American Journal of Medical Genetics 42:586-592 (1992)
Evidence for Processing of Dolichol-Linked Oligosaccharides in Patients With Neuronal Ceroid-Lipofuscinosis Peter F. Daniel, Derrick L. Sauls, and Rose-Mary N. Boustany Department of Biochemistry, Eunice Kennedy Shriver Center for Mental Retardation, Waltham and Department of Neurology, Harvard Medical School and Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts
In agreement with reports from other laboratories, we have shown that patients with the juvenile or late infantile forms of neuronal ceroid-lipofuscinosis (NCL) have greatly increased levels (5-fold to 20-fold) of dolichyl pyrophosphoryl oligosaccharides in their cerebral gray matter. Oligosaccharidescontaining 2 GlcNAc residues and 3 to 9 mannose residues were liberated by mild acid hydrolysis. The oligosaccharide profile given by brain tissue from 2 patients with infantile NCL was markedly different from that of late infantile and juvenile NCL brain, with Man,GlcNAc, as the most abundant component and decreasing amounts of Man,- Man,and MaQGlcNAc,. By contrast, Man,GlcNAc, was the most abundant oligosaccharide present in all juvenile NCL brain samples analyzed. Both the susceptibility of the isolated Man,GlcNAc, to endoglucosaminidase H digestion and permethylation analysis clearly indicated that it is not an intermediate in the biosynthesis of Glc,MmGlcNAc,-PP-dolichol but has undergone catabolism, probably either in the endoplasmic reticulum or in the Golgi apparatus. Treatment of cultured skin fibroblasts for 7 days with N-methyldeoxynojirimycin, a potent inhibitor of the endoplasmic reticulum processing enzymes glucosidase I and 11, resulted in an accumulation of the same Man,GlcNAc,-PP-dolichol species that was elevated in juvenile NCL brain. The level in untreated fibroblasts was undetectable, suggesting that inhibition of processing glu-
cosidases has interfered with the regulation and compartmentalization of lipid-linked oligosaccharides.
KEY WORDS: dolichyl pyrophosphoryl oligosaccharides, Batten disease, fibroblasts, brain, Nmethyldeoxynojirimycin, glucosidase-I, oligosaccharide processing
INTRODUCTION A significant elevation of dolichyl phosphate in brains from English setter dogs homozygous for NCL, and in brain from a late infantile (LINCL) and a juvenile (JNCL) patient, was first reported by Keller et al. [19841. Elevated dolichyl phosphate was subsequently confirmed in brain, liver, muscle, and pancreas from a number of patients with LINCL and JNCL by Hall and Patrick [1985, 19871. This dolichyl phosphate has now been shown to be produced from dolichyl pyrophosphoryl oligosaccharide (Dol-PP-OS) by the alkaline saponification step used as a part of the isolation procedure [Hall and Patrick, 19881. We have confirmed that there is a marked elevation in Dol-PP-OS species in human NCL brain tissue [Daniel e t al., 19891 and so has Pullarkat et al. [19881. Recently, Hall et al. [19891 have reported very high concentrations of Dol-PP-OS in storage cytosomes isolated from brain tissue of a sheep model of ceroid-lipofuscinosis. The regulation of the amount of Dol-PP-OS available for N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is imperfectly understood. Spiro and Spiro [19861 have shown that N-linked glycan synthesis in thyroid ER is controlled by membrane organiReceived for publication April 5,1991; revision received July 25, zation and by availability of dolichyl phosphate. Cacan 1991. et al. [19871 have demonstrated that there is further Address reprint requests to Dr. P. F. Daniel, Department of Biochemistry, E. K. Shriver Center, 200 "rapelo Road, Waltham, control at the level of the completed Dol-PP-OS, which is regulated by the activity of a Dol-PP-OS specific pyMA 02254. Rose-Mary N. Boustany is now at Department of Pediatric Neu- rophosphatase that produces dolichyl phosphate and phospho-oligosaccharide. The phospho-oligosaccharide rology, Duke University Medical Center, Durham, NC 27710. 0 1992 Wiley-Liss, Inc.
Abnormal Dolichol Metabolism in Batten Disease is rapidly dephosphorylated and deglucosylated and then undergoes endoglucosaminidase cleavage and stepwise removal of mannose residues, after translocation to a cytoplasmic compartment [Cacan et al., 19891. Normal levels of this pyrophosphatase have been demonstrated in LINCL and JNCL fibroblasts [Daniel et al., 19881, suggesting that it is unlikely that the elevated lipid-linked oligosaccharidesobserved in patients’ brain tissue is due to a genetic deficiency of this enzyme. Regardless of whether this elevation in brain from patients with NCL is due to a primary defect or is merely a secondary phenomenon, the increase is real and reproducible; it is found not only in human patients but also in sheep and dogs with NCL. Such lipid-linked oligosaccharides are thus worthy of study for any insight they may shed on the various forms of NCL in particular, and on factors regulating glycoprotein biosynthesis in general.
