Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Abstract

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken

The subcellular localization of the Niemann-Pick Type C proteins depends on the adaptor complex AP-3

“…These results support the hypothesis that, in addition to the AP-1/GGA sorting machinery (see Fig. 2) (Willenborg et al, 2005), the subcellular fate of mammalian NPC2 is also regulated by the AP-3 complex, probably by an indirect mechanism.…”

“…mentioned above, hNPC2 is modified by the addition of a mannose-6-phosphate group (Naureckiene et al, 2000), which is a signal for targeting soluble molecules to the lysosome when recognized by the mannose-6-phosphate receptor (Kornfeld and Mellman, 1989;Pohlmann et al, 1995). Consistent with this modification, the targeting of NPC2 is regulated by mannose-6-phosphate receptors (Willenborg et al, 2005).…”

“…These results suggest that scNpc2p is trafficked through prevacuolar compartments prior to reaching the vacuole, similar to what is observed for other proteins dependent upon AP-1 or the GGA proteins in yeast (Boehm and Bonifacino, 2002). Furthermore, these results highlight the conservation between the NPC2 orthologs, because both are targeted by AP-1/GGA-controlled pathways in both yeast (this work) and mammals (Willenborg et al, 2005).…”

“…We assessed the co-localization of the functional fusion protein hNPC2-GFP, expressed from an adenovirus construct (Berger et al, 2005b), with AP-3 in AP-3 +/+ cells or, as a control, AP-3 -/-cells. As an additional control, we colocalized hNPC2 with the adaptor complex AP-1, an adaptor that binds to the cytosolic domains of the mannose-6-phosphate receptors that preferentially deliver NPC2 from the Golgi complex to lysosomal organelles (Willenborg et al, 2005). Protein distribution was analyzed by indirect immunofluorescence confocal microscopy.…”

NPC2, the Protein Deficient in Niemann-Pick C2 Disease, Consists of Multiple Glycoforms That Bind a Variety of Sterols

“…We also found that Asn-39 is always linked to an Endo H-sensitive oligosaccharide, whereas Asn-116 is variably glycosylated both in terms of the presence and processing of the oligosaccharide. Given that NPC2 is targeted via the mannose 6-phosphate lysosomal targeting pathway (8,26,27), Endo H sensitivity of the NPC2 oligosaccharides is likely to reflect the presence of mannose 6-phosphate (23); this modification occurs temporally prior to and blocks the actions of Golgi ␣-mannosidase, which is required for conversion of N-linked glycans to Endo H-resistant forms. Thus, Asn-39 represents the primary substrate of lysosomal protein UDP-N-acetyglucosamine phosphotransferase.…”