Báo cáo khoa học: The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells pdf

To study intracellular mannose salvage pathways, we used an N-linked glycosylation assay system using DNase I [19] in PMI-null cells.. We also show that, when cells were incubated in med

glycosylation in PMI-deficient mouse embryonic fibroblast cells

Naonobu Fujita1, Ayako Tamura1, Aya Higashidani1, Takashi Tonozuka1, Hudson H Freeze2and Atsushi Nishikawa1

1 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Japan

2 Tumor Microenvironment Program, Burnham Institute for Medical Research, La Jolla, CA, USA

Eukaryotic cells contain mannose mainly in the form

of N-linked oligosaccharides and glycophospholipid

anchors [1,2] The only known pathway providing

GDP-mannose for these molecules requires the

follow-ing conversions: mannose 6-phosphate to mannose

1-phosphate to GDP-mannose and dolichyl-P-mannose

[3,4] In mammals, mannose 6-phosphate can be

formed in several ways The first, primary, and by far

the best-known, way is via the following conversions: Glc to Glc6P to Fru6P to mannose 6-phosphate Phosphomannose isomerase (PMI) (which catalyzes the Fru6P to mannose 6-phosphate conversion) has an important role in this pathway The second is by direct phosphorylation of exogenous mannose that is trans-ported by a mannose transporter [5] The third is by direct phosphorylation of endogenous mannose that is

Keywords

congenital disorders of glycosylation;

lipid-linked oligosaccharide; mannose; N-lipid-linked

oligosaccharide; phosphomannose

isomerase

Correspondence

A Nishikawa, Department of Applied

Biological Science, Tokyo University of

Agriculture and Technology, 3-5-8

Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan

Fax: +81 42 367 5705

Tel: +81 42 367 5905

E-mail: nishikaw@cc.tuat.ac.jp

(Received 22 October 2007, revised 6

December 2007, accepted 17 December

2007)

doi:10.1111/j.1742-4658.2008.06246.x

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans All pathways must generate mannose 6-phosphate, the activated form of mannose Imported or salvaged mannose is directly phosphory-lated by hexokinase, whereas fructose 6-phosphate from glucose is con-verted to mannose 6-phosphate by phosphomannose isomerase (PMI) Normally, PMI provides the majority of mannose for glycan synthesis To assess the contribution of PMI-independent pathways, we used PMI-null fi-broblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein In PMI-null cells, imported mannose and salvaged mannose make

a significant contribution to N-glycosylation When these cells were grown

in mannose-free medium along with the mannosidase inhibitor, swainso-nine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated Adding 13 lm mannose to the medium completely restored normal glycosylation Treatment with bafilomycin A1,

an inhibitor of lysosomal acidification, also markedly reduced N-glycosyla-tion of DNase I, but in this case only 8 lm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of man-nose made a significant contribution Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane traf-ficking was blocked by expression of a mutant SKD1 From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways

Abbreviations

CDG, congenital disorder of glycosylation; GFP, green fluorescent protein; LLO, lipid-linked oligosaccharide; MOI, multiplicity of infection; PMI, phosphomannose isomerase.

salvaged from glycoconjugates that are degraded

within the same cell An additional minor pathway

may be conversion of GDP-fucose to GDP-mannose

via unstable intermediate

GDP-4-keto-6-deoxy-man-nose [6] It has previously been assumed that most of

the mannose in macromolecules is derived from

glu-cose [7] This assumption was based on the universal

distribution of PMI [8], and the fact that PMI is

essen-tial for yeast growth in the absence of mannose [9]

However, exogenous mannose can contribute

signifi-cantly to glycosylation in some cells [10]

The congenital disorders of glycosylation (CDGs)

