Abstract
Cosmc is an endoplasmic reticulum chaperone necessary for normal protein O-GalNAc glycosylation through regulation of T-synthase, its single client. Loss-of-function of Cosmc results in expression of the Tn antigen, which is associated with multiple human diseases including cancer. Despite intense interest in dysregulated expression of the Tn antigen, little is known about the structure and function of Cosmc, including domain organization, secondary structure, oligomerization, and co-factors. Limited proteolysis experiments show that Cosmc contains a structured N-terminal domain (CosmcΔ256), and biochemical characterization of CosmcΔ256 reveals wild type chaperone activity. Interestingly, CosmcE152K, which shows loss of function in vivo, exhibits wild type-like activity in vitro. Cosmc and CosmcE152K heterogeneously oligomerize and form monomeric, dimeric, trimeric, and tetrameric species, while CosmcΔ256 is predominantly monomeric as characterized by chemical crosslinking and blue native page electrophoresis. Additionally, Cosmc selectively binds divalent cations in thermal shift assays and metal binding is abrogated by the CosmcΔ256 truncation, and perturbed by the E152K mutation. Therefore, the N-terminal domain of Cosmc mediates T-synthase binding and chaperone function, whereas the C-terminal domain is necessary for oligomerization and metal binding. Our results provide new structure-function insight to Cosmc, indicate that Cosmc behaves as a modular protein and suggests points of modulation or regulation of in vivo chaperone function.
Highlights
Cosmc is an endoplasmic reticulum (ER) chaperone that is required for the activity of Tsynthase, an essential glycosyltransferase (GT), and its only known client [1]
Obtaining large amounts of highly purified material is a common bottleneck for biochemical and biophysical studies, and though our previous work relied upon a baculovirus insect cell expression system for expressing recombinant Cosmc, it is not ideal because it is low yield [17, 19, 21]
We pursued a variety of constructs for Cosmc expression in E. coli, including co-expression with bacterial chaperones, specialized host strains, periplasmic localization, fusion proteins, and low temperature expression
Summary
Cosmc is an endoplasmic reticulum (ER) chaperone that is required for the activity of Tsynthase, an essential glycosyltransferase (GT), and its only known client [1]. T-synthase (Core 1 β1−3 galactosyltransferase–C1GALT1) is necessary for normal O-glycoprotein biogenesis and elongates the Tn antigen (GalNAcα1-Ser/Thr glycopeptide) by the addition of galactose to form the core 1 glycan structure (Galβ1−3GalNAcα1-Ser/Thr) [2]. This type of O-GalNAc glycosylation is a ubiquitous and complex posttranslational modification, where ~80% of proteins that enter the secretory pathway are predicted to contain one or more O-GalNAc modifications, which are temporally and spatially regulated [3, 4]. Biochemical characterization of Cosmc decision to publish, or preparation of the manuscript
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