EMBO J. doi:10.15252/embj.201488375

N-linked glycosylation and subsequent tailoring steps of the attached glycans occur in the endoplasmic reticulum and Golgi, mediated by a series of glycosyltransferases and glycosidases. Some of the glycosyltransferases involved are also secreted, and recent work implicated γ-secretase as the protease initiating secretion, but the full details of this process and the functional importance of secretion are unknown. Voss et al. now identify signal peptide peptidase-like 3 (SPPL3) as the key protease in this process, linking glycosyltransferase secretion to reduced enzyme function. SPPL3 was expected to function similarly to its better-characterized family members such as SPP, but identifying substrates has proven challenging. The authors first observed that inducible expression of SPPL3, but not an inactive mutant, in HEK293 cells reduced the molecular weight of representative glycoproteins; in contrast, SPPL3 knockdown resulted in increased weight. Treatment of the cells with glycosidases specific to O- or N-linked modifications defined SPPL3-induced changes as associated with N-linked glycosylation, whereas treatment with an inhibitor of α-mannosidase I—an enzyme involved in N-glycan tailoring—indicated that SPPL3's impact was downstream of this activity. Analysis of Sppl3-deficient mice recapitulated the cellular experiments, confirming the link between SPPL3 and N-glycosylation. A comparison of cell supernatants and lysates pointed to GnT-V as the primary substrate of SPPL3, with other N-glycan–modifying enzymes β3GnT1 and β4GalT1 serving as substrates to a lesser extent. Analytical characterization of glycans excised from proteins supported these results, demonstrating that the branched glycan chains normally generated by GnT-V were strongly diminished in cells overexpressing SPPL3. Finally, the authors confirmed that GnT-V secretion is dependent on proteolysis between Leu29 and Thr33 and in a γ-secretase–independent manner, as a secretase inhibitor did not alter GnT-V localization. Although the direct proteolysis of the glycosyltransferases by SPPL3 remains to be shown, these results set the stage for further investigations of this important system.