are metabolic syndromes with a wide symptomatology

and severity, stemming from deficient N-glycosylation

of proteins [11] CDG type I defects are due to

insuffi-cient synthesis or poor transfer of the lipid-linked

oligosaccharide (LLO) precursor sugar chain to

pro-teins, leaving many glycosylation sites unoccupied

One of these, CDG-Ib, is due to a deficiency in PMI

that reduces the endogeneous production of

man-nose 6-phosphate [11,12] As these patients can be

treated with dietary mannose supplements, it is evident

that abnormalities in the mannose metabolic pathways

have serious medical consequences Therefore, there is

currently great interest in understanding mannose

metabolism, including salvage pathways

Although the contribution of monosaccharide

sal-vage pathways to glycosylation may be substantial

[13–16], mannose salvage pathways have received

rela-tively little attention and have yet to be systematically

investigated [17] Sources of salvaged mannose and its

relative contribution to glycoprotein synthesis are

poorly understood The main difficulty in the study of

mannose salvage pathways is that mannose

6-phos-phate derived from mannose-salvaging pathways are

indistinguishable from those derived from glucose

We constructed PMI-knockout mice, and PMI-null

cells were established from embryonic cells [18] To

study intracellular mannose salvage pathways, we used

an N-linked glycosylation assay system using DNase I

[19] in PMI-null cells Wild-type DNase I has two

potential N-linked glycosylation sites Although the

N-terminal Asn18–Ala–Thr sequon is fully

glycosylat-ed, the C-terminal Asn106–Asp–Ser sequon differs in

tissues and cultured cells [19] The occupation of the

C-terminal sequon might reflect the glycosylation

capa-bility of cells Using PMI-null cells, we can examine the

mannose salvage pathways independently of glucose

interconversion at a physiological concentration of

glucose In this article, we demonstrate that mannose

salvage pathways make significant contributions to

glycosylation, and that the degradation of

glycoconju-gates occurs mainly at low pH in the lysosomes We

also show that, when cells were incubated in medium supplemented with 10% fetal bovine serum, the pre-dominant source of salvaged mannose was degradation

of endogenous glycoconjugates

Results Determination of glycosylation efficiency using the mutant DNase I expression method

Adenovirus for expression of bovine mutant DNase I that has only one N-linked glycosylation site was infected into various cultured cells, and the glycosyla-tion efficiency of expressed DNase I was determined [19] As shown in Fig 1A, the percentage of glycosy-lated molecules showed an inherent value depending

on the cells When another mutant DNase I was expressed in these cells, e.g with the Asn106–Ser–Thr sequon instead of the Asn106–Asp–Ser sequon, the glycosylation efficiency in these cells was almost 100% (data not shown) We previously reported that the glycosylation efficiency of the Asn106–Asp–Ser sequon on DNase I depended on the tissue of origin [19]

To confirm that the glycosylation percentage of expressed mutant DNase I reflected the glycosylation capability of the cells, we next investigated glycosyla-tion of integrin b1 Fibroblasts derived from CDG patients were infected by adenovirus expressing mutant DNase I and labeled metabolically with [35S]Met and [35S]Cys After harvesting of the condi-tioned medium for the DNase I glycosylation assay, the cells were lysed and integrin b1 was immunopre-cipitated As shown in Fig 1B, the molecular mass of the precipitated protein differed according to the type

of CDG It seems that these differences mainly depended on the number of N-linked oligosaccha-rides It was hard to calculate the exact glycosylation efficiency of integrin b1, however, as integrin b1 has more than 10 potential N-glycosylation sites It is noteworthy that the percentage of glycosylated DNase I correlated well with that of the immunopre-cipitated protein Thus, the glycosylation efficiency of this mutant DNase I with the Asn106–Asp–Ser se-quon seemed to reflect the glycosylation capability of the cells

Glycosylation efficiency of mutant DNase I expressed in CDG-Ib fibroblast cells

We measured the glycosylation efficiency of DNase I

in CDG-Ib fibroblast cells, mouse wild-type fibroblast cells, and PMI-null embryonic fibroblast cells CDG-Ib

cells, owing to a substantial deficiency in PMI activity,

do not have enough LLO, which results in unoccupied

N-linked glycosylation sequons In the absence of

exogenously supplied mannose, GDP-mannose levels

are markedly lower in CDG-Ib cells [20] In PMI-null

cells, mannose 6-phosphate cannot be supplied from

the glycolytic pathway via PMI, so when PMI-null

cells are cultured in mannose-free medium, mannose

6-phosphate for glycosylation must be salvaged from

elsewhere To remove the effects of free mannose in

the medium, cells were cultured in MEM supplemented

with 10% dialyzed fetal bovine serum for 24 h, and glycosylation analyses were then performed We pre-dicted that the glycosylation efficiency would be lower

in CDG-Ib cells and decreased markedly in PMI-null cells However, CDG-Ib cells maintained sufficient gly-cosylation ability, and surprisingly, PMI-null cells showed only a slight reduction in glycosylation effi-ciency (Fig 2) From these results, we speculated that substantial amounts of mannose are supplied by the salvage pathways

72

Huh6 A549

PC12

A

B

1 2 3

82 55 0

]

Fig 1 Determination of glycosylation efficiency using a mutant

DNase I expression method Glycosylation efficiencies of mutant

DNase I expressed in various types of cells were determined by

infecting cells with adenovirus carrying mutant DNase I and then

incubated with [ 35 S]Met and [ 35 S]Cys (A) The protein was

immuno-precipitated from conditioned medium with rabbit anti-DNase I

serum and protein A–agarose beads, and the eluant was subjected

to SDS ⁄ PAGE In the first panel, lane 1 is intact

immunoprecipitat-ed protein, and lanes 2 and 3 are

endo-b-N-acetylglucosamini-dase-treated and PNGase F-treated samples, respectively DNase I

having different types of N-glycan is usually detected as a

broad band [19] The arrow indicates the location of

non-glycosylat-ed DNase I, and ‘]’ shows the migration of the singly glycosylatnon-glycosylat-ed

DNase I The percentage of glycosylated molecules is indicated

under the picture (B) Upper panel: determination of glycosylation

efficiency of mutant DNase I in several fibroblast cells derived from

CDG type I patients 42F is a human control fibroblast cell line The

percentage of glycosylated molecules, quantified by densitometry,

is indicated under the picture Lower panel: SDS ⁄ PAGE of

immuno-precipitated integrin b1 derived from several CDG type I cells.

