Identification and characterization of endogenous shed ICOSL from mouse sera, human sera and active systemic lupus erythematosus patient sera

摘要

ICOSL is a type I cell surface B7 family member (Fig. 1) expressed on B cells, dendritic cells and macrophages. ICOSL exerts its function by co-stimulating effector and memory T cells via its receptor ICOS and plays a critical role in T helper cell and B cell differentiation, isotype switching, germinal center formation and memory B cell expansion. Cell surface ICOSL is known to be shed following ICOS binding or B cell receptor engagement. This shedding is reported to occur through a proteolytic cleavage that releases the extracellular region of ICOSL. Here we report for the first time the identification of two distinct C-termini for circulating shed ICOSL in normal mouse, normal human, and active SLE patient sera. In order to identify the endogenous circulating C-terminus of shed ICOSL, we used a proteomics approach consisting of an immunoprecipitation step followed by deglycosylation, SDS-PAGE (Fig. 2), and in-gel digestion to generate peptides. Reversed-phase nano-LC coupled with high resolution Orbitrap mass spectrometer was employed for peptide separation and identification. Database searching was used with the enzyme specificity set to "partially enzymatic - cleaves at either end" in order to detect the endogenous non-tryptic N or C-termini. Oxygen-18 labeling during digestion was utilized to make certain the C-termini that were identified were in fact endogenous and not aberrant trypsin digestion products (fig. 4).