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Biochemical characterization of functional domains of the chaperone Cosmc.

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 (Cosmc?256), and biochemical characterization of Cosmc?256 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 Cosmc?256 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 Cosmc?256 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.

SUBMITTER: Hanes MS

PROVIDER: S-EPMC5493369 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Biochemical characterization of functional domains of the chaperone Cosmc.

Hanes Melinda S MS Moremen Kelley W KW Cummings Richard D RD

PloS one 20170630 6

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. Li ...[more]

PMID: 28665962

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Project description:The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.

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Biochemical characterization of functional domains of the chaperone Cosmc.

Publications

Biochemical characterization of functional domains of the chaperone Cosmc.

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