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N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells*

  • Marko Kosicek
    Footnotes
    Affiliations
    From the Laboratory for Neurodegenerative Disease Research, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia;
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  • Ivan Gudelj
    Footnotes
    Affiliations
    Genos Glycoscience Research Laboratory, Zagreb, Croatia;
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  • Anita Horvatic
    Affiliations
    ERA Chair team, Internal Diseases Clinic, University of Zagreb, Faculty of Veterinary Medicine, Heinzelova 55, 10000 Zagreb, Croatia;
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  • Tanja Jovic
    Affiliations
    From the Laboratory for Neurodegenerative Disease Research, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia;

    University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
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  • Frano Vuckovic
    Affiliations
    Genos Glycoscience Research Laboratory, Zagreb, Croatia;
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  • Gordan Lauc
    Affiliations
    Genos Glycoscience Research Laboratory, Zagreb, Croatia;

    University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
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  • Silva Hecimovic
    Correspondence
    To whom correspondence should be addressed: Laboratory for Neurodegenerative Disease Research, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia. Tel.:+385-1-4571-284;.
    Affiliations
    From the Laboratory for Neurodegenerative Disease Research, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia;
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  • Author Footnotes
    * This work was funded by the Unity Through Knowledge Fund (S.H.), by the European Commission FP7 grants MIMOmics (contract #305280, G.L.), HTP-GlycoMet (contract #324400, G.L.), VetMedZg (contract #621394, A.H.), H2020 grants GlySign (contract #722095, G.L.), SYSCID (contract #733100, G.L.) and IMforFuture (contract #721815, G.L.) as well as by funding for the Croatian National Centre of Research Excellence in Personalized Healthcare and IRI project Nova generacija visokoprotočnih glikoservisa.
    This article contains supplemental material.
    ‡‡ These authors contributed equally to this work.
Open AccessPublished:January 24, 2018DOI:https://doi.org/10.1074/mcp.RA117.000129
      Increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4–5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration.
      Since their first description scientists have tried to fully characterize lysosomal composition and function. Today, many facts are known about lysosomal physiology. The acidic pH, ionic gradients and the membrane potential make lysosomes an ideal environment for activity of luminal lysosomal hydrolases and a cellular center for nutrient sensing and recycling (
      • Xu H.
      • Ren D.
      Lysosomal Physiology.
      ). Lysosome's primary role is to digest a cargo from endocytic, phagocytic or autophagocytic pathways. More than 50 lysosomal hydrolases have been characterized and dysfunction in their activity/levels leads to accumulation of the lysosomal cargo which causes lysosomal storage disorders (LSDs)
      The abbreviations used are: LSD, Lysosomal storage disorder; AD, Alzheimer's disease; APP, β-amyloid precursor protein; BACE1, β-secretase; CHO, Chinese hamster ovary; HCD, Higher energy collisional dissociation; HILIC, Hydrophilic interaction liquid chromatography; EEA1, Early Endosome Antigen 1 - early endosomal marker; ESI, Electrospray ionization; FASP, Filter aided sample preparation; GO, Gene ontology; LAMP1, Lysosomal Associated Membrane Protein 1 - lysosomal marker; MALDI, Matrix-assisted laser desorption/ionization; NAG, N-Acetyl-β-D-glucosaminidase; NPC, Niemann-Pick Type C; PD, Parkinson's disease; Rab7, RAS-related GTP-binding protein 7- late endosomal marker; SPE, Solid-phase extraction; TfR, Transferrin receptor – a marker of recycling endosomes; TOF, Time-of-flight; UPLC, Ultra-performance liquid chromatography.
