Impact of the NiemannâPick c1 Gene Mutation on the Total Cellular

Subscriber access provided by University of Rochester | River Campus & Miner Libraries

Article

Impact of the Niemann–Pick c1 gene mutation on the total cellular glycomics of CHO cells Jun-ichi Furukawa, Minami Soga, Kazue Okada, Ikuko Yokota, Jinhua Piao, Tetsumi Irie, Takumi Era, and Yasuro Shinohara J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00070 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 21, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Impact of the N iemann– iemann – P ick c1 gene mutation on the total cellular glycomics of CHO cells

Jun-ichi Furukawa † ¶ , Minami Soga ‡ , Kazue Okada † ¶ , Ikuko Yokota † ¶ , Jinhua Piao † , Tetsumi Irie § * , Takumi Era ‡ * , and Yasuro Shinohara † # * † Graduate

School of Advanced Life Science, Hokkaido University, Sapporo 001-0021,

Japan ‡ Department

of Cell Modulation, Institute of Molecular Embryology and Genetics,

Kumamoto University, Kumamoto, 860-0811, Japan § Department

of Clinical Chemistry and Informatics, Graduate School of Pharmaceutical

Sciences, Kumamoto University, Kumamoto, 862–0973, Japan ¶ Department

of Orthopaedic Orthopaedic Surgery, Graduate School of Medicine,

Hokkaido University, Sapporo, Japan. # Department

of Pharmacy, Kinjo Gakuin University, Nagoya 463-8521, Japan

*Corresponding authors: YS; Phone: +81 52 798 0180; Fax: +81 52 798 0754; e-mail: [email protected]. TE; Phone: +81 96 373 6589; Fax: +81 96 373 6590; e-mail: tera@kumamoto-u. ac.jp TI; Phone: +81 96 371 4552; Fax: +81 96 371 4552; e-mail: [email protected]

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Niemann–Pick disease type C (NPC) is an autosomal recessive lipid storage disorder, and the majority of cases are caused by mutations in the NPC1 gene. In this study, we clarified how a single gene mutation in the NPC1 gene impacts the cellular glycome by analyzing the total glycomic expression profile of Chinese hamster ovary cell mutants defective in the Npc1 gene ( Npc1 KO CHO cells). A number of glycomic alterations were identified, including increased expression of lactosylceramide, GM1, GM2, GD1, various neolacto-series glycosphingolipids, and sialyl-T (O-glycan), which was found to be the major sialylated protein-bound glycan, as well as various N-glycans, which were commonly both fucosylated and sialylated. We also observed significant increases in the total amounts of free oligosaccharides (fOSs), especially in the unique complex- and hybrid-types fOSs. Treatment of Npc1 KO CHO cells with 2-hydroxypropyl-β-cyclodextrin (HPBCD), which can reduce cholesterol and glycosphingolipid (GSL) storage, did not affect the glycomic alterations observed in the GSL-, N- and O-glycans of Npc1 KO CHO cells. However, HPBCD treatment corrected the glycomic alterations observed in fOSs to levels observed in wild-type cells. Keywords: Niemann-Pick disease; glycomics; N-glycan; O-glycan; glycosaminoglycan; glycosphingolipids; free oligosaccharide; cyclodextrin

2


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research Introduction Niemann–Pick disease type C (NPC) is an inborn error of metabolism caused by mutations in the lipid transporter genes, NPC1 and NPC2 . 1 -2 In cooperation with NPC2, NPC1 is involved in the transport of cholesterol between lysosomes and the endoplasmic reticulum (ER). Mutations in the NPC1 and NPC2 genes disrupt this transport mechanism, resulting in the accumulation of free cholesterol and glycolipids in lysosomes. NPC patients suffer from hepatological and neurological dysfunctions, and eventually die due to respiratory and hepatic failure. 3 Miglustat is a lipogenesis inhibitor indicated for NPC; however, its effects are so limited that patients eagerly await new advances in drug development for NPC therapy. 4 A Phase IIb/III clinical trial of