35 S-labeled cells obtained from above experiment were lysed

(100 m M Tris ⁄ HCl, 150 m M NaCl, 1% NP-40 buffer, pH 8.0,

con-taining Roche protease inhibitor cocktail) and immunoprecipitated

using monoclonal antibody to integrin b1 and protein A–agarose

beads The precipitate was subjected to SDS ⁄ PAGE using 7%

polyacrylamide gel.

WT

A

B PMI-null

Baf A1

SW

CDG-Ib

WT PMI-null

CDG-Ib

Control

0

20

40

60

80

100

Control

SW Baf A1

] ] ]

Fig 2 Effect of swainsonine and bafilomycin A1on glycosylation CDG-Ib fibroblast cells, mouse wild-type embryonic fibroblast cells and PMI-null fibroblast cells were incubated for 24 h in MEM sup-plemented with 10% dialyzed fetal bovine serum with or without

10 l M swainsonine (SW) or 100 n M bafilomycin A1(Baf.A1) prior to metabolic labeling The percentages of glycosylated molecules were analyzed as described in Experimental procedures (A) SDS ⁄ PAGE analysis representative of three experiments with simi-lar results The arrow indicates the location of nonglycosylated DNase I (B) The bands in (A) were quantified by densitometry, and percentages of the glycosylated molecules were calculated Values shown are means of five independent experiments.

To investigate the relative contribution of lysosomal

a-mannosidases to mannose salvage pathways in

PMI-null cells, they were treated with either swainsonine or

bafilomycin A1, and glycosylation analysis was

per-formed Swainsonine is an indolizidine alkaloid that

acts as a reversible inhibitor of lysosomal

a-mannosi-dase and of the Golgi complex a-mannosia-mannosi-dase II [21]

Bafilomycin A1 is a highly specific inhibitor of

vacuo-lar-type H+-ATPase [22], and inhibits acidification

and degradation in lysosomes of cultured cells [23]

Treatment with these drugs did not affect the

percent-age of glycosylated molecules in normal and CDG-Ib

cells, whereas the values in PMI-null cells were reduced

almost to zero These results indicate that, in PMI-null

cells, almost all mannose 6-phosphate for glycosylation

is supplied by salvage pathways that involve

lyso-somal a-mannosidases

Supplemental mannose corrected glycosylation

deficiency

If swainsonine and bafilomycin A1 block the mannose

salvage pathways, then supplemental mannose should

be sufficient to correct the glycosylation deficiency in

swainsonine-treated or bafilomycin A1-treated

PMI-null cells We thus performed mannose titration

experiments in swainsonine-treated and bafilomycin

A1-treated PMI-null cells Cells were treated with

10 lm swainsonine or 100 nm bafilomycin A1 along

with the indicated concentration of mannose for 12 h

prior to metabolic labeling The percentages of

glycosylated molecules were then measured (Fig 3) As

expected, the glycosylation efficiencies increased in

swainsonine-treated and bafilomycin A1-treated

PMI-null cells with increasing mannose concentrations in

the medium About 13 lm and 8 lm mannose were

sufficient to fully restore glycosylation in

swainsonine-treated and bafilomycin A1-treated PMI-null cells,

respectively From these results, we conclude that the

reduction in glycosylation efficiency in

swainso-nine-treated and bafilomycin A1-treated PMI-null cells

is caused by blockade of the mannose salvage

path-ways and not of the glycoprotein maturation steps

Overexpression of SKD1E235Qstrongly inhibited

core glycosylation in PMI-null cells

In mannose salvage pathways, important sources of

mannose are probably glycoconjugates, which are

transported to lysosomes by a membrane trafficking

process To test the involvement of membrane

traffick-ing in mannose salvage pathways, we examined the

effect of dominant-negative SKD1 mutants on the

mannose supply in PMI-null cells SKD1 is a member

of the ATPase family associated with cellular activities, and for which the yeast homolog Vps4p has been shown to be involved in endosomal⁄ vacuolar mem-brane transport [24] Expression of a mutant SKD1 molecule, named SKD1E235Q, that lacks ATPase activ-ity in mammalian cells exerted dominant-negative effects on various membrane-transport processes that involve endosomes [25] As shown in Fig 4A, when an adenovirus delivery system was used, almost 100% of the cell population overexpressed green fluorescent protein (GFP)–SKD1E235Q [26] When the glycosyla-tion assay was performed in GFP–SKD1E235Q -overex-pressing cells, the percentage of glycosylated molecules

in wild-type cells was unchanged, but the percentage in

PMI-null

0 2 5 5 7 5 1 0 12.5 15 2 0 (µ M )

