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

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.

Syndecan-1 shedding by meprin ß impairs keratinocyte adhesion and differentiation in hyperkeratosis.

Dysregulation of proteolytic enzymes has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as fibrosis or Netherton syndrome. The metalloprotease meprin ß was found to be upregulated in hyperproliferative skin diseases. AP-1 transcription factor complex has been reported to induce Mep1b expression. Since AP-1 and its subunit fos-related antigen 2 (fra-2) are associated with the onset and progression of psoriasis, we wanted to investigate if this could partially be attributed to increased meprin ß activity. Here, we demonstrate that fra-2 transgenic mice show increased meprin ß expression and proteolytic activity in the epidermis. To avoid influence by other fra-2 regulated genes, we additionally generated a mouse model that enabled tamoxifen-inducible expression of meprin ß under the Krt5-promotor to mimic the pathological condition. Interestingly, induced meprin ß expression in the epidermis resulted in hyperkeratosis, hair loss and mottled pigmentation of the skin. Employing N-terminomics revealed syndecan-1 as a substrate of meprin ß in skin. Shedding of syndecan-1 at the cell surface caused delayed calcium-induced differentiation and impaired adhesion of keratinocytes, which was blocked by the meprin ß inhibitor fetuin-B.