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

Laura Hobohm,Fenja H Bahr,Jule Truberg,Andreas Tholey,Daniel Meisinger,Marion Mengel,Tomas Koudelka,Matthias Voss,Lukas Heintz,Sebastian Kapell,Anna Hofmann,Sarah-Sophie Schacht,Alexander Jochimsen

doi:10.1007/s00018-022-04163-y

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.

Highlights

The Golgi apparatus is an integral part of the eukaryotic secretory pathway
Given that GTf catalysis is strictly dependent on nucleoside-conjugated monosaccharide building blocks, which are scarcely available in the extracellular space, the prevailing hypothesis is that Golgi enzymes do not actively contribute to glycan synthesis or remodelling once secreted [7] making it conceivable that GTf secretion could rather serve the purpose of Golgi proteostasis
We found that the majority of type II membrane protein-derived peptides more abundant in CM of parental cells did originate within annotated transmembrane domain (TMD), suggesting they are likely liberated by intramembrane proteolysis (Fig. 2f, g)

Summary

Introduction

The Golgi apparatus is an integral part of the eukaryotic secretory pathway. Golgi cargo arrives from the endoplasmic reticulum (ER), is trafficked through the Golgi and eventually sorted into vesicles destined for the cell surface, the extracellular space or the endolysosomal compartment [2]. While the intra-Golgi function of GMEs in glycan synthesis is evident, the (patho-)physiological relevance of Golgi GME secretion is incompletely understood [4, 7]. Given that GTf catalysis is strictly dependent on nucleoside-conjugated monosaccharide building blocks, which are scarcely available in the extracellular space, the prevailing hypothesis is that Golgi enzymes do not actively contribute to glycan synthesis or remodelling once secreted [7] making it conceivable that GTf secretion could rather serve the purpose of Golgi proteostasis. More recent observations corroborate enzyme-catalysed extracellular glycosylation in certain physiological settings and challenge the hypothesis that secreted Golgi GTfs do generally not display catalytic activity in the extracellular space. Platelets, for example, can release soluble GTfs along with sugar nucleotides to fuel glycosylation [8–10] and a growing body of evidence supports a physiological role of extracellular sialylation in the hematopoietic system [11–13]

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Results

Conclusion