WT Man conc

Baf.A 1

0 1 2 5 5 7.5 1 0 (µ M )

PMI-null

WT Man conc

SW

A

B

0

20

40

60

80

100

Mannose conc (µ M )

PMI-null (SW)

WT (SW)

PMI-null (Baf.A1)

WT (Baf.A1)

Fig 3 Mannose titration in swainsonine-treated or bafilomycin A 1 -treated PMI-null cells PMI-null cells were cultured with 10 l M swainsonine or 100 n M bafilomycin A1 for 12 h prior to labeling The indicated concentrations of mannose (0–20 l M ) were also added to the media simultaneously (A) Glycosylation analysis The data shown are from a single experiment that is representative of three replicates The arrow indicates the location of

nonglycosylat-ed DNase I (B) The graph shows the mannose titration curve in wild-type (WT) and PMI-null cells.

PMI-null cells was markedly reduced (Fig 4B) These

results demonstrate that most salvaged mannose comes

from material transported to lysosomes via endosomal

trafficking pathways

The contribution of serum glycoproteins and

endogenous glycoconjugates to mannose salvage

pathways

When PMI-null cells are incubated in media

supple-mented with 10% fetal bovine serum, the

glycoconju-gates used in mannose salvage pathways could be

derived from either endogenous or exogenous

mole-cules To identify the relative contributions of

exoge-nous and endogeexoge-nous glycoconjugates, we performed

the glycosylation assay under serum-free conditions

To remove glycoconjugates from the culture medium,

10% fetal bovine serum was replaced with 1% BSA or

2% TCH, which is a serum replacement product and

contains extremely low concentrations of glycoprotein

Cells were incubated for 12 h prior to metabolic

label-ing in MEM supplemented with 1% BSA or 2% TCH, and the glycosylation assay was then performed As shown in Fig 5A, although glycoconjugates were almost completely removed from the culture medium, the percentage of glycosylated molecules in PMI-null cells was only slightly reduced We obtained the same result when preincubation in serum-free medium was extended to 24 h (data not shown) This indicates that, when the culture medium does not contain fetal bovine serum, substantial amounts of mannose can be derived from endogenous rather than exogenous glycoconju-gates

If endogenous glycoconjugates are the main source

of mannose, the percentage of glycosylated molecules should be reduced with time when PMI-null cells are cultured in mannose-free medium, Prior to metabolic labeling, PMI-null cells were incubated with MEM supplemented with 10% dialyzed fetal bovine serum for 0, 12, 24, 36 or 48 h (Fig 5B), and glycosylation assays were then performed As shown in Fig 5C, increasing the preincubation time reduced the percentage

0

20

40

60

80

100

120

MOI = 0 MOI = 1000 MOI = 2000

SKD1 E235Q MOI = 1000

SKD1 E235Q MOI = 2000 MOI = 0

DIC

Fluorescence

A

B

Fig 4 Overexpression of SKD1 E235Q inhib-ited mannose salvage pathways To gener-ate the overexpression of SKD1E235Q, cells were coinfected with AxCALSKD EQ at an MOI of 0, 1000, or 2000, and AxCANCre at

an MOI of 150 (A) Twenty-four hours post-infection, the cells were washed twice with NaCl ⁄ P i and fixed with 4% paraformalde-hyde for 15 min Fluorescent images were obtained with a Zeiss Axiocam controlled by AXIOVISON software (B) Prior to metabolic labeling, the cells were incubated with MEM supplemented with 10% dialyzed fetal bovine serum, containing AxCALSKD EQ

and AxCANCre at the indicated MOI The glycosylation analysis was performed as described in Experimental procedures The results are presented as percentage of untreated cells, which was defined as 100%.

MEM+ 20% FBS

MEM + 10% dialyzed FBS

0 12 24 36 48 60

Pre-incubation time (h)

0

20

40

60

80

100

120

A

B

E

C

D

Control

(10 % dialyzed FBS)

1 % BSA 2 % TCH

Glycosylation efficiency (%) 0

0 10 20 30 40 50

20 40 60 80 100

Pre-incubation time (h)

Retention time (min)

0 h

24 h

48 h

G0 1 2 3

Glycoprotein Total protein

G R 1 0.94 0.64 (Glycoprotein/Total protein)

