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.

Background: SPPL proteases are intramembrane-cleaving aspartyl proteases of the GxGD type. Results: Under certain circumstances, SPPL3 cleaves FVenv independent of prior shedding, generating substrates for subsequent intramembrane proteolysis. Conclusion: Unlike other known GxGD proteases, SPPL3 can act as a sheddase and an intramembrane protease within the regulated intramembrane proteolysis cascade. Significance: This initial biochemical characterization of SPPL3 will help to address its physiological role in later studies.

Background: Bri2 is a substrate for intramembrane proteolysis by SPPL2b. Results: Reducing the ␣-helical content of the Bri2 transmembrane domain increases its intramembrane cleavage by SPPL2b. Destabilization of the Bri2 transmembrane domain is predominantly mediated by Gly-60. Conclusion: A single helix-destabilizing glycine residue of the transmembrane domain of Bri2 is sufficient for efficient intramembrane proteolysis by SPPL2b. Significance: Understanding substrate requirements of intramembrane-cleaving proteases.

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the ␥-secretase complex, cause aberrant amyloid ␤-peptide (A␤) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. ␥-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor ␣ and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of ␤-amyloid precursor protein (␤APP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by ␥-secretase containing PS1 with the G384A mutation. As compared with wildtype PS1, the mutation selectively slowed A␤40 production, whereas A␤42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign A␤40.

Incubation of the furoquinoline alkaloid dictamnine with microsomal fractions from phenobarbital-induced rat liver resulted in a metabolite mixture from which demethyldictamnine and dictamnic acid were identified by a GC-MS technique. The identity of the two compounds was confirmed by synthesis. Other metabolites were characterized by their mass spectra only. The pattern of the metabolites suggested a possible pathway of metabolism in vitro. We assume that the metabolism of dictamnine is analogous to the metabolism of the related 8-methoxypsoralen and takes place via an unstable epoxide and subsequent oxidative opening of the furan ring. Direct evidence for the formation of an epoxide was, however, not obtained. The identified compounds as well as some putative metabolites were shown to be non-mutagenic in Salmonella typhimurium TA98 except for 8-hydroxydictamnine, which, however, was not detected in the metabolite mixture.