3Department of Biochemistry, Osaka University Medical School, Osaka 565-0871, Japan, 4Department of Internal Medicine and Molecular Science, Osaka University Medical School, Osaka 565-0871, Japan, 5Osaka International University for Women, Osaka 570-0014, Japan, 6Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan, and 7Division of Human Genetics, Childrens Hospital Research Foundation, Cincinnati, OH 45229-3039, USA
Received on July 25, 2000; revised on September 29, 2000; accepted on September 29, 2000.
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Abstract |
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Key words: carbohydrate function/fucosyltransferase/glycosyltransferase/N-glycan/transgenic mice
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Introduction |
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GDP-L-Fuc:N-acetyl-ß-D-glucosaminide 1,6 fucosyltransferase (
1,6 FucT) catalyzes the transfer of a fucose residue from GDP-fucose to the position 6 of the innermost GlcNAc residue of N-glycans and is involved in the biosynthesis of hybrid and complex types of N-linked oligosaccharides in glycoproteins. The reaction products of this enzyme,
1,6 fucosylated oligosaccharides, are widely distributed in mammalian tissues. It is generally believed that
1,6 fucosylation plays an important role in fetal development (Bakkers et al., 1997
). Under some pathological conditions, the expression of
1,6 FucT and the extent of fucosylation are altered. For example, the level of
1,6 FucT is elevated in both liver and serum during the process of hepatocarcinogenesis (Hutchinson et al., 1991
). The presence of fucosylated
-fetoprotein is a good marker for distinguishing patients with hepatocarcinoma from those with chronic hepatitis and liver cirrhosis (Sato et al., 1993
; Taketa et al., 1993
).
Followed by the development of convenient assay method for the enzyme activity (Uozumi et al., 1996),
1,6 FucT was homogeneously purified, and its cDNA was cloned from porcine brain and human gastric tumor cells in our laboratory (Uozumi et al., 1996
; Yanagidani et al., 1997
). The
1,6 FucT gene was found to be expressed in most rat organs (Miyoshi et al., 1997
). A relatively high level of expression was observed in brain and small intestine, but only trace levels were found in liver. The molecular cloning of the
1,6 FucT gene enabled us to manipulate the gene and to remodel the N-linked glycans in individual cells and some animal models. We previously produced transgenic mice that overexpressed the N-acetylglucosaminyltransferase III (GnT III) genes, in an attempt to elucidate the biological roles of the bisecting GlcNAc in N-linked glycans (Ihara et al., 1998
). The N-linked glycans that were attached to apolipoprotein B in the liver of GnT III transgenic mice underwent a change, and the mice developed a fatty liver due to aberrant apolipoprotein B secretion. In the present paper, we report on a study of transgenic mice that overexpress human
1,6 FucT gene, in an attempt to study the biological roles of the core
1,6 fucose residue in N-linked glycans. The
1,6 FucT transgenic mice showed a unique phenotype of steatosis.
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Results |
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Changes of N-linked glycans in 1,6 FucT transgenic mice
To determine the manner in which the glycans of proteins are fucosylated in the 1,6 FucT transgenic mice, a liver extract was subjected to aleuria aurantia lectin (AAL) blot analysis to detect the
1,6 fucose in N-linked glycans (Fukumori et al., 1989
). When whole homogenates were used, the observed difference between the transgenic mice and wild mice was very slight. However, significant changes were found after isolation of the light mitochondrial and microsomal fractions (Figure 2A). When the light mitochondrial fraction was subjected to two-dimensional electrophoresis and stained with AAL, additional specific bands with
1,6 fucose structures were detected (Figure 2B). Immunoblot with a monoclonal antibody CAB4, which recognizes the
1,6 fucose structure in the core of N-glycans (Srikrishna et al., 1997
), showed patterns similar to the AAL blots (data not shown). AAL blots of serum and kidney proteins also showed additional components and stronger signals in the
1,6 FucT transgenic mice compared to the control mice (data not shown). These observations indicate that the introduced
1,6 FucT in fact catalyzes the addition of fucose residues to the core of N-linked oligosaccharides of a large number of glycoproteins in liver and kidney.
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Accumulated lipids in 1,6 FucT transgenic mice
To determine which step is damaged in the transgenic mice, we attempted to characterize the accumulated lipids. Triglycerides, total cholesterol, and free fatty acid levels were found to be increased in the liver of the transgenic mice (Table I). Furthermore, thin-layer chromatography revealed that the amount of triglyceride and cholesterol ester increased significantly in the transgenic mice, compared with their wild littermates (Figure 7). These findings indicate that triglycerides and cholesterol ester had accumulated in the transgenic mice. A similar storage pattern of triglycerides and cholesterol ester was observed in the kidney (data not shown).