      1The abbreviations used are: LSD, Lysosomal storage disorder; AD, Alzheimer's disease; APP, β-amyloid precursor protein; BACE1, β-secretase; CHO, Chinese hamster ovary; HCD, Higher energy collisional dissociation; HILIC, Hydrophilic interaction liquid chromatography; EEA1, Early Endosome Antigen 1 - early endosomal marker; ESI, Electrospray ionization; FASP, Filter aided sample preparation; GO, Gene ontology; LAMP1, Lysosomal Associated Membrane Protein 1 - lysosomal marker; MALDI, Matrix-assisted laser desorption/ionization; NAG, N-Acetyl-β-D-glucosaminidase; NPC, Niemann-Pick Type C; PD, Parkinson's disease; Rab7, RAS-related GTP-binding protein 7- late endosomal marker; SPE, Solid-phase extraction; TfR, Transferrin receptor – a marker of recycling endosomes; TOF, Time-of-flight; UPLC, Ultra-performance liquid chromatography.
      (
      • Futerman A.H.
      • van Meer G.
      The cell biology of lysosomal storage disorders.
      ). Besides luminal hydrolases, integral lysosomal membrane proteins are also important for proper function of lysosomes. Mutations in genes encoding these proteins lead to defects in the transport of lysosomal cargo and/or ions across the lysosomal membrane also causing the LSDs (
      • Saftig P.
      • Klumperman J.
      Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function.
      ). Individuals with lysosomal storage disorders often develop symptoms early in life and in majority of LSDs the brain, especially neurons, are affected (
      • Schultz M.L.
      • Tecedor L.
      • Chang M.
      • Davidson B.L.
      Clarifying lysosomal storage diseases.
      ).
      Niemann-Pick Type C (NPC) disease is a rare, autosomal recessive, progressive and fatal disorder characterized by abnormal cholesterol trafficking and intracellular accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. NPC is caused by loss of function of either NPC1, a multi-transmembrane lysosomal protein, or NPC2, a small luminal lysosomal protein that mediate intracellular cholesterol transport (
      • Pacheco C.D.
      • Lieberman A.P.
      The pathogenesis of Niemann-Pick type C disease: a role for autophagy?.
      ). Recently, NPC disease has shown to share several pathological features with the most common and complex Alzheimer's disease (AD) (
      • Malnar M.
      • Hecimovic S.
      • Mattsson N.
      • Zetterberg H.
      Bidirectional links between Alzheimer's disease and Niemann-Pick type C disease.
      ). Our previous work has suggested that dysfunction of the late endosomal/lysosomal compartments in NPC1-cellular model is, most likely, responsible for the AD-like features in NPC (
      • Malnar M.
      • Kosicek M.
      • Lisica A.
      • Posavec M.
      • Krolo A.
      • Njavro J.
      • Omerbasic D.
      • Tahirovic S.
      • Hecimovic S.
      Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells.
      ,
      • Malnar M.
      • Kosicek M.
      • Mitterreiter S.
      • Omerbasic D.
      • Lichtenthaler S.F.
      • Goate A.
      • Hecimovic S.
      Niemann-Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the β-secretase pathway.
      ), and that increased levels of free cholesterol in NPC play an important role in compartmentalization of the key AD-proteins (β-amyloid precursor protein - APP and β-secretase - BACE1) within lipid rafts (
      • Kosicek M.
      • Malnar M.
      • Goate A.
      • Hecimovic S.
      Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts.
      ) or in modulation of membrane stiffness lading to sequestration of both APP and BACE1 within the endolysosomal pathway (
      • von Einem B.
      • Weber P.
      • Wagner M.
      • Malnar M.
      • Kosicek M.
      • Hecimovic S.
      • von Arnim C.A.F.
      • Schneckenburger H.
      Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-pick type C disease cells.
      ). Besides cholesterol, other lipids, especially phospholipids and sphingolipids are also involved in these processes (
      • Kosicek M.
      • Hecimovic S.
      Phospholipids and Alzheimer's disease: Alterations, mechanisms and potential biomarkers.
      ).
