The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells Naonobu Fujita 1 , Ayako Tamura 1 , Aya Higashidani 1 , Takashi Tonozuka 1 , Hudson H. Freeze 2 and Atsushi Nishikawa 1 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-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 A 1 , 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. 788 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 b 1 . Fibroblasts derived from CDG patients were infected by adenovirus expressing mutant DNase I and labeled metabolically with [ 35 S]Met and [ 35 S]Cys. After harvesting of the condi- tioned medium for the DNase I glycosylation assay, the cells were lysed and integrin b 1 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 b 1 , however, as integrin b 1 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 N. Fujita et al. Intracellular mannose salvage pathways FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 789 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. 42 F Ic Ie Ig Ii 1 8 4 2 5 0 70 (%) 72 Huh6 A549 HeLa COS I 82 62 88 78 (%) 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 glycosylated 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 b 1 derived from several CDG type I cells. 35 S-labeled cells obtained from above experiment were lysed (100 m M Tris ⁄ HCl, 150 mM NaCl, 1% NP-40 buffer, pH 8.0, con- taining Roche protease inhibitor cocktail) and immunoprecipitated using monoclonal antibody to integrin b 1 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 W T PMI-n u ll Glycosylation efficiency (%) C D G -I b Control 0 20 40 60 80 100 Control SW Baf. A1 ] ] ] Fig. 2. Effect of swainsonine and bafilomycin A 1 on 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 nM bafilomycin A 1 (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. Intracellular mannose salvage pathways N. Fujita et al. 790 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 A 1 , 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-mannosidase II [21]. Bafilomycin A 1 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 A 1 block the mannose salvage pathways, then supplemental mannose should be sufficient to correct the glycosylation deficiency in swainsonine-treated or bafilomycin A 1 -treated PMI- null cells. We thus performed mannose titration experiments in swainsonine-treated and bafilomycin A 1 -treated PMI-null cells. Cells were treated with 10 lm swainsonine or 100 nm bafilomycin A 1 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 A 1 -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 A 1 -treated PMI-null cells, respectively. From these results, we conclude that the reduction in glycosylation efficiency in swainso- nine-treated and bafilomycin A 1 -treated PMI-null cells is caused by blockade of the mannose salvage path- ways and not of the glycoprotein maturation steps. Overexpression of SKD1 E235Q strongly 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 SKD1 E235Q , 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)–SKD1 E235Q [26]. When the glycosyla- tion assay was performed in GFP–SKD1 E235Q -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 0 5 10 15 20 Mannose conc. (µ M) Glycosylation efficiency (%) 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 nM bafilomycin A 1 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. N. Fujita et al. Intracellular mannose salvage pathways FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 791 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 endogenous 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 W T PMI n u ll MOI = 0 MOI = 1000 MOI = 2000 SKD1 E 23 5 Q MOI = 1000 SKD1 E 23 5 Q MOI = 2000 MOI = 0 DIC Fluorescence A B Glycosylation efficiency (%) Fig. 4. Overexpression of SKD1 E235Q inhib- ited mannose salvage pathways. To gener- ate the overexpression of SKD1 E235Q , 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%. Intracellular mannose salvage pathways N. Fujita et al. 792 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS MEM+ 20% FBS MEM + 10% dialyzed FBS 012243648 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 (%) Glycosylation efficiency (%) 0 0 1020304050 20 40 60 80 100 Pre-incubation time (h) Fluorescence Retention time (min) 04 812 16 0 h 24 h 48 h G0 1 2 3 Glycoprotein Total protein G. R. 1 0.94 0.64 (Gl y co p rotein/Total p rotein) F. I. 100 87 56 % 100 93 87 % M0 24 48 h M0 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. N. Fujita et al. Intracellular mannose salvage pathways FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 793 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 Glucose Mannose GDP- -6-P -6-P -1-P Fru-6-P Lysosome PMI 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). Intracellular mannose salvage pathways N. Fujita et al. 794 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 A 1 treatment, 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 A 1 . Swainsonine inhibits both lyso- somal and nonlysosomal mannosidases, whereas bafilo- mycin A 1 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 b 1 (SG ⁄ 19) were from Seikagaku Cor- poration (Tokyo, Japan), bafilomycin A 1 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 [ 35 S]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 C 18 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 Lipofectamine 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 (TICD 50 ) 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 · 10 5 cells were plated into six-well plates and incubated at 37 °C for 16 h in a CO 2 incubator. 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 N. Fujita et al. Intracellular mannose salvage pathways FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 795 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 ⁄ P i and incu- bated for 4 h at 37 °C with 0.7 mL of MEM without Met ⁄ Cys but supplemented with 10% dialyzed fetal bovine serum and 2.65 MBq of [ 35 S]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 using 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 37 °C for 30 min. Following incubation, the samples were subjected to SDS ⁄ PAGE. Swainsonine and bafilomycin A 1 treatment Cells were treated with 10 lm swainsonine, 100 nm bafilo- mycin A 1 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 A 1 ,or20lm 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 A 1 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 A 1 and the indicated concentrations of mannose. The glycosylation analysis was then performed as described above. GFP–SKD1 E235Q overexpression A Cre ⁄ loxP inducible system was utilized to express SKD1 E235Q [26], because constitutive expression of SKD1 E235Q 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 SKD1 E235Q (AxCALSKD EQ ) 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 SKD1 E235Q by coinfection with AxCALSKD EQ at 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 ⁄ P i and fixed with 4% parafor- maldehyde in NaCl ⁄ P i 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 )1 protein and no sugars. Cells were preincubated for 12 h at 37 °C 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, Intracellular mannose salvage pathways N. Fujita et al. 796 FEBS Journal 275 (2008) 788–798 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 gauge v2.1 software (Fuji Film), the stained proteins were imaged. Analysis of LLOs Approximately 1.0 · 10 7 harvested cells were suspended in methanol and dried under N 2 . 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 C 18 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 H 2 O. For elution of the oligosaccharides, the C 18 SepPak column was removed and the oligosaccharides were eluted from the ENVI-CARB tube with 6 mL of H 2 O ⁄ 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 NH 2 P- 50 4D column (150 · 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–SKD1 E235 , 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). References 1 Kornfeld R & Kornfeld S (1985) Assembly of aspara- gine-linked oligosaccharides. Annu Rev Biochem 54, 631–664. 2 Menon AK (1994) Structural analysis of glycosylphos- phatidylinositol anchors. Methods Enzymol 230, 418– 442. 3 Varki A (1991) Radioactive tracer techniques in the sequencing of glycoprotein oligosaccharides. FASEB J 5, 226–235. 4 Varki A (1994) Metabolic radiolabeling of glycoconju- gates. 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Miwako I, Ohashi M, Ohsumi M & Ohsumi Y (2000) The mouse SKD1, a homologue 798 26 27 28 29 30 31 32 33 34 of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells Mol Biol Cell 11, 747–763 Nara A, Mizushima N, Yamamoto A, Kabeya Y, Ohsumi Y & Yoshimori T (2002) SKD1 AAA ATPasedependent endosomal transport is involved in autolysosome formation Cell Struct Funct 27, 29–37 . 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. 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. 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,