F I 100 87 56 % 100 93 87 %

M 0 24 48 h M 0 24 48 h

82 k

42 k

180 k

Fig 5 The contribution of serum glycoproteins in culture medium to N-glycosylation (A) To remove glycoproteins from the culture medium, 10% dialyzed fetal bovine serum was replaced with 1% BSA or 2% TCH PMI-null cells were incubated for 12 h in MEM supplemented with 1% BSA or 2% TCH prior to metabolic labeling The percentages of glycosylated molecules were analyzed as described in Experimental proce-dures The number of glycosylated molecules is presented as percentage of the value in 10% dialyzed fetal bovine serum (B) Scheme of the time course for an experiment After 0, 12, 24, 36 or 48 h of incubation with MEM supplemented with 10% dialyzed fetal bovine serum (indi-cated with dotted line), the glycosylation analyses were performed (C) Time course of the reduction in glycosylation efficiency of DNase I in PMI-null cells The medium was replaced every 4 h with fresh MEM supplemented with 10% dialyzed fetal bovine serum Values represent mean ± SD of three independent experiments (D) LLO patterns of cells harvested after 0, 24 and 48 h of incubation with MEM supplemented with 10% dialyzed fetal bovine serum The cells were collected, and LLOs were labeled with 2-aminopyridine and analyzed using HPLC accord-ing to the method described in Experimental procedures Ten per cent volume of the product of each sample was injected into the column The arrows indicate the elution positions of standard pyridylaminated oligosaccharides, G0 is Man 9 GlcNAc 2 -PA, and G3 is Glc 3 Man 9 GlcNAc 2 -PA (2-aminopyrimidine) (E) Glycosylation analysis of total cell protein About 30% of each sample was harvested after 0, 24 and 48 h of incubation with 10% dialyzed fetal bovine serum, and subjected to SDS ⁄ PAGE using 12% polyacrylamide gel The gel was first stained with Pro-Q Emer-ald 300 for glycoprotein determination, and next with SYPRO Ruby for total protein determination, according to the manufacturer’s instructions Total fluorescence intensity (FI) in each lane was calculated; the amount after 0 h of incubation with 10% dialyzed fetal bovine serum was 100% The total FI of glycoprotein ⁄ total protein is shown as glycosylation ratio (GR) The standard protein (M) molecular masses are indicated.

of glycosylated molecules in PMI-null cells In

contrast, the percentage of glycosylated molecules in

wild-type cells was unchanged (data not shown) As

the amount of LLO also decreased, depending on the

increase in preincubation time (Fig 5D), this seems to

be attributable to the lack of mannose salvage and an

inability to derive sufficient mannose from other

sources We then determined the variation of amount

of glycoprotein in cells As shown in Fig 5E, although

the total amount of protein in cells decreased to 87%

after 48 h of incubation, the amount of glycoprotein

decreased more The results of culturing for 48 h

showed that the percentage of glycosylated protein in

cells decreased to 64% and the total amount of

glyco-protein dropped to nearly half the initial amount On

the other hand, the amount of glycoprotein in the

medium did not differ substantially from the beginning

to the end of the experiment, because the medium was

changed every 4 h From these findings, we conclude

that PMI-null cells incubated in medium supplemented

with 10% fetal bovine serum can salvage mannose

from endogenous glycoconjugates, but that the amount

is insufficient to maintain normal protein synthesis

(growth) or normal levels of LLO

Discussion

In this study, the mannose salvage pathways have been

systematically investigated in PMI-null cells The four

main findings are as follows: (a) the contribution of

mannose salvage pathways to glycosylation is quite

substantial; (b) glycoconjugate degradation mainly

occurs in lysosomes under low-pH conditions; (c)

gly-coconjugates are transported to lysosomes via

endo-somal trafficking pathways; and (d) when cells are

incubated in medium supplemented with 10% fetal

bovine serum, mannose can be derived from

endoge-nous glycoconjugates

The unexpected finding that normal and CDG-Ib

cells showed nearly equal levels of glycosylation

(Fig 2) led us to investigate the mannose salvage

path-way in mammalian cells PMI-null cells cannot

gener-ate mannose from glucose, and they are forced to rely

on mannose salvage pathways This allowed us

exam-ine the mannose salvage pathways at physiological

concentrations of glucose

We consider that the glycosylation analysis method

using DNase I works well because N-glycosylation

may not be crucial for the correct folding of DNase I

In the pulse-chase experiment, the percentage of

gly-cosylated molecules of DNase I was fairly constant

over the time course of the experiments (data not

shown) We also observed that the percentage of

glycosylated molecules was not affected by treatment with the proteasome inhibitor MG132 [27] (data not shown) These results, however, indicate that DNase I

is a glycoprotein, so the glycosylation analysis method using DNase I may be unaffected by endoplasmic reticulum quality control mechanisms

One of the most important findings of this study is that glycosylation deficiency in swainsonine-treated PMI-null cells was completely restored by only 13 lm supplemental mannose (Fig 3) This concentration is equivalent to about 25% of the normal blood level of mannose in humans [28,29] It has been reported that, under physiological concentrations of glucose and mannose, human fibroblasts can derive about 70% of the mannose in N-linked chains from mannose, and about 10% of the transported mannose is used for gly-cosylation, whereas the remainder is isomerized to Fru6P [10] Therefore, we consider the results of the mannose titration to be reasonable