      Glycosylation is one of the most common co-translational and post-translational modification which regulates the structure, stability, localization and function of various proteins, and N-glycosylation has been the most studied type of it (

      Stanley, P., Schachter, H., and Taniguchi, N., (2009) Chapter 8. N-Glycans, Essentials of Glycobiology, 2nd Edition,

      ). N-glycans are known to be versatile and responsive to environmental stimuli and undergo significant changes in numerous diseases including those of central nervous system (
      • Vučković F.
      • Krištić J.
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      • Teruel M.
      • Keser T.
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      • Barrios C.
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      • Menni C.
      • Wang Y.
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      • Song H.
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      • Spector T.D.
      • Harjaček M.
      • Alarcon-Riquelme M.
      • Molokhia M.
      • Wang W.
      • Lauc G.
      Association of systemic lupus erythematosus with decreased immunosuppressive potential of the IgG glycome.
      ,
      • Gudelj I.
      • Baciarello M.
      • Ugrina I.
      • De Gregori M.
      • Napolioni V.
      • Ingelmo P.M.
      • Bugada D.
      • De Gregori S.
      • Ðerek L.
      • Pučić-Baković M.
      • Novokmet M.
      • Gornik O.
      • Saccani Jotti G.
      • Meschi T.
      • Lauc G.
      • Allegri M.
      Changes in total plasma and serum N-glycome composition and patient-controlled analgesia after major abdominal surgery.
      ,
      • Barrios C.
      • Zierer J.
      • Gudelj I.
      • Štambuk J.
      • Ugrina I.
      • Rodríguez E.
      • Soler M.J.
      • Pavić T.
      • Šimurina M.
      • Keser T.
      • Pučić-Baković M.
      • Mangino M.
      • Pascual J.
      • Spector T.D.
      • Lauc G.
      • Menni C.
      Glycosylation Profile of IgG in Moderate Kidney Dysfunction.
      ,
      • Freidin M.B.
      • Keser T.
      • Gudelj I.
      • Štambuk J.
      • Vučenović D.
      • Allegri M.
      • Pavić T.
      • Šimurina M.
      • Fabiane S.M.
      • Lauc G.
      • Williams F.M.K.
      The Association Between Low Back Pain and Composition of IgG Glycome.
      ,
      • Bieberich E.
      Synthesis, Processing, and Function of N-glycans in N-glycoproteins.
      ). Moreover, it has been recently shown that modulation of glycosylation of APP, a key protein in the pathogenesis of AD, may represent a potential target for AD therapy (
      • Jacobsen K.T.
      • Iverfeldt K.
      O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-β precursor protein (APP).
      ). It has been previously shown that glycoproteins accumulate in NPC model (
      • Mbua N.E.
      • Flanagan-Steet H.
      • Johnson S.
      • Wolfert M.A.
      • Boons G.-J.
      • Steet R.
      Abnormal accumulation and recycling of glycoproteins visualized in Niemann-Pick type C cells using the chemical reporter strategy.
      ), and that blocking the O-linked glycosylation lowers cholesterol levels and increases the number of lysosomes (
      • Li J.
      • Deffieu M.S.
      • Lee P.L.
      • Saha P.
      • Pfeffer S.R.
      Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells.
      ), thus rescuing the NPC cellular defects. In the present work, we studied N-glycosylation profile of the lysosomal membrane proteins in NPC1-null cells versus wt-cells. We tested the hypothesis that alteration of the lysosomal glycocalyx is an additional feature of the lysosomal dysfunction in NPC disease, as well as in other LSDs.
      To the best of our knowledge, here we describe the first complete N-glycome of the lysosomal glycocalyx in NPC disease cellular model, which potentially could be useful for restoring lysosomal storage defects in NPC disease and other LSDs as well as for rescuing pathological processes occurring in AD.

      DISCUSSION

      In this work we describe changes in the lysosomal glycocalyx in a cellular model of a lysosomal storage disease Niemann-Pick type C (NPC). Lysosomal impairment is considered to play an important role in the pathogenesis of neurodegenerative diseases including the rare inherited lysosomal storage disorders, like Niemann-Pick type C disease (NPC), as well as the most common and complex Alzheimer's and Parkinson's disease (AD and PD). Here, we have characterized the N-glycan profile of the lysosomal glycocalyx of NPC1-null versus CHOwt cells. Notably, in our previous studies we have substantially used this NPC disease cellular model to elucidate the molecular and cellular details of an AD-like phenotype in NPC (
      • Malnar M.
