DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex, Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

Abstract

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPP␣ secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the ␥-secretase complex, resulting in a reduction of the PS1 active fragment blocking A␤ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active ␥-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of A␤ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the ␥-secretase and affect the APP processing pathway. Alzheimer disease (AD)2 is the primary cause of adult onset dementia, with a dramatic increase in the incidence of AD apparent in our aging population. AD is pathologically characterized by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-␤ (A␤) peptides, A␤40 and A␤42 (1). The early onset familial form of AD is linked to three genes, amyloid precursor protein (APP) and presenilin (PS1 and PS2) (2, 3), strongly suggesting that the production of A␤ is a key factor in the pathogenesis of AD. A␤ is generated by proteolysis of APP, driven by the secretases found in the cell. Prior to proteolysis, APP undergoes a number of post-translational modifications, including N-glycosylation in the endoplasmic reticulum (ER) and O-glycosylation in the Golgi apparatus. In order to generate A␤40 and A␤42, APP is first cleaved by ␤-secretase and then by ␥-secretase. For the cleavage of APP, ␤-secretase competes with ␣-secretase, which produces non-amyloidogenic peptides (4). ␥-Secretase is an aspartyl protease complex composed of four core components, including presenilins (PS1/PS2), presenilin enhancer 2 (PEN2), nicastrin, and anterior pharynx-defective 1 (APH1) (2). Presenilin is the catalytic core of the ␥-secretase complex consisting of nine transmembrane domains (5) and is cleaved by an unknown protease called "presenilinase" or self-cleavage stimulated by PEN2 binding of the cytosolic loop between transmembrane domains 6 and 7, releasing N-and C-terminal PS1 fragments, th...

The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation

“…Furthermore, the knockdown of KCP2 resulted in a mild yet specific perturbation of pSAP N-glycosylation, confirming the effect of KCP2 depletion we observed in vitro and showing that KCP2 is a bone fide functional component of the mammalian OST. Although a recent study concluded that KCP2 is dispensable for mammalian OST activity, only one authentic protein substrate was analysed in vitro (Wilson et al, 2011). We now provide the first evidence that KCP2 levels influence protein N-glycosylation in a substrate-specific manner, with the most striking effect observed upon the biogenesis of endogenous pSAP in HeLa cells.…”

“…Whilst mammalian DC2 shows weak homology to S. cerevisiae Ost3p and Ost6p (Shibatani et al, 2005), a search for conserved domains in KCP2 found no homology to known protein families. Hence, the potential function of KCP2 during protein N-glycosylation is unclear (Kelleher and Gilmore, 2006;Roboti and High, 2012), and lacking any experimental support (Wilson et al, 2011).…”

Sortilin mediates the release and transfer of exosomes in concert with two tyrosine kinase receptors

“…We used a number of compounds that are known to affect protein transport and stimulate secretion at different stages. Brefeldin A (BFA) is known to affect the classical protein transport pathway, preventing anterograde traffic from the endoplasmic reticulum (ER) to the Golgi and resulting in retrograde transport of proteins from the Golgi to the ER (Lippincott-Schwartz et al, 1989;Wilson et al, 2011). As shown in Fig.…”

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

“…Isoforms of the complex containing either STT3A or STT3B were described . The newly identified subunits KCP2 and DC2 seem to associate with mammalian OST complexes (Shibatani et al 2005;Wilson et al 2011;Roboti and High 2012a).…”

“…KCP2 associates mainly, or even exclusively, with STT3A complex isoforms, and knockdown experiments led to the conclusion that KCP2 may facilitate the glycosylation of selected substrate proteins (Roboti and High 2012a,b). DC2 might interact with the g-secretase and seems not to be directly involved in Nglycosylation (Wilson et al 2011). …”