Signal peptide peptidase-like 3 (SPPL3) is a type II membrane protein-selective sheddase that regulates cellular N-glycosylation

Intramembrane proteolysis - hydrolysis of membrane proteins within
or close to their membrane-spanning regions - is a crucial cellular
process that is conserved throughout all kingdoms of life. It is
executed by distinct classes of polytopic membrane proteins, the
intramembrane-cleaving proteases, that provide a hydrophilic,
proteinaceous environment accommodating membrane protein substrates
as well as water molecules within the hydrophobic membrane interior
and catalyse peptide bond hydrolysis. In particular,
intramembrane-cleaving aspartyl proteases have received attention
as the presenilins, the catalytic subunits of the γ-secretase
complex, were identified as key players in Alzheimer's disease
pathophysiology. In addition to presenilins, mammalian genomes
harbour presenilin homologues which include signal peptide
peptidase (SPP) and SPP-like (SPPL) proteases. Among these, the
Golgi-resident protease SPPL3 stands out as it is highly conserved
among metazoa and SPPL3 orthologues are also found in plants.
However, due to the lack of known substrates, SPPL3 has thus far
hardly been characterised. Hence, the purpose of this study was to
identify its substrates and elucidate its physiological
function(s). In the first part of this study, the foamy virus
envelope glycoprotein (FVenv) was identified as the first substrate
of SPPL3. This allowed to study SPPL3's proteolytic activity in
detail, with a focus on its substrate selectivity and sensitivity
towards previously characterised inhibitors of
intramembrane-cleaving aspartyl proteases. Importantly, this study
revealed in addition that two other intramembrane-cleaving
proteases, SPPL2a and SPPL2b, also endoproteolyse FVenv. SPPL2b in
particular had been studied in detail before and therefore SPPL3-
and SPPL2b- mediated endoproteolysis of FVenv were examined in
parallel to directly compare these phylogenetically related
intramembrane-cleaving proteases. This uncovered an unexpected
idiosyncrasy of SPPL3 that clearly sets SPPL3 apart from other
intramembrane-cleaving aspartyl proteases: SPPL3 endoproteolysed
full-length FVenv and did not require the substrate's prior
tailoring by another proteolytic activity - an otherwise common
phenomenon among intramembrane-cleaving aspartyl proteases. In the
second part, the physiological function of SPPL3 was investigated.
Alterations in the cellular levels of proteolytically active SPPL3
turned out to impact the composition of N-glycans attached to
endogenous cellular glycoproteins. SPPL3 over-expression was
accompanied by a decrease in glycoprotein molecular weight, i.e. a
hypoglycosylation phenotype, while loss of SPPL3 expression in cell
culture models but also in vivo resulted in a hyperglycosylation
phenotype. This led to the identification of Golgi glycan-modifying
enzymes such as GnT-V and β3GnT1 as novel physiological substrates
of SPPL3. Loss or reduction of SPPL3 expression, for instance, led
to a marked intracellular accumulation of these enzymes, explaining
the more extensive N-glycan elaboration and the hyperglycosylation
phenotype observed under these conditions. At the same time
secretion of these enzymes was reduced under these conditions.
Together with additional observations such as the mapping of the
SPPL3 cleavage site to the membrane-spanning region of GnT-V, this
study demonstrates that SPPL3-mediated intramembrane proteolysis of
such glycan-modifying enzymes liberates their active
site-harbouring ectodomains. Acting in this manner, SPPL3 controls
the intracellular pool of active glycan-modifying enzymes.
Importantly, the finding that SPPL3 proteolytically cleaves
full-length glycan-modifying enzymes and sheds their ectodomains is
well in line with the observations made for FVenv and suggested
that SPPL3 acts functionally equivalent to classical sheddases or
rhomboid proteases but much unlike all other characterised
mammalian intramembrane-cleaving aspartyl proteases. To examine
whether these observations hold also true on a global cellular
scale, a proteomic approach was undertaken in the third part of the
study to define the SPPL3 degradome of HEK293 cells in conditions
of SPPL3 over-expression. On the one hand, this led to the
identification of numerous novel, mostly Golgi-resident candidate
SPPL3 substrates and, considering the physiological implications,
suggests that SPPL3 is very intricately linked to Golgi function.
On the other hand, this approach supports the initial hypothesis
that SPPL3 acts as a cellular type II membrane protein-selective
sheddase. Taken together, this study provides the first in-depth
characterisation of the intramembrane protease SPPL3 and reveals
the cellular function of SPPL3. SPPL3 displays considerable and
marked differences to other intramembrane-cleaving aspartyl
proteases and emerges as a fundamental cellular sheddase that
exhibits strong selectivity for type II-oriented, Golgi-resident
membrane proteins. Products of SPPL3-mediated endoproteolysis of
these Golgi factors are secreted and/or may be subject to
intracellular degradation which compromises their catalytic
activity. Thus, SPPL3 indirectly controls protein glycosylation in
the Golgi apparatus.