      • Kosicek M.
      • Lisica A.
      • Posavec M.
      • Krolo A.
      • Njavro J.
      • Omerbasic D.
      • Tahirovic S.
      • Hecimovic S.
      Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells.
      ,
      • Malnar M.
      • Kosicek M.
      • Mitterreiter S.
      • Omerbasic D.
      • Lichtenthaler S.F.
      • Goate A.
      • Hecimovic S.
      Niemann-Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the β-secretase pathway.
      ,
      • Kosicek M.
      • Malnar M.
      • Goate A.
      • Hecimovic S.
      Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts.
      ,
      • Kosicek M.
      • Wunderlich P.
      • Walter J.
      • Hecimovic S.
      GGA1 overexpression attenuates amyloidogenic processing of the amyloid precursor protein in Niemann-Pick type C cells.
      ). Based on our results we speculate that changes in the N-glycome of the lysosomal membrane proteins may contribute to lysosomal (dys)function in NPC disease.
      Lysosomes were isolated using the magnetic ferrofluid nanoparticles instead of the traditionally used ultracentrifugation fractionation procedures because the isolation using the magnetic beads reveals more pure lysosomal fractions. Indeed, our Western blot analysis of endosomal and lysosomal marker proteins in the magnetically isolated lysosomal fractions confirmed that they were mainly LAMP1-positive, indicating that we have successfully isolated lysosomal organelles. However, we noticed that the yield of the isolated lysosomes was substantially lower in NPC1-null versus CHOwt cells. This contrasts with the recently reported lysosomal isolation from HeLa NPC1-KO cells using similar approach (
      • Tharkeshwar A.K.
      • Trekker J.
      • Vermeire W.
      • Pauwels J.
      • Sannerud R.
      • Priestman D.A.
      • te Vruchte D.
      • Vints K.
      • Baatsen P.
      • Decuypere J.-P.
      • Lu H.
      • Martin S.
      • Vangheluwe P.
      • Swinnen J.V.
      • Lagae L.
      • Impens F.
      • Platt F.M.
      • Gevaert K.
      • Annaert W.
      A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.
      ). In contrast to our dextran-coated ferrofluid nanoparticles, Tharkeshwar et al. used much smaller size coated ferrofluid nanoparticles which may be the reason of the different cellular behavior of the NPC1-lacking lysosomes. Our further analysis of the dextran uptake and its lysosomal accumulation between CHOwt and NPC1-null cells revealed that leakage of lysosomes may likely explain decreased lysosomal isolation efficiency in NPC1-null versus wt-cells by the “dextran-coated ferrofluid nanoparticles” method. Indeed, lysosomal leakage has been proposed to occur in the most common neurodegenerative disorders, such as AD (
      • Cataldo A.M.
      • Barnett J.L.
      • Berman S.a.
      • Li J.
      • Quarless S.
      • Bursztajn S.
      • Lippa C.
      • Nixon R.a.
      Gene Expression and Cellular Content of Cathepsin D in Alzheimer's Disease Brain: Evidence for Early Up-Regulation of the EndosomaI-Lysosomal System.
      ), as well as the rare LSDs, such as NPC (
      • Amritraj A.
      • Peake K.
      • Kodam A.
      • Salio C.
      • Merighi A.
      • Vance J.E.
      • Kar S.
      Increased Activity and Altered Subcellular Distribution of Lysosomal Enzymes Determine Neuronal Vulnerability in Niemann-Pick Type C1-Deficient Mice.