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Discussion |
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We previously reported on the ectopic overexpression of GnT III, which resulted in the addition of the bisecting GlcNAc, which regulates the branching of N-glycans, disrupting apolipoprotein B secretion (Ihara et al., 1998). Unlike the GnT III transgenic mice, no significant changes in serum lipids in
1,6 FucT transgenic mice were observed, suggesting that no problem exists in terms of the secretion of lipids out of the hepatocytes. The electron microscopic observation revealed that lipid droplets had accumulated in the lysosomes in hepatocytes and renal tubular cells of
1,6 FucT transgenic mice. These data, along with that on the accumulation of cholesterol ester and triglyceride, suggest that the hydrolysis of lipid esters in the lysosomes is abrogated in the transgenic mice.
Lysosomes are important organelles involved in lipid metabolism (Lusa et al., 1998). Triglycerides and cholesterol ester carried by VLDL and low-density lipoproteins (LDLs) are endocytosed into cells via LDL receptors. The endosomes join with primary lysosomes to become secondary lysosomes, in which triglycerides and cholesterol ester are hydrolyzed by hydrolases. If the balance of load and degradation is disturbed, these lipids may accumulate in the lysosomes and finally form membrane-bound lipid droplets (Lough et al., 1970
). Lysosomes with accumulated lipids, called lipolysosomes, are regarded as a specific feature of Wolmans disease (Lough et al., 1970
). Lipolysosomes can be occasionally found in some other liver disorders (Hayashi et al., 1977
), but the ratios of membrane-bound to naked lipid droplets were <3.1%, which is much less than in Wolmans disease and CESD (Hayashi et al., 1983
). Wolmans disease is an autosomal recessive disorder with an inherited deficiency of LAL (Anderson et al., 1994
; Pagani et al., 1998
). LAL catalyzes the hydrolysis of cholesterol ester and triglycerides in the lysosomes. In the case of the
1,6 FucT transgenic mice, cholesterol ester and triglycerides had accumulated and LAL activity was significantly reduced, suggesting that a reduced level of hydrolysis is at least partly responsible for the accumulation of such lipids.
Mouse and human LALs contain five conserved potential N-linked glycosylation sites. The potential role of glycosylation of LAL in the formation or maintenance of a catalytically active enzyme has been a controversial issue. Some investigators have suggested that glycosylation might not be essential for catalytic function by demonstrating that enzyme activity, after treatment with endoglycosidase H, was unchanged (Sando and Rosenbaum, 1985; Ameis et al., 1994
). Others have concluded that glycosylation is important by showing that the activity is reduced, as the result of the same treatment (Pariyarath et al., 1996
) and that tunicamycin treatment led to the production of inactive LAL and that an active form of LAL could not be expressed in a bacterial system (Sheriff et al., 1995
). In the present study, we found that the specific activity of LAL was greatly reduced when LAL became highly fucosylated via the introduction of the
1,6 FucT gene. This finding supports the conclusion that the glycosylation of LAL regulates its activity.
Organs affected in Wolmans disease include mainly liver, spleen, intestine, and the adrenal gland. Unlike Wolmans disease, lipid accumulation is confined to hepatocytes and proximal renal tubular cells in the 1,6 FucT transgenic mice. This may reflect the high expression of the transgene in liver and kidney (Figure 1). The deficient state of LAL is expressed in two major phenotypes in the clinic (Yoshida and Kuriyama, 1990
; Nakagawa et al., 1995
). One is Wolmans disease and the other is designated CESD, in which only cholesteryl esters are stored (Sloan and Frederickson, 1972
; Pagani et al., 1998
). Wolmans disease is the more severe form; it is nearly always fatal in the first year of life. CESD is more benign; these patients may survive to adulthood. The molecular basis of the different phenotypes is actually not yet clear and may be due to residual enzyme activity (Anderson et al., 1994
; Pagani et al., 1998
). The LAL-knockout mice share many features of Wolmans disease but have a milder phenotype and are fertile, although they undergo massive cholesterol ester and triglyceride storage with complete loss of LAL activity (Du et al., 1998
).