2-hydroxypropyl-β-cyclodextrin

(HPBCD),

which

can

reduce

cholesterol

and

glycosphingolipid (GSL) storage in neurons, is currently being conducted in the United States and Europe. Furthermore, we recently found that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) is more effective than HPBCD in reducing the cholesterol accumulation and restoring the functional and molecular abnormalities in cells derived from NPC patients. Because of the availability of disease modifying treatments 5 and the development of new therapies, there is an urgent need for reliable and robust biomarkers. As most cellular processes are generally regulated by different feedback loops and alterations, and glycomic profiles are likely to be amplified compared to upstream genetic variations, a glycomic study of NPC may provide a good opportunity to identify novel biomarkers to monitor disease progression or serve as outcome measures in future clinical trials. Indeed, several pioneering studies reported various glycomic alterations in

NPC

involving

various

glycosphingolipids

(glucosylceramides

(GlcCer),

lactosylceramide (LacCer), GM2, GM3, and asiao-GM2). 6 -11 In addition, a recent study reported that accumulated monohexosylceramides and the GM2 ganglioside in the liver, and GM2 and GM3 gangliosides in the brain of the Npc1 -/ - mouse were markedly decreased in response to HPBCD treatment. 1 2 The disease-specific accumulation of various sialylated glycoconjugates within endocytic compartments of NPC1 -null and

NPC2 -deficient fibroblasts is caused by impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1 -null or NPC2 -deficient cells with cyclodextrin was effective in reducing cholesterol storage, as well as the endocytic accumulation of sialylated glycoconjugates, 1 3 although structure intensive analysis was not performed in this study. In this study, we aimed to clarify the effects of a mutation in the Npc1 gene on the total glycome of Chinese hamster ovary (CHO) cells ( Npc1 KO CHO cells). The employed qualitative

and

quantitative

methods

enabled

mass

spectrometry-based

and

structurally-intensive analyses of lipid-linked glycans (i.e., glycosphingolipids (GSLs)) and protein-linked glycans (i.e., N- and O-linked glycans, glycosaminoglycans such as chondroitin sulfate (CS) and heparan sulfate (HS)), as well as free oligosaccharides (fOSs). We observed various unique glycomic alterations in all classes of sub-glycomes. 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In addition, we analyzed the effects of HPBCD treatment on the expression of the total glycome in Npc1 KO CHO cells. Our glycomic approach clarified the increased expression of specific glycoforms in Npc1 -deficient cells for all classes of glycoconjugated glycans as well as free oligosaccharides.

4


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research Experimental Section

Reagents HPBCD with an average degree of substitution of 4.7 was kindly donated by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Trypsin, heparinase, and heparitinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Peptide: N-glycanase F was obtained from Roche Diagnostics K.K., (Tokyo, Japan). Rhodococcus endoglycoceramidase I was prepared as described previously. 1 4, 1 5 Glucose oxidase was procured from Wako Chemicals Co., Ltd. (Osaka, Japan). Pronase was purchased from Calbiochem (San Diego, CA, USA). Hyaluronidase SD Streptococcus dysgalactiae and chondroitinase ABC were obtained from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). BlotGlyco ® beads and Nα-((aminooxy)acetyl)tryptophanyl arginine methyl ester (aoWR) were procured from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). The MultiScreen Solvinert Low-Binding Hydrophilic PTFE plate (pore size, 0.45 µm) was purchased from Merck Millipore (Darmstadt,

Germany).

Disialyloctasaccharide

(A2GN1),

PMP,

and

N,

N’,N’’,N’’’-tetraacetyl chitotetraose (GN4) were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The MassPrep HILIC µElution plate was purchased from Waters Corp. (Milford, MA, USA). Other solvents and reagents were of the highest grade commercially available.

Cells Wild-type and Npc1 null Chinese hamster ovary (CHO) cells were kindly donated by Dr. Katsumi Higaki, Tottori University. 1 6 CHO cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Sigma-Aldrich) supplemented with 10% fetal bovine serum. For HPBCD treatment, the cells were cultured in the presence of 1 mM HPBCD for 4 days.

Extraction of cellular glycoproteins and free oligosaccharides For the N-glycan and fOS analyses, glycoproteins and fOSs were extracted as previously described. 1 7 Approximately 1 × 10 6 cells suspended in 100 µL of 100 mM Tris-acetate buffer (pH 7.4) containing 2% sodium dodecyl sulfate, which served as a surfactant for the complete dissolution of cells, were homogenized using an Ultrasonic Homogenizer (Taitec Corp., Saitama, Japan). Reductive alkylation of the cellular proteins was performed, followed by the precipitation of proteins in the presence of a 4-fold volume of ice-cold ethanol. The precipitates equivalent to 2.5 × 10 5 cellular proteins were dried, dissolved in 100 mM ammonium bicarbonate, and digested with trypsin. Deglycosylation was performed by the addition of 2 U of PNGase F. The supernatants containing cellular fOSs were completely desiccated using a centrifugal evaporator and dissolved in deionized water. The samples were directly subjected to the glycoblotting procedure.