      ). This defect was fully recovered upon NPC1-back expression in NPC1-null cells, suggesting that the observed feature of decreased lysosomal isolation in the NPC1-null cells is dependent on NPC1-function. Moreover, particles in the isolated lysosomal fractions from NPC1-null cells presented with substantially increased radius (4–5 fold) and contained 3-fold more cholesterol than the lysosomal fraction of CHOwt cells. Altogether, these results (enlarged lysosomal organelles together with cholesterol accumulation) reveal that the magnetically isolated lysosomal fractions contain the key features of lysosomal impairment in NPC disease, indicating that these lysosomes could be further used for the organelle profiling.
      Next, we sought to analyze the N-glycome of the lysosomal membrane proteins from the isolated lysosomal fractions in order to elucidate if the N-glycosylation pattern of the lysosomal glycocalyx is altered in NPC1-null versus CHOwt cells and, thus, may contribute to the lysosomal dysfunction. Indeed, our findings support this as we observed both changes in the high-mannose and sialylated N-glycome patterns of the lysosomal membrane proteins between the two cell lines. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx. All of the detected structures have the characteristics of N-glycosylation in CHO cell line; myriad of LacNAc structures, core fucosylation, α2,3 linked sialic acid and higher abundance of N-acetylneuraminic than of N- glycolylneuraminic acid (
      • Xu X.
      • Nagarajan H.
      • Lewis N.E.
      • Pan S.
      • Cai Z.
      • Liu X.
      • Chen W.
      • Xie M.
      • Wang W.
      • Hammond S.
      • Andersen M.R.
      • Neff N.
      • Passarelli B.
      • Koh W.
      • Fan H.C.
      • Wang J.
      • Gui Y.
      • Lee K.H.
      • Betenbaugh M.J.
      • Quake S.R.
      • Famili I.
      • Palsson B.O.
      • Wang J.
      The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line.
      ,
      • Lin N.
      • Mascarenhas J.
      • Sealover N.R.
      • George H.J.
      • Brooks J.
      • Kayser K.J.
      • Gau B.
      • Yasa I.
      • Azadi P.
      • Archer-Hartmann S.
      Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.
      ,
      • North S.J.
      • Huang H.H.
      • Sundaram S.
      • Jang-Lee J.
      • Etienne A.T.
      • Trollope A.
      • Chalabi S.
      • Dell A.
      • Stanley P.
      • Haslam S.M.
      Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.
      ). Compared with CHO cellular glycocalyx where the main N-glycan structures were a series of core-fucosylated asialoglycans (
      • North S.J.
      • Huang H.H.
      • Sundaram S.
      • Jang-Lee J.
      • Etienne A.T.
      • Trollope A.
      • Chalabi S.
      • Dell A.
      • Stanley P.
      • Haslam S.M.
      Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.
      ), we detected that the main structures of the lysosomal N-glycome belong to the high-mannose glycans with H9N2 as the most abundant structure, regardless of the cell line, whereas core-fucosylated sialylated N-glycans were the second most abundant group of the glycans. Although the function of the lysosomal glycocalyx remains to be elucidated, it is known that sialic acids can protect molecules from attack by glycosidases and proteases (
      • Umeda T.
      • Tomiyama T.
      • Sakama N.
      • Tanaka S.
      • Lambert M.P.
      • Klein W.L.
      • Mori H.
      Intraneuronal amyloid β oligomers cause cell death via endoplasmic reticulum stress, endosomal/lysosomal leakage, and mitochondrial dysfunction in vivo.
      ) which may indicate that higher level of sialylation plays a role in protecting the limiting lysosomal membrane from the action of degradative lysosomal enzymes in the lumen. Yet, this is the first study where the specific changes of N-glycosylation of the lysosomal glycocalyx were observed between the NPC1-null (NPC disease cellular model) and CHOwt cell lines. More precisely, we identified higher abundance of more complex sialylated and smaller high-mannose N-glycans in the lysosomes derived from NPC1-null versus wt-cells. Higher glycosylation of the lysosomal proteins in NPC disease was previously observed by two independent studies (
      • Chen F.W.
      • Gordon R.E.
