N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion

Abstract

Golgi membrane proteins such as glycosyltransferases and other glycan-modifying enzymes are key to glycosylation of proteins and lipids. Secretion of soluble Golgi enzymes that are released from their membrane anchor by endoprotease activity is a wide-spread yet largely unexplored phenomenon. The intramembrane protease SPPL3 can specifically cleave select Golgi enzymes, enabling their secretion and concomitantly altering global cellular glycosylation, yet the entire range of Golgi enzymes cleaved by SPPL3 under physiological conditions remains to be defined. Here, we established isogenic SPPL3-deficient HEK293 and HeLa cell lines and applied N-terminomics to identify substrates cleaved by SPPL3 and released into cell culture supernatants. With high confidence, our study identifies more than 20 substrates of SPPL3, including entirely novel substrates. Notably, our N-terminome analyses provide a comprehensive list of SPPL3 cleavage sites demonstrating that SPPL3-mediated shedding of Golgi enzymes occurs through intramembrane proteolysis. Through the use of chimeric glycosyltransferase constructs we show that transmembrane domains can determine cleavage by SPPL3. Using our cleavage site data, we surveyed public proteome data and found that SPPL3 cleavage products are present in human blood. We also generated HEK293 knock-in cells expressing the active site mutant D271A from the endogenous SPPL3 locus. Immunoblot analyses revealed that secretion of select novel substrates such as the key mucin-type O-glycosylation enzyme GALNT2 is dependent on endogenous SPPL3 protease activity. In sum, our study expands the spectrum of known physiological substrates of SPPL3 corroborating its significant role in Golgi enzyme turnover and secretion as well as in the regulation of global glycosylation pathways.

C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly

“…Although this does not fully exclude binding of Frey to potential exosites on the enzyme required for substrate recruitment, these data are suggestive of a model in which Frey specifically interacts with the catalytic center of SPPL2c with high affinity, thereby blocking access of potential substrate molecules. How this high affinity can be obtained and whether an inhibitory motif within the Frey TMD might contribute to this will be a central question of further research, especially since analysis of cleavage sites of aspartic intramembrane proteases has not revealed any obvious consensus cleavage sites yet (21,23,30). Stabilization of Frey by SPPL2c represents the first described nonproteolytic function of an SPPL2c protease.…”

Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b

“…S7 A ). We also tested whether the GnT-IVb smaller band is detected in the medium, because most glycosyltransferases are cleaved near the transmembrane border and secreted ( 33 ). As a result, the smaller band was not detected in the medium ( Fig.…”

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling

“…In line with this, a proline mutation in a proposed hinge region in the TM domain of GNT‐V, a substrate of SPPL3, resulted in increased initial cleavage of the substrate [55]. Computational analysis of 23 SPPL3 substrates indicates a modest enrichment of glycine residues in the middle of the cleavable TM domain [118]. A recent study including deuterium exchange experiments on the APP substrate's TM domain analyzed the effect of mutations on the N‐and C‐terminal part of the substrate's TM domain as well as of a hinge region between them.…”

“…Sequence analysis of cleavage regions so far has not led to the identification of consensus recognition sequences within the substrate's TM domains [2,55,117]. For SPPL3, it was suggested that an M or Y in position P1 might be favorable [33]; however, a recent N-terminomics study on the enzyme did not detect a consensus sequence [118]. Yet, the authors found that when substrate and SPPL3 are located in the same compartment, the exchange of the TM domain can turn a nonsubstrate to a substrate [119].…”