We also found lipid accumulation within lysosomes in the proximal renal tubular cells of our 1,6 FucT transgenic mice. It is unique that the lipid vacuoles are mainly located in the basolateral compartments of the epithelial cells. No previous study has been found that describes this type of lipid accumulation. According to the few available reports on the ultrastructure of kidney of patients with microvesicular fatty liver, lipid accumulation in kidney proximal tubular cells may occasionally be found but in different manners (Slater and Hague, 1984
; Jung et al., 1993
). In several experimental animal models of liver steatosis (Fan et al., 1996
; Shimano et al., 1996
; Reue and Doolittle, 1996
; Hashimoto et al., 1999
) no accumulation of lipid in the lysosomes of renal tubules has been reported. The basis for lipid accumulation in the lysosomes in the basolateral compartments of the epithelial cells might be due to the fact that the nutrition of epithelial cells of the proximal tubule is derived from outside of the basal membrane, where the lipids within VLDL and LDL were endocytosed via the receptors and then fused with nearby lysosomes. Because of the deficiency of hydrolysis, the esterified lipids accumulated in this location. The lysosomes in the apical compartments might be less responsible for lipid metabolism and therefore be less affected.
A number of proteins may be involved in the lysosomal transport and digestion of triglycerides and cholesterol ester. For instance, LAL-inhibitory proteins have been reported (Kubo et al., 1981; Gorin et al., 1982
), and a physiological detergent, such as saposins (Bierfreund et al., 2000
), could help LAL digest those lipids. Little is known about how the degradation products of lipid hydrolysis exit the lysosomes. Any dysfunction in these processes may lead to an accumulation of lipids in the organelle. Because the lipid storage that is actually observed by microscopic analysis appears to be more severe than that expected by the reduction of LAL activity, other factor(s) in these processes may be blocked in
1,6 FucT transgenic mice. Alternatively, the accumulated inactive LAL may have a dominant negative effect on the hydrolysis of lipids in the lysosomes.
In conclusion, we report the development of an experimental model mouse with steatosis in the liver and kidney by ectopic expression of 1,6 FucT. A novel mechanism for lipid storage due to down-regulation of lysosomal acid lipase activity by remodeling of its glycosylation is proposed.
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Materials and methods |
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Mice
DNA was extracted from tails of mice developed from the above-mentioned fertilized eggs and analyzed by Southern blotting for the incorporation of human 1,6 FucT cDNA. Six out of 30 mice were found to be positive and were mated with C57BL/6 mice. Northern blot analysis of RNAs from the tail, liver, and kidney and lectin blots of serum proteins were carried out in order to detect the expression of the trans-
1,6 FucT cDNA. Two mouse lines with high levels of expression of
1,6 FucT were established. These animals were maintained in 1212 h light-dark cycles (light from 8 AM to 8 PM) and fed with a chow diet (Oriental Corp, Osaka), which contained 75 mg/kg cholesterol and 3.7 g/kg fat.
Lectin and immunoblotting
Biotin-labeled AAL was obtained from the Honen Corp (Japan). Affinity purified rabbit anti-human LAL IgG, which cross-reacts with mouse LAL, was used for the detection and immunoprecipitation of mouse LAL (Du et al., 1996). The CAB4 monoclonal antibody that recognizes the
1,6 fucose residue in the core of N-linked glycans was kindly provided by Dr. Freeze (Srikrishna et al., 1997
). A monoclonal antibody 15C6 against human
1,6 FucT was obtained from Fujirebio Inc. (Japan). Goat polyclonal anti-human cathepsin D antibodies were prepared in our laboratory. This antibody cross-reacts with mouse cathepsin D. Proteins from serum, liver, or other organs were subjected to SDSPAGE and transferred to PVDF membranes. Western blots and lectin blots were carried out as described previously (Miyoshi et al., 1997
; Ihara et al., 1998
).
1,6 FucT activity assay
1,6 FucT activity was assayed by the method of Uozumi et al. (1996)
. Briefly, cell homogenates were mixed with the assay buffer in a total volume of 15 µl, containing 1020 µg protein, 200 mM MES, pH 6.2, 1% Triton X-100, 500 µM GDP-fucose, and 5 µM
1,6 FucT acceptor. After 1 h of incubation at 37°C, the mixture was boiled for 3 min and centrifuged at high speed for 10 min. Ten microliters of the supernatant were subjected to HPLC. Activity was expressed as pmols of GDP-fucose transferred to the acceptor per h per mg protein.
