Identifying C‐mannosylated Peptides Involved In Hsc70 Mediated Inflammation

Glycosylation functions in a variety of ways including ligand recognition and to ensure only proteins which are properly folded are transferred to the Golgi apparatus. Glycoproteins are proteins in which a carbohydrate binds a specific motif of amino acid residues located on the protein. There are three binding types, including O‐linked, N‐linked, and the less commonly detected C‐linked. C‐mannosylation is a form of glycosylation in which mannose is attached via a carbon‐carbon bond with the first tryptophan in trp‐x‐x‐trp sequences. Many proteins that are instrumental in the process of inflammation have been found to possess C‐mannosyltryptophan. The primary antibody 1B5, IgG class has been shown to recognize Hsc70, a heat shock family protein that interacts with mannosylated peptides. Our objective is to use the 1B5 antibody to isolate Hsc70 ligands from cell culture. We have applied this technique to probe Hsc70 from lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages. LPS stimulates the production of Hsc70 in macrophage cultures, allowing for analysis of ligands that associate with Hsc70. After isolating Hsc70, identification of mannosylated ligands can occur. Both LPS and non‐LPS treated cells are grown in parallel and collected at 0, 2, 24, and 48 hour time periods. GNL‐pulldowns are then used to isolate C‐mannosylated proteins and anti‐Hsc70 immunoprecipitation was utilized to isolate all Hsc70‐binding proteins. An interaction between C‐mannosylated proteins and heat shock protein 70 (Hsp70) has been found to increase the production of TNF‐α in macrophages, which can result in a strong inflammatory response, potentially leading to septicemia if unregulated. Our goal is to identify what C‐mannosylated proteins are interacting with Hsc70 by probing Hsc70 ligands and C‐mannosylated proteins.