Analysis of C‐Mannosylated Proteins, Hsc70, and Inflammation

Abstract

C‐mannosylation is a unique type of post‐translational modification composed of a mannose attached to the indole functional group, most commonly to the amino acid sequence Trp‐xaa‐xaa‐Trp through a carbon‐carbon bond. Many of the functions of C‐linked glycosylation have yet to be defined. Some proteins with the C‐mannosylation composition have been found to be key factors in the process of inflammation (Tuoen et al., 2012). Heat Shock Cognate protein 70 (HSC70) is a molecular chaperone that is constitutively expressed, and has been classified as having a high affinity for C‐mannosylated peptides (Ihara et al., 2010). While HSC70 is known to regulate various cellular functions, its interactions with many other molecules remains unknown. However, HSC70 has been discovered to have a vital role in the myocardial inflammatory response, especially after ischemia and reperfusion injury. (Ning et al., 2008). In a study conducted by Amgen Incorporated, in vitro models were designed to examine the effects of ischemia on myocytes using coverslip hypoxia. Myocytes cultured in these conditions showed metabolic activity that caused inflammation and necrosis, but more importantly showed an effective model of ischemic conditions (Pitts et al., 2004).The aim of this experiment is to use similar methods to induce hypoxic conditions to stimulate the upregulation of HSC70 in RAW264.7 cells. By upregulating and isolating HSC70 in ischemic conditions, there is possibility of extracting C‐mannosylated glycoproteins associated with the protein. We use immunoprecipitation of HSC70 to identify ligands that are upregulated in ischemic conditions which are sequenced by tandem mass spectrometry.Support or Funding Information