We then considered the relative contributions of diet, salvaging and glucose interconversion to glyco-protein synthesis (Fig 6) The mannose titration results and those of a previous report [10] indicate that

a low concentration of mannose is sufficient for proper glycosylation in fibroblast cells Furthermore, we also observed that a substantial amount of mannose can be

-1-P Fru-6-P

Lysosome

PMI

Glycolysis/

TCA cycle

LLO synthesis/

Glycosylation/

Processing

Glycoprotein degradation

ER

Glucose transporter Mannose transporter

M

M

M M M

G

G G

(A)

(B)

(D)

Oligosaccharide degradation (C)

Oligosaccharide processing

Golgi

M

(C) Endocytosis

(D1) (D2)

Fig 6 Metabolic pathways of mannose 6-phosphate in mammalian cells Mannose 6-phosphate for N-linked glycosylation may be obtained via the following routes (A) From glucose via a pathway involving PMI (B) Plasma mannose may be transported inside the cell (C) It may be generated in the cytosol, and transported through the endoplasmic reticulum (ER) and Golgi apparatus via a degradation and processing pathway (D) It may be salvaged from lysosomal degradation of oligosaccharide (D1) and glycoconjugates via membrane-traffic-dependent salvage pathways (D2).

supplied by the salvage pathways in PMI-null cells.

The relative contributions made by exogenous sugar,

salvage pathways and interconversions probably vary

with cell type and amount of glycoprotein synthesized;

therefore, further research is required to clarify these

points

Although this study was focused on the lysosomal

mannose salvage pathway, which is dependent on

membrane trafficking, there are also two membrane

traffic-independent mannose salvage pathways [30] The

first involves the free mannose that is generated during

glycoprotein maturation steps As mammalian cells

generally contain higher proportions of complex

oligo-saccharides than unprocessed high-mannose-type

chains, it is clear that most of the nine mannose residues

initially incorporated into the LLO precursor will be lost

as free mannose The second involves the free

oligo-saccharides that are generated in the cytosol, and are

products of LLO breakdown, glycopeptides and

incor-rectly folded glycoproteins generated during quality

control screening of the biosynthesis of glycoproteins in

the endoplasmic reticulum [31] Indeed, the

glycosyla-tion efficiency in PMI-null cells was reduced to almost

zero both by swainsonine and bafilomycin A1treatment,

but there were considerable differences in the mannose

titration curves (Fig 3B) This distinction may be due

to differences in the mechanism of action of swainsonine

and bafilomycin A1 Swainsonine inhibits both

lyso-somal and nonlysolyso-somal mannosidases, whereas

bafilo-mycin A1 blocks only lysosomal mannosidases

Therefore, some mannose used in glycosylation can

come from membrane traffic-independent salvage

path-ways Studies are underway to clearly define the relative

contributions of these mannose salvage pathways

Experimental procedures

Materials

The ViraPower Adenoviral Gateway Expression kit,

Lipofec-tamine 2000, Opti-MEM, LR Clonase Enzyme mix and the

Pro-Q Emerald 300 Glycoprotein Gel Stain with SYPRO

Ruby protein gel stain kit were all purchased from Invitrogen

Life Technologies (Carlsbad, CA, USA) Swainsonine and

antibody to integrin b1(SG⁄ 19) were from Seikagaku

Cor-poration (Tokyo, Japan), bafilomycin A1 was from Wako

Pure Chemicals (Tokyo, Japan),

endo-b-N-acetylglucosa-minidase and PNGase F were from New England Biolabs

(Ipswich, MA, USA), Pro-mix [35S]Met⁄ Cys labeling mixture

was from GE Healthcare Bioscience (Little Chalfont, UK),

the serum replacement medium TCH was from CELOX

Labolatories, Inc (St Paul, MN, USA), the C18 SepPak

column and ENVI-CARB solid-phase extraction tube

were from Waters (Milford, MA, USA) and Supelco (Belle-fonte, PA, USA) respectively, and fetal bovine serum was obtained from ICN Biomedicals (Costa Mesa, CA, USA) Other media and reagents were from Sigma (St Louis, MO, USA)

Plasmid and adenovirus preparation

Previously, we described an N-glycosylation efficiency assay [19] In this work, we used 1-delta wild-type bovine DNase I, in which Asn18 was exchanged for Glu, with only one glycosylation site, Asn106–Asp–Ser To obtain higher reproducibility, an adenovirus expression system of mutant DNase I was constructed as follows Adenoviruses bearing mutant bovine DNase I was prepared using the ViraPower Adenovirus Expression System according to the manufac-turer’s instructions Briefly, the mutant bovine DNase I cDNAs were subcloned into the pENTER 1A vector After purification of the plasmids, the cDNA inserts were trans-ferred to the pAd⁄ CMV ⁄ V5-DEST vector by means of the Gateway system using LR clonase The plasmids were puri-fied and digested with the PacI restriction enzyme One microgram of linearized plasmid was diluted with 200 lL

of Opti-MEM, and then mixed with 200 lL of lipofecta-mine solution, dissolving 4 lL of Lipofectalipofecta-mine 2000 in Opti-MEM, and transfected into subconfluent 293A cells in