      • Ioannou Y.a.
      NPC1 late endosomes contain elevated levels of non-esterified (‘free‘) fatty acids and an abnormally glycosylated form of the NPC2 protein.
      ,
      • Dixit S.S.
      • Jadot M.
      • Sohar I.
      • Sleat D.E.
      • Stock A.M.
      • Lobel P.
      Loss of niemann-pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.
      ). Chen et al. suggested that only NPC2 protein is more glycosylated whereas others remain unchanged. However, Dixit et al. proved that higher level of glycosylation is not limited to NPC2 but rather widely present in the lysosomal proteome. Indeed, in this study we detected that more complex and sialylated N-glycans are present in the lysosomal glycocalyx of the NPC1-null versus wt-cells whereas the same cannot be said for the whole-cell-lysate N-glycome. Even though, the changes were solely related to the lysosomal N-glycome, the changes of the high-mannose N-glycans were equally distributed in N-glycans of the NPC1-null cells regardless of their origin, i.e. cellular versus lysosomal. It was noticed in previous studies that expression of α-mannosidase is higher in NPC disease which can explain higher abundance of smaller high-mannose glycans that we observed in NPC1-null cells. This is further supported by findings which showed that lysosomal α-mannosidase is capable of mannose digestion of the native proteins (
      • Dixit S.S.
      • Jadot M.
      • Sohar I.
      • Sleat D.E.
      • Stock A.M.
      • Lobel P.
      Loss of niemann-pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.
      ,
      • Damme M.
      • Morelle W.
      • Schmidt B.
      • Andersson C.
      • Fogh J.
      • Michalski J.-C.
      • Lubke T.
      Impaired Lysosomal Trimming of N-Linked Oligosaccharides Leads to Hyperglycosylation of Native Lysosomal Proteins in Mice with -Mannosidosis.
      ). Moreover, higher abundance of H5N2 can be explain by lower expression of dolichol-phosphate-mannose (DPM) biosynthesis regulatory protein in NPC disease (
      • Vázquez M.C.
      • del Pozo T.
      • Robledo F.A.
      • Carrasco G.
      • Pavez L.
      • Olivares F.
      • González M.
      • Zanlungo S.
      Alteration of gene expression profile in niemann-pick type C mice correlates with tissue damage and oxidative stress.
      ) as this protein is needed to stabilize expression and localization of DPM1, a catalytic subunit of DPM synthase (
      • Maeda Y.
      • Tomita S.
      • Watanabe R.
      • Ohishi K.
      • Kinoshita T.
      DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: Correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate.
      ). Indeed, an accumulation of H5N2 was observed in the cells with a mutation in DPM synthase (
      • Stoll J.
      • Robbins A.R.
      • Krag S.S.
      Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol.
      ). Interestingly, it has been recently reported that decreasing the lysosomal glycocalyx density causes cells to become less dependent on NPC1 protein function to transfer cholesterol across the lysosomal membrane. Namely, inhibition of O-glycosylation reduced accumulation of cholesterol in NPC1-null cells which is the primary feature of NPC disease (
      • Li J.
      • Deffieu M.S.
      • Lee P.L.
      • Saha P.
      • Pfeffer S.R.
      Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells.
      ). Because in this study we observed higher level of glycosylation in the NPC1-null versus wt-cells this may indicate that glycosylation could be actively involved in cholesterol accumulation and NPC disease progression.
      Thus, modulation of the lysosomal glycocalyx may not only cause impairment of the lysosomal function but may also be used as a tool to bypass certain lysosomal defects such as cholesterol accumulation in NPC disease. Further glycome profiling of the lysosomal glycocalyx in other lysosomal storage disorders as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the function of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Vizcaíno J.A.
      • Csordas A.
      • Del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ) partner repository with the dataset identifier PXD008438.

      Acknowledgments

      We would like to thank Lucija Horvat for the assistance with confocal microscopy and Dr. Maja Dutour Sikiric for assistance with Zetasizer instrument.

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