Complex N-Linked Glycosylation: A Potential Modifier of Niemann–Pick Disease, Type C1 Pathology

Abstract

Complex asparagine-linked glycosylation plays key roles in cellular functions, including cellular signaling, protein stability, and immune response. Previously, we characterized the appearance of a complex asparagine-linked glycosylated form of lysosome-associated membrane protein 1 (LAMP1) in the cerebellum of Npc1−/− mice. This LAMP1 form was found on activated microglia, and its appearance correlated both spatially and temporally with cerebellar Purkinje neuron loss. To test the importance of complex asparagine-linked glycosylation in NPC1 pathology, we generated NPC1 knock-out mice deficient in MGAT5, a key Golgi-resident glycosyl transferase involved in complex asparagine-linked glycosylation. Our results show that Mgat5−/−:Npc1−/− mice were smaller than Mgat5+/+:Npc1−/− mice, and exhibited earlier NPC1 disease onset and reduced lifespan. Western blot and lectin binding analyses of cerebellar extracts confirmed the reduction in complex asparagine-linked glycosylation, and the absence of the hyper-glycosylated LAMP1 previously observed. Western blot analysis of cerebellar extracts demonstrated reduced calbindin staining in Mgat5−/−:Npc1−/− mice compared to Mgat5+/+:Npc1−/− mutant mice, and immunofluorescent staining of cerebellar sections indicated decreased levels of Purkinje neurons and increased astrogliosis in Mgat5−/−:Npc1−/− mice. Our results suggest that reduced asparagine-linked glycosylation increases NPC1 disease severity in mice, and leads to the hypothesis that mutations in genes involved in asparagine-linked glycosylation may contribute to disease severity progression in individuals with NPC1. To examine this with respect to MGAT5, we analyzed 111 NPC1 patients for two MGAT5 SNPs associated with multiple sclerosis; however, we did not identify an association with NPC1 phenotypic severity.

Niemann-Pick Disease Type C (NPDC) by Mutation of NPC1 and NPC2: Aberrant Lysosomal Cholesterol Trafficking and Oxidative Stress

“…LAMP-1 is expressed in the lysosomal membrane to modulate lysosomal activity. Deficiencies in NPC1 not only increased LAMP-1 expression but also LAMP-1 glycosylation [55]. Glycosylation is an essential process for lysosomal membrane proteins for vesicle trafficking, and the abnormal glycosylation of LAMP-1 possesses the potential to induce NPDC [56].…”

“…Glycosylation is an essential process for lysosomal membrane proteins for vesicle trafficking, and the abnormal glycosylation of LAMP-1 possesses the potential to induce NPDC [56]. LAMP-1 hyper-glycosylation was observed in the cerebellum of NPC1 knock-out mice [55]. In addition, lysosomal membrane stabilization is associated with ion transportation through the lysosomal membranes.…”

Alterations in Proteostasis Mechanisms in Niemann–Pick Type C Disease

“…Such accumulation of sphingosine in NPC may be due to the observed reduction in sphingosine kinase 1 (SphK1), which is one of the enzymes that phosphorylates sphingosine [87]. In addition, alterations in lysosome-associated membrane protein 1 (LAMP1), a protein with highly glycosylated luminal domains that contributes to the integrity of the lysosomal membrane and glycocalyx, have also been found in NPC [88,89]. In the cerebellum of Npc1 −/− mice, differences in LAMP1 glycosylation have been observed that are associated with disease progression and the death of Purkinje neurons [89].…”

“…In addition, alterations in lysosome-associated membrane protein 1 (LAMP1), a protein with highly glycosylated luminal domains that contributes to the integrity of the lysosomal membrane and glycocalyx, have also been found in NPC [88,89]. In the cerebellum of Npc1 −/− mice, differences in LAMP1 glycosylation have been observed that are associated with disease progression and the death of Purkinje neurons [89]. Changes in LAMP1 glycosylation patterns have also been observed in studies with NPC1−/− cells, as they had high levels of mannose and sialylates, which can lead to changes in the lysosomal glycocalyx [35].…”