Shedding of glycan‐modifying enzymes by signal peptide peptidase‐like 3 (SPPL3) regulates cellular N‐glycosylation

Abstract

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, b-1,3 N-acetylglucosaminyltransferase 1 and b-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

Proteolytic Processing of Neuregulin 1 Type III by Three Intramembrane-cleaving Proteases

“…Western blot and mRNA analysis confirms efficient knockdown of SPPL2a/b and SPPL3 (lower panel). Note that upon SPPL3 knockdown SPPL2a is hyperglycosylated (C) in line with previous findings (19). Quantification from three independent experiments: mean Ϯ S.D., *, p Ͻ 0.05, two-tailed unpaired Student's t test.…”

“…Note that upon SPPL3 knockdown, SPPL2a is hyperglycosylated (Fig. 8C), which is in line with previous findings (19). Efficient knockdown of SPPL2b was confirmed by mRNA analysis (Fig.…”

“…The construct was generated by PCR and cloned into the pcDNA3.1hygroϩ vector (Life Technologies, Inc.). For matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry (MS) analysis of the NRG1 C-peptides, the V5-NRG-NTF-FLAG construct was generated by inverting the tags of FNRG-NTF-V5 and adding an alanine-proline motif at the C terminus to prevent degradation by carboxypeptidases in cell culture supernatants (19). The BACE1 cDNA construct was described previously (31) as were the lentiviral expression construct NRG1 type III and the production of lentiviral particles (24).…”

“…Identification of the Intramembrane Cleavage Sites of the NRG1 Type III NTFЈ-To unambiguously demonstrate that NRG1 type III is subject to endoproteolysis within its N-terminal TMD, we determined the SPPL2a/SPPL2b cleavage site(s) in NRG1 type III by MALDI-TOF MS. We previously utilized truncated substrate constructs that include an N-terminal V5 and a C-terminal FLAG epitope tag to determine SPP/SPPL cleavage sites and have also included an alanine-proline motif at the very C terminus to prevent degradation by carboxypeptidases (19). We now generated a similar NRG1 type III (amino acids 2-114)-derived construct (V5-IIINRG-NTF-FLAG) and expressed this in HEK293 cells.…”

“…For SPPL3, a rather unexpected and exceptional function has been shown very recently. SPPL3 sheds glycosyltransferases and thereby regulates their intracellular activity (19).…”