Preparation of tissue homogenates and subcellular fractionation
The liver from each mouse was perfused through the portal vein with an ice-cold sucrose medium (0.25 M sucrose in 10 mM TrisCl buffer, pH 7.4, and 1 mM EDTA) and homogenized in 10 vol of the ice-cold sucrose medium using a Potter-Elvehjem-type homogenizer with six strokes of a loose-fitting Teflon pestle. Subcellular fractions were separated by differential centrifugation using OptiprepTM (Nycomed Amersham, Norway) according to the manufacturers instruction. Marker enzymes of each organelle were used for identification of the fractions. Protein concentration was determined with a BCA protein assay kit (Pierce).
Analysis of lipids
Total lipids were extracted with 10 vol of chloroform/methanol (2:1, v/v). After the solvent was evaporated, the residue was dissolved in either a minimum vol of chloroform/methanol (2:1, v/v) for thin-layer chromatography or 1% Triton X-100 for the determination of total cholesterol, triglycerides, and free fatty acids. For the separation of lipids, the samples were applied to a thin-layer plate (10 x 10 cm, silica gel 60, Merck, Germany) and developed with hexane/ether/formic acid (80:20:2 v/v/v). After drying, the plate was submerged in a solution containing 3% copper acetate and 8% phosphoric acid for 5 min and then baked at 200°C for visualization of lipids. For the determination of total cholesterol, triglycerides, and free fatty acids, Monotest kit (Boehringer Mannheim, Germany), TG I kit (Wako, Japan), and NEFA IC kit (Wako, Japan) were used, respectively.
Analysis of serum lipoproteins
Fresh mouse serum in sample buffer was loaded onto a MultiGel-Lipo ready-made acrylamide gel (Daiichi Pure Chemicals Co., Ltd., Japan) for electrophoresis according to the manufacturers recommended protocol. The gel was stained with Sudan black to reveal VLDL and HDL components.
Histochemical examination
Fresh tissues were fixed in a 10% formaldehyde in 0.1 M phosphate buffer (pH 7.4). Paraffin sections and frozen sections were prepared for hematoxylin-eosin staining and for Oil red O or Sudan III staining to reveal neutral lipids, respectively.
Electron microscopic observation
Anesthetized mice were perfused via the left ventricle with a 3% glutaraldehyde solution buffered at pH 7.4 with 0.1 M Millonigs phosphate buffer. The liver and kidney were excised as described previously (Miyagawa et al., 1995). Briefly, the liver and kidney were cut into small pieces and fixed in the same fixative for 2 h at 4°C. After a secondary fixation with 1% osmium tetroxide buffered at pH 7.4 with 0.1 M Millonigs phosphate buffer for 1 h at 4°C, specimens were dehydrated and embedded in Epon (epoxy resin). Ultra-thin sections, cut on a Reichert-Jung Ultracut E ultramicrotome, were doubly stained with aqueous uranyl acetate (3.0%) and Reynoldss lead citrate and then subjected to electron microscopy using a Hitachi H-7000 apparatus.
Lysosomal enzyme assays
LAL activity was assayed using cholesterol-[1-14C]-oleate (American Radiolabled Chemicals, Inc., USA) as described previously (Ishii et al., 1995; Merkel et al., 1999
) with slight modifications. First, a substrate stock solution was made by mixing 0.57 ml of nonradioactive cholesteryl-oleate (10 mg/ml in hexane) with 50 µCi of cholesteryl-[1-14C]-oleate and adding hexane to 2 ml. For 20 reactions, 100 µl of substrate stock solution was mixed with 100 µl of 18.4 mg/ml lysolecithin in chloroform/methanol (1:1, v/v). After the solution was dried under a stream of nitrogen, 0.8 ml 0.9% NaCl was added, and the resulting mixture was sonicated for 10 min in an ice-water bath. For assays, a 40 µl aliquot of this substrate was mixed with 0.11.0 mg of protein and a solution containing 100 mM sodium acetate (pH 5.0) and 1% Triton X-100 in a final volume of 200 µl. The reaction mixture was incubated at 37°C for 3060 min, and the reaction was stopped by adding 3.25 ml of chloroform/methanol/heptane (1.42:1.25:1.00, v/v/v), followed by vortexing for 10 s. One milliliter of 1 N NaOH was then added, and the samples were vortexed for 30 s. After centrifugation for 10 min at 1000 x g, 1 ml of the upper layer was transferred to a vial, mixed with 5 ml of scintillation fluid, and counted for radioactivity with a liquid scintillation counter. Activity was expressed as pmol of free fatty acid released by 1 mg of protein per h.
-Fucosidase activity was assayed as described previously (Lovell et al., 1994
; Prasad and Pullarkat, 1996
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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