Identifying C-mannosylatedproteins in RAW264.7 cells via in vitro HSC70 Co-immunoprecipitation strategies

C-mannosylation is a specific type of glycosylation in which α-mannose is covalently bound to the C2 atom of a tryptophan via a carbon-carbon bond. Presently, it is known to occur on proteins that contain the W-x-x-W-x-x-W/C motif found within the Thrombospondin Type-1 Repeat (TSR), with ‘x’ denoting any amino acid. C-mannosylation has been implicated in a number of cellular functions, including protein secretion and type I cytokine receptor function. Previous research has shown that exogenous C-mannosylated peptides derived from TSR-containing proteins upregulate the production of the pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNF-α) in RAW264.7 macrophage cells. This upregulation is substantially increased in the presence of Hsc70. When these peptides are internalized by RAW264.7 cells, a binding interaction occurs between the two proteins, which also correlates with increased TNF-α levels. With the knowledge that exogenous C-mannosylated peptides and Hsc70 can influence cytokine production, this study has focused on identifying endogenous C-mannosylated proteins in RAW264.7 cells that interact with Hsc70. The potential of a naturally occurring interaction between these two proteins could elucidate the importance of C-mannosylation in the innate immune response. Through Hsc70 co-immunoprecipitation, a potentially C-mannosylated protein has been identified in Junctophilin-1 (JPH1). JPH1 forms a junctional membrane complex (JMC) between the endoplasmic reticulum and plasma membrane of excitable cells, facilitating communication between calcium ion release channels. Tandem mass spectrometry data suggests that the hexose appears on a single tryptophan in a peptide bearing the sequence: E-G-E-W-A-N-N-K. This indicates that JPH1 may be an example of noncanonical C-mannosylation. To further explore the status of JPH1 as a C-mannosylated protein, the protein was overexpressed in HEK293T cells. This experiment showed that JPH1 was not C-mannosylated in HEK293T cells. Noting the lack of modification, reverse transcriptase polymerase chain reaction (RT-PCR) was performed with both RAW264.7 and HEK293T RNA. These experiments showed that all known C-mannosyltransferase homologs (DPY19L1-4) were expressed in HEK293T cells, while only DPY19L1 and L4 were expressed in RAW cells. Further work is being conducted to overexpress JPH1 in RAW264.7 cells to determine why, or if, JPH1 is C-mannosylated in this particular cell line