Host expression system modulates recombinant Hsp70 activity through post‐translational modifications. (25th March 2020)

• Record Type: Journal Article
• Authors: Rigo, Mauricio M.; Borges, Thiago J.; Lang, Benjamin J.; Murshid, Ayesha; , Nitika; Wolfgeher, Donald; Calderwood, Stuart K.; Truman, Andrew W.; Bonorino, Cristina
• Is Part Of: FEBS journal. Volume 287:Number 22(2020)
• Page Start: 4902
• Page End: 4916
• Publication Date: 2020-03-25
• Subjects: heat shock protein -- post‐translational modifications -- DnaK -- Escherichia coli -- Pichia pastoris; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15279
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : The use of model organisms for recombinant protein production results in the addition of model‐specific post‐translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70‐kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g., as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry of the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities. Database: Mass spectrometry proteomic data are available in the PRIDE database under the accession number PXD011583. Abstract : Recombinant Hsp70 used in studies can have different functions, sometimes presenting opposite effects. Here, we report that the host expression system modifies the chaperone code of Hsp70, hence its structure and function.