1 mL of Opti-MEM in six-well plates The 293A cells were then cultured for 8 days in DMEM supplemented with 10% fetal bovine serum The medium was replaced every

2 days When most cells became detached from the plates, the cells and culture medium were harvested together, freeze–thawed three times, and centrifuged (1500 g for

20 min) to obtain the adenovirus-enriched supernatants Aliquots of the supernatants were then added to fresh 293A cells and cultured for 3 days to amplify adenoviruses Viral titers were determined by tissue culture infective dose

50 (TICD50) methods with 293A cells

Cell culture and infection

PMI-deficient mouse embryonic fibroblast cells (PMI-null cells) [18] and wild-type mouse embryonic fibroblast cells (WT cells) were grown in DMEM containing 20% fetal bovine serum CDG-Ib patient cells were grown in a-MEM containing 10% fetal bovine serum All media were supplemented with 10 UÆmL)1 penicillin and

100 mgÆmL)1 streptomycin The cells were infected with adenoviruses bearing mutant bovine DNase I as follows One day before infection, approximately 2· 105cells were plated into six-well plates and incubated at 37C for 16 h

in a CO2incubator The medium was replaced with 1 mL

of culture medium containing adenoviruses bearing mutant bovine DNase I at a multiplicity of infection (MOI) of

300 After incubation for 24 h, the medium containing

adenoviruses was replaced with 1 mL of MEM

supple-mented with 10% dialyzed fetal bovine serum Following

an additional incubation for 12 h, the medium was again

replaced with 1 mL of MEM supplemented with 10%

dia-lyzed fetal bovine serum After incubation for another

12 h, the cells were washed twice with NaCl⁄ Piand

incu-bated for 4 h at 37C with 0.7 mL of MEM without

Met⁄ Cys but supplemented with 10% dialyzed fetal bovine

serum and 2.65 MBq of [35S]Met⁄ Cys labeling mixture

The culture medium was then harvested

Immunoprecipitation and glycosylation analysis

The extent of glycosylation of mutant DNase I was

mea-sured as previously described [19] Briefly, the harvested cell

culture medium was incubated with 1 lL of anti-DNase I

serum overnight Then, 20 lL of a 50% protein A–agarose

bead suspension was added After 1 h of additional

rota-tion, the beads were washed three times The

immunopre-cipitated protein was then eluted from the beads in 2X

SDS⁄ PAGE sample buffer by boiling for 5 min The eluted

samples were separated by SDS⁄ PAGE using 12%

poly-acrylamide gel, and the intensities of the bands

correspond-ing to DNase I were quantified uscorrespond-ing a BAS1000 bioimage

analyzer (Fuji Film Co., Tokyo, Japan)

Glycosidase digestion

Immunoprecipitated DNase I was released from the beads

by boiling for 10 min in 0.5% SDS⁄ 1.0%

2-mercaptoetha-nol solution After centrifugation (15 000 g for 1 min), the

concentrated reaction mixtures were adjusted to contain

50 mm sodium phosphate (pH 7.5) and 1.0% NP-40 for

PNGase F digestion, or 50 mm sodium citrate (pH 5.5) for

endo-b-N-acetylglucosaminidase digestion, according to the

manufacturer’s instructions PNGase F (2 units) or

endo-b-N-acetylglucosaminidase (2 units) was added, and the

reactions were incubated at 37C for 30 min Following

incubation, the samples were subjected to SDS⁄ PAGE

Swainsonine and bafilomycin A1treatment

Cells were treated with 10 lm swainsonine, 100 nm

bafilo-mycin A1 or 20 lm mannose for 24 h prior to metabolic

labeling in 1 mL of MEM supplemented with 10% dialyzed

fetal bovine serum Metabolic labeling was then performed

in the presence of 10 lm swainsonine, 100 nm

bafilo-mycin A1, or 20 lm mannose, respectively Glycosylation

analysis was performed as described above

Mannose titration

Prior to metabolic labeling, cells were incubated with 10 lm

swainsonine or 100 nm bafilomycin A1 and different

concentrations of mannose (0–20 lm) for 12 h Metabolic labeling was also performed in the presence of 10 lm swainsonine or 100 nm bafilomycin A1 and the indicated concentrations of mannose The glycosylation analysis was then performed as described above

GFP–SKD1E235Qoverexpression

A Cre⁄ loxP inducible system was utilized to express SKD1E235Q [26], because constitutive expression of SKD1E235Q is toxic for 293A cells, in which recombinant adenoviruses are grown Twenty-four hours after infection with adenoviruses bearing mutant DNase I at an MOI of