Extraction of GSLs and GAGs 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The GAG and GSL extraction procedures were similar to those previously described. 1 0 Delipidation was performed by the addition of 450 µL of a chloroform/methanol (C/M) solution (2/1, v/v) to the cell pellet, followed by sonication at room temperature as previously described for the extraction of glycoproteins and N-glycans. Methanol (150 µL) was then added, yielding a solvent composition of C/M = 1/1 (v/v), and the sample was sonicated. Thereafter, methanol (300 µL) was added (C/M = 1/2, v/v), and the sample was sonicated once again. The resulting extracts were centrifuged at 20,000 × g for 10 min, and the supernatant and pellet were subjected to GSL-glycan and GAG analysis, respectively. For the GSL-glycan analysis, the supernatant containing crude cellular lipids was completely dried using a centrifugal evaporator. Crude cellular lipids were suspended in 50 µL of 50 mM sodium acetate buffer (pH 5.5) containing 0.2% Triton X-100 (Sigma-Aldrich) as a surfactant, followed by the addition of 25 mU of EGCase I to release the intact glycans from the GSLs. For the GAG analysis, the pellet was subjected to an overnight non-specific protease digestion at 37 °C by the addition of 100 µg of Pronase in 50 mM Tris-acetate buffer (pH 7.5) containing 10 mM CaCl 2 . To precipitate the peptides linked to GAG chains, ethanol precipitation was carried out as previously described for the extraction of glycoproteins and fOSs. The ethanol-precipitated sample was completely digested to disaccharidic GAGs using 5 mU each of a mixture of heparinase, heparitinase, hyaluronidase SD, and chondroitinase ABC. The sample and enzymes were then incubated overnight at 37 °C in 100 µL of 100 mM ammonium acetate buffer (pH 7.5) containing 5 mM calcium acetate. The digested sample was centrifuged, and the supernatant was collected, dried using a centrifugal evaporator, and dissolved in 25 µL of deionized water containing 50 pmol of an internal standard, isomaltotriose (Sigma-Aldrich). The samples were directly subjected to the glycoblotting procedure.

Cellular O-glycomics The protein pellet was prepared from approximately 1 × 10 6 cells as previously described for the extraction of glycoproteins and fOSs. The ethanol-precipitated proteins were subjected to O-glycomic analysis. The proteins were concentrated using an Amicon Ultra Centrifugal Filter unit (molecular weight cut-off, 3000 Da) (Millipore). The concentrated proteins were subjected to the microwave-assisted BEP reaction using the Monowave 300 microwave reactor. After the BEP reaction, bis-PMP-labeled GN4 was added as an external standard, and the mixture was neutralized with 1.0 M hydrochloric acid. Chloroform was added, and the mixture was vigorously vortexed. The chloroform layer was discarded, and the PMP-labeled glycans in the resultant aqueous layer were purified by passage through a graphitized carbon column and an Iatrobeads silica gel column as previously described. 1 7

Glycoblotting procedure N-glycans, fOSs, GAGs, and GSL-glycans were subjected to the glycoblotting procedure. Detailed procedures and materials are provided elsewhere. 17 ,18 6


Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MALDI-TOF/TOF MS analysis Purified N-glycans, O-glycans, fOSs, and GSL-glycan solutions were combined with 2,5-dihydrobenzoic acid (10 mg/mL in 30% acetonitrile) and subsequently subjected to MALDI-TOF MS analysis as previously described. 17 All measurements were performed using an Ultraflex II TOF/TOF mass spectrometer equipped with a reflector and controlled by the FlexControl 3.0 software package (Bruker Daltonics GmbH, Bremen, Germany) according to established protocols. All spectra were obtained in the reflectron mode with an acceleration voltage of 25 kV, a reflector voltage of 26.3 kV, and a pulsed ion extraction of 160 ns in the positive ion mode. The masses were annotated using the FlexAnalysis 3.0 software package. The GlycoSuiteDB (http://glycosuitedb.expasy.org/ glycosuite/glycodb) and SphinGOMAP (http://www.sphingomap.org/) online databases were used for the structural identification of GSL-glycans. Absolute quantification was performed by comparative analyses between the areas of the MS signals derived from each

