar Roy; Tom Patapoff; Salina Louie; Brad Snedecor; Shahram Misaghi

doi:10.1083/jcb.201908087

UBR E3 ligases and the PDIA3 protease control degradation of unfolded antibody heavy chain by ERAD

J Cell Biol. 2020 Jul 6;219(7):e201908087. doi: 10.1083/jcb.201908087.

Authors

Affiliations

¹ Cell Culture and Bioprocess Operations Department, Genentech Inc., South San Francisco, CA.
² Department of Microchemistry, Proteomics and Lipidomics, Genentech Inc., South San Francisco, CA.
³ Department of Molecular Biology, Genentech Inc., South San Francisco, CA.
⁴ Department of Research Biology, Genentech Inc., South San Francisco, CA.
⁵ Department of Early Stage Pharmaceutical Development, Genentech Inc., South San Francisco, CA.

Abstract

Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.

MeSH terms

Amino Acid Sequence
Animals
B-Lymphocytes / cytology
B-Lymphocytes / enzymology
CHO Cells
Calmodulin-Binding Proteins / genetics*
Calmodulin-Binding Proteins / metabolism
Cloning, Molecular
Cricetulus
Endoplasmic Reticulum-Associated Degradation / genetics*
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Humans
Immunoglobulin Heavy Chains / biosynthesis*
Immunoglobulin Heavy Chains / chemistry
Immunoglobulin Heavy Chains / genetics
Mice
Mice, Inbred C57BL
Models, Molecular
Protein Biosynthesis
Protein Disulfide-Isomerases / genetics*
Protein Disulfide-Isomerases / metabolism
Protein Interaction Domains and Motifs
Protein Structure, Secondary
Proteolysis
Recombinant Proteins / biosynthesis
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Ubiquitin-Protein Ligases / genetics*
Ubiquitin-Protein Ligases / metabolism
Ubiquitination

Substances

Calmodulin-Binding Proteins
Immunoglobulin Heavy Chains
Recombinant Proteins
UBR5 protein, human
UBR4 protein, human
Ubiquitin-Protein Ligases
Protein Disulfide-Isomerases
PDIA3 protein, human