Signal peptide peptidase– and SPP-like 3–dependent shedding of α1,6-fucosyltransferase differentially affects core fucosylation

Alpha1,6-fucosyltransferase (FUT8) biosynthesizes core fucose on N-glycans, which plays essential roles in various biological processes, including immunity and development. Although FUT8 is a Golgi-resident type II membrane protein, it is also secreted by an unknown mechanism. Here, we demonstrate that signal peptide peptidase (SPP) and signal peptide peptidase-like 3 (SPPL3), members of an intramembrane protease family, both cleave FUT8 for secretion. Knockout of SPP or SPPL3 in cells partially impaired FUT8 secretion, and double KO led to more drastic impairment in secretion, indicating that SPP and SPPL3 independently cleave FUT8. Sequencing analysis revealed that the N terminus of FUT8 in the media was mapped in the stem region, which is far from the expected cleavage site for SPP/SPPL3, suggesting that FUT8 undergoes two-step proteolytic processing, initially by SPP/SPPL3 and subsequently by another protease. Moreover, glycoproteomics suggested that the substrate glycoprotein preference of FUT8 was altered by knocking out SPP or SPPL3, highlighting the importance of FUT8 shedding in core fucosylation.