glycan

and

10

pmol

of

the

internal

standard

NeuAc2Gal2GlcNAc2

+

Man3GlcNAc1(A2GN1), which was added to the sample solution prior to glycoblotting. To obtain TOF/TOF mode measurements for fragment ion analysis, precursor ions were accelerated to 8 kV and selected in a timed ion gate. Fragment ions generated by the laser-induced decomposition of the precursor were further accelerated by 19 kV in the LIFT cell. The fragment ion masses were analyzed after passing the ions through the ion reflector.

HPLC analysis 2AB-labeled GAG disaccharides were analyzed by HPLC. Detailed procedures and materials are provided elsewhere. 1 7 ,1 9

Statistical analysis All data obtained for N-, O-, GSL-glycans and fOSs were presented as the mean ± SD of three experiments performed in triplicate. The statistical differences between the two groups were evaluated using a two-tailed Student’s t test. In case of GAG disaccharide analysis, values were shown from a single experiment, thus statistical analysis was not performed.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Results

1. Lipid-bound glycans (GSL-glycnas) There were no significant differences in the total amounts of GSLs between the wild-type and Npc1 KO CHO cells. However, the levels of ten out of 31 GSL glycans identified in this study were significantly higher in Npc1 KO CHO cells than in wild-type cells (Fig. S1). Three of these GSL glycans were sialylated GSLs, and seven were neutral GSLs. According to the SphinGOMAP database, 2 0 many of these GSL glycans can adopt more than one isomeric structure, namely, the ganglio (Gg)-, globo (Gb)-, and/or (neo)lacto (nLc)-series GSLs, with the same m/z value as shown in Table S1. Considering that the observed glycomic alterations were mutually related to one another in the GSL biosynthetic pathway, nLc-series GSL glycans were the only sub-GSL glycome among the three types of GSL glycomes to include all ten GSL glycans (Fig. 1a). However, it is likely that several of the GSL glycans also exist as Gb- and/or Gg-series GSL glycans as shown in Figure 1b, c. Although the detailed structure determination of GSL-glycans is an important task, we did not conduct a more detailed structure analysis as it was very difficult to perform, and the purpose of this study was to clarify the effects of the Npc1 mutation on the total glycome of cells. The treatment of Npc1 KO CHO cells with HPBCD did not alter the glycome in these cells.

8


Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research a) neolacto-series

β4

α3

β3

β4

β4

β3

βCer 231

β4

β3 β4 β3 β4 nLc6Cer/i antigen

β3 α6

α8

α3

β4

β3

β4

α3

βCer 44

β4

β3

β4

β3

β4

β3 β4 nLc5Cer

β4 β4

α3

53

β4

βCer

β4

βCer

β3

β4

α3

β3 247

247 β4

βCer

β4

52

α3

β4

β4

β3

β4

βCer

βCer 40

β4 β3 β4 βCer Sialyl paragloboside 42

α?

β4

β4

βCer

βCer

β3 407

43 β4

β3 β4 nLc4 Cer

β3

βCer 169

β4

βCer

β4

βCer

β3 12

β4 βCer Lc3 Cer

210

4

β4 βCer LacCer 3

b) globo-series

b) ganglio-series α8 β4

β3 β3

α4

β4

α3

α6

β6

β4 β4 GD1β

β3

β4

βCer α3 128

βCer

β4 βCer GD1a 75

β3 23

β3

α6 β3

β6 β3

α4

β4

β4 β4 GM1α

β4

βCer α3 127

βCer

β4 βCer GM1 72

β3 β3

β3

α4

β4

and/or α3 β3 α4 β4 Forssman antigen

22

βCer 20

α6

β4 β4 GM2α

β3

19

β3 α4 β4 Gb5Cer, SSEA-3

β4

βCer

βCer

α3

126

β4 βCer GM2 69

βCer 21

β4 β4 βCer asialo-GM2 17

β3 α4 β4 βCer Gb4 Cer, P antigen 18

α3

β4 βCer GM3 15

β4 βCer LacCer 3

Figure 1. GSL glycan content significantly increased in Npc1 KO CHO cells compared to wild-type

cells.