300, cells were washed and coinfected with adenoviruses bearing SKD1E235Q(AxCALSKDEQ) at an MOI of 0, 500,

1000, or 2000, and Cre recombinase (AxCANCre) at an MOI of 150 After incubation for 12 h, the medium con-taining adenoviruses was replaced with 1 mL of MEM containing 10% dialyzed fetal bovine serum Following an additional incubation for 12 h, the glycosylation analysis was performed as described above

Fluorescence microscopy

PMI-null cells were grown in eight-well chamber slides (Nalge Nunc International, Rochester, NY, USA) and stimu-lated to overexpress SKD1E235Q by coinfection with AxCALSKDEQat an MOI of 0, 1000, or 2000, and AxCAN-Cre at an MOI of 150 After 24 h of incubation, the cells were washed twice with NaCl⁄ Piand fixed with 4% parafor-maldehyde in NaCl⁄ Pi for 15 min The cells were then analyzed by fluorescence microscopy using a Zeiss Axiovert

200 (Carl Zeiss Inc., Thornwood, NY, USA)

Measurement condition in serum-free media

TCH is a serum replacement product containing 0.6 mgÆmL)1protein and no sugars Cells were preincubated for 12 h at 37C in 1 mL of MEM supplemented with 1% BSA or 2% TCH Metabolic labeling was then performed in MEM supplemented with 1% BSA or 2% TCH, respectively

Prolonged incubation with mannose-free media

After incubation for 0, 12, 24, 36 or 48 h in MEM supple-mented with 10% dialyzed fetal bovine serum, the glycosyl-ation efficiency of DNase I and total cell protein were analyzed In this experiment, medium was replaced every

4 h with fresh MEM supplemented with 10% dialyzed fetal bovine serum For measurement of glycosylation of total cell protein, collected cells were lysed by 8 m urea contain-ing 2% Chaps, and then each lysate was subjected to SDS⁄ PAGE using 12% polyacrylamide gel First, glycopro-teins were stained with Pro-Q Emerald 300, and second,

total proteins were stained with the SYPRO Ruby protein

gel stain kit according to the manufacturer’s instructions

Using a Lumino LAS-3000 imaging analyzer and multi

gaugev2.1 software (Fuji Film), the stained proteins were

imaged

Analysis of LLOs

Approximately 1.0· 107

harvested cells were suspended

in methanol and dried under N2 Afterwards, LLOs

were extracted as previously described [32] Briefly, LLOs

were extracted in chloroform⁄ methanol ⁄ water (CMW;

10 : 10 : 3), and the materials in the CMW extract were

treated with weak acid to generate soluble

oligosaccha-rides The hydrolysates were then loaded onto a C18

SepPak column directly connected to a 3 mL ENVI-CARB

solid-phase extraction tube to remove residual salt and

lip-ids, as previously described [33] After loading of the

sam-ple, the columns were washed with 9 mL of 2%

acetonitrile⁄ 0.1 m ammonium acetate in H2O For elution

of the oligosaccharides, the C18 SepPak column was

removed and the oligosaccharides were eluted from the

ENVI-CARB tube with 6 mL of H2O⁄ acetonitrile (3 : 1,

v⁄ v) Then, the dried samples were labeled with

2-amino-pyridine for HPLC analysis Pyridylamination was

per-formed as described previously [34] Pyridylaminated

oligosaccharides were further purified with a Cellulose

Cartridge Glycan preparation kit (Takara Bio Inc., Shiga,

Japan), and separated by HPLC using an Asahipak NH2

P-50 4D column (1P-50· 4.6 mm; Shodex, Showa Denko KK,

Tokyo, Japan) Solvent A was 97% acetonitrile adjusted to

pH 7.0 with 0.3% acetic acid Solvent B was 10%

acetoni-trile adjusted to pH 7.0 with 0.3% acetic acid Gradient

conditions were a linear gradient of 70% solvent A and

30% solvent B to 40% solvent A and 60% solvent B over

20 min at a flow rate of 0.8 mLÆmin)1, followed by 5 min

of 40% solvent A and 60% solvent B to 100% solvent B

at the same flow rate Elution was monitored by

fluores-cence (excitation wavelength, 320 nm; emission wavelength,

400 nm) Each peak was identified by comparison with a

standard pyridylaminated oligosaccharide elution time

Acknowledgements

We express our gratitude to Dr Tamotsu Yoshimori

for providing adenoviruses bearing Cre-recombinase

and GFP–SKD1E235, to Dr Sumihiro Hase for

pro-viding standard pyridylaminated oligosaccharides, and

to Mr Kazuyuki Iimura for technical assistance To

Dr Nobuhiro Takahashi for permission to use his

laboratory for fluorescence microscopy This work

was supported by CREST of JST, the National

Insti-tutes of Health grant R01 DK55695, and the Rocket

Williams Fund (HHF)

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