Glycan

structures,

which

were

estimated

by

referring

to

the

SphinGOMAP database, are shown for each GSL subclass (i.e., (a) neolacto-, (b) globo-, and (c) ganglio-series). Quadrilaterals of the same color indicate that they were isomers whose molecular weights were identical and indistinguishable by this study. Structures without quadrilaterals indicate that their expressions were not significantly altered between the wild-type and Npc1 KO CHO cells.

2. Protein-bound glycans (N- and O-glycans) 2.1.

N-glycans

The total amount of N-glycans significantly increased by ~50%. The levels of 19 out of 58 N-glycans identified in this study were significantly higher in Npc1 KO CHO cells than in wild-type cells (Table S2, Fig. S2). N-glycans

can

be

structurally

classified

into

pauci-mannose

(PM;

Man 1 -4 GlcNAc 2 Fuc 0 -1 ), high-mannose (HM; Man 5 -9 GlcNAc 2 ), hybrid- and complex-types. Among these four types, the most significant change was the increased expression of HM-type N-glycans (1.6-fold) (Figure 2). The expression of hybrid-type N-glycans also 9


Journal of Proteome Research increased moderately by ~10%. As hybrid- and complex-type N-glycans are often modified with fucose and/or sialic acid residue(s), glycomic alterations were analyzed based on the status of fucosylation and sialylation. We found that the levels of complex-type N-glycans modified with both fucose(s) and sialic acid(s) were significantly higher in Npc1 KO CHO cells than in wild-type cells. Fucosylated and sialylated hybrid-type N-glycans also tended to increase; however, the increase was not statistically significant. Although the total amount of PM-type N-glycans was not affected by the Npc1 mutation, the expression levels of Man3F, Man4, and Man4F were significantly higher in Npc1 KO CHO cells than in wild-type cells (Fig. S2). None of the above-mentioned glycomic alterations in Npc1 KO CHO cells were affected by HPBCD treatment.

PM type

a)

?

?

HM type

Hybrid type

Complex type

Hybrid Fuc(-)Sia(-)

Complex Fuc(-)Sia(-)

Hybrid Fuc(+)Sia(-)

Hybrid Fuc(-)Sia(+)

Complex Fuc(+)Sia(-)

Hybrid Fuc(+)Sia(+)

c)

100

** **

90 80 70

WT(-)

0.6

NPC(-)

0.4

NPC(+)

60 50 40 30

*

20

0.2 0

8

*

*

*

HM

Hyb

Com

2.5 2

3

1.5

1.5

2

1

1

1

0.5

0.5

0 WT(-) NPC(-) NPC(+)

0

Com Fuc(-)Sia(-) 2.5

4

2

3

1.5

2

1

1

0.5

0 WT(-) NPC(-) NPC(+)

WT(-) NPC(-) NPC(+)

Com Fuc(-)Sia(+) 5

5

WT(-) NPC(-) NPC(+)

0 WT(-) NPC(-) NPC(+)

Com Fuc(+)Sia(-) 15 10

0

PM

2.5 2

2

0

Hyb Fuc(-)Sia(-) 3

4

Com Fuc(+)Sia(+) 10

Hyb Fuc(-)Sia(+) 3

0

4

* *

5

WT(-) NPC(-) NPC(+)

6

10

Hyb Fuc(+)Sia(-) 6

**

Complex Fuc(-)Sia(+)

Complex Fuc(+)Sia(+)

Hyb Fuc(+)Sia(+) 0.8

pmol/100µg protein

b)

pmol/100µg protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 20

0 WT(-) NPC(-) NPC(+)

WT(-) NPC(-) NPC(+)

Figure 2. Alterations in the N-glycome of Npc1 KO CHO cells compared to wild-type cells, and the effects of HPBCD treatment on Npc1 KO CHO cells. (a) N-glycan biosynthetic pathway in CHO cells. (b) Expression of PM, HM, hybrid (Hyb)- and complex (Com)-type N-glycans in wild-type and Npc1 KO CHO cells. (c) Expression profiles of hybrid- and complex-type N-glycans in wild-type and Npc1 KO CHO cells depending on the presence of fucose and/or sialic acid residues. Values are shown as mean ± S.D. (N=3, * P

Impact of the NiemannâPick c1 Gene Mutation on the Total Cellular

Recommend Documents

Impact of the NiemannâPick c1 Gene Mutation on the Total Cellular