587
Labelled oligosaccharides were analyzed on an amino column (4.6 x 250 mm; Supelco, Inc., Bellefonte, PA) in acetonitrile/water at a flow rate of 1 ml/min. Oligosaccharides were eluted with a linear gradient of acetonitrile/water changing from 65 :35 to 45 :55 over 65 min. Separation of oligosaccharides was monitored by continuous flow detection using a Flo-One f3 radioactivity flow detector (Radiomatic Instruments, Tampa, FL). Permethylation Analysis Man,GlcNAc was isolated by semi-preparative HPLC on a 5 p,m C8 column (10 x 250 mm; Rainin Instruments) at a flow rate of 4 ml/min. Because preparative columns do not possess as great a resolving power as 3 p,m analytical columns, a second, isocratic separation on the preparative column was necessary to achieve sufficient purity for structural characterization (>95% pure). Purified Man,GlcNAc was permethylated by the use of solid sodium hydroxide in dimethyl sulphoxide by the method of Gunnarson 119871, purified by partition chromatography on a C18 Bond Elut cartridge (Analytichem International, Harbor City, CA), and then hydrolysed in 2 M aqueous trifluoroacetic acid and converted to permethylated alditol acetates [Waeghe et al., 19831. Permethylated alditol acetates were analyzed by capillary GC-MS with ammonia chemical ionization to increase sensitivity and specificity [Geyer et al., 19821.
MATERIALS AND METHODS Isolation of Dolichyl Oligosaccharides From Brain Dol-PP-OSwere isolated from up to 1 g of brain tissue by the procedure of Hall and Patrick [19881. Dissected cerebral cortex grey matter was homogenized in distilled water (0.5 g tissue/ml) using a Polytron homogenizer. The homogenate was first delipidated by extraction twice with 2 volumes of butanol/diisopropyl ether Metabolic Labelling Studies (2 :3 vlv). Chloroform (3 vol) and methanol (1.5vol) were then added to the lower phase yielding two phases after Cultured fibroblasts from JNCL and LINCL patients centrifugation. The upper phase was discarded, and the and controls (1-4 x lo6 cells/ml) were metabolically lower phase was washed twice with methanollwater labelled with 3.7 MBq/ml of [2-3Hlmannose(NEN Re(1 : 1 v/v; 1.5 vol) and then mixed with methanol (1.3vol) search Products, Boston, MA) for 1 h in glucose-free and water (0.3 vol) to produce a single phase solvent of DMEM medium, as described by Cacan et al. [19871. compositionchlorofordmethanol/water (10 : 10 : 3). Two Cells were pre-treated with either N-methyldeoxynofurther extracts of the tissue residue with 2.5 vol of jirimycin or mannojirimycin in complete DMEM me10 : 10 :3were combined with the initial 10 :10 :3 extract dium containing 15%fetal calf serum for either 1h or for and concentrated by nitrogen evaporation. 7 days. Inhibitors were purchased from Genzyme Corporation (Boston, MA). Analysis by HPLC After incubation, the cells were pelleted and sequenOligosaccharides were quantitatively released from tial lipid extraction was performed as described by Cathe 10 : 10 :3 extract by mild acid hydrolysis as described can et al. [19871. The soluble oligosaccharides released by Chalifour and Spiro [ 19841. Water-soluble material into the aqueous phase were separated from precursor was deionized on coupled columns of Dowex 50 (H + 1and mannose by gel filtration on Bio-Gel P-2. Radioactivity Dowex 1 (acetate) in a pasteur pipette and then digested in the chlorofodmethanol fraction (Man-P-Dol),in the with endoglucosaminidase H (Endo H; Genzyme Corpo- chlorofordmethanol/water 10: 10 :3 fraction (Dol-PPration, Boston, MA). The Endo H digested oligosac- OS), and in the glycoprotein pellet was determined by charides were desalted on a column of Bio-Gel P-2, re- liquid scintillation counting. Analysis of the oligosacduced with sodium borohydride, perbenzoylated, and charide moiety from Dol-PP-OS was performed after analyzed by reversed phase HPLC on a 3 micron OC- mild acid hydrolysis (0.01N HC1, 100°C, 15 min) of the tylsilica column (3.2 x 100 mm; Rainin Instruments, chloroform/methanol/water fraction, followedby Endo H Woburn, MA), as described by Daniel [1987]. Oligosac- treatment overnight in 50 mM citrate buffer, pH 5.5, charides were eluted at a flow rate of 1 ml/min with a and gel filtration on Bio-Gel P-2. gradient of acetonitrile-water (80 :20) changing to pure RESULTS acetonitrile over 20 min. Oligosaccharideswere detected Quantification of Dolichyl Oligosaccharides in by their UV absorbance at 230 nm using an Applied Brain Biosystems model 757 variable wavelength detector and Patients with JNCL or LINCL have greatly increased chromatograms were plotted on a Spectra Physics SP4270 integratorlplotter. Oligosaccharides isolated levels (5-fold to 20-fold) of Dol-PP-OS in their cerebral from a sheep fed “locoweed” (Astragalus lentiginosus) gray matter (Table I). Diseased controls consisted of 2 Hurler, a Tay-Sachs, and a Fabry patient. Oligosacwere used as standards [Warren et al., 19881.
588
Daniel et al.
TABLE I. Measurement of Dolichyl-PP-Oligosaccharidesin Brain
Normal controls (N = 14) Diseased controls (N = 4) Infantile NCL ( N = 2 ) Late-infantile NCL (N = 4) Juvenile NCL (N = 7)
nmol/g tissue f S.D. 2.1 f 1.9 4.2 ? 3.9 13.7 10.8 f 5.4 41.8 13.2
*
9
A
Range
Evidence for Processing of Dolichol-Linked Oligosaccharides in Patients With Neuronal Ceroid-Lipofuscinosis Peter F. Daniel, Derrick L. Sauls, and Rose-Mary N. Boustany Department of Biochemistry, Eunice Kennedy Shriver Center for Mental Retardation, Waltham and Department of Neurology, Harvard Medical School and Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts
In agreement with reports from other laboratories, we have shown that patients with the juvenile or late infantile forms of neuronal ceroid-lipofuscinosis (NCL) have greatly increased levels (5-fold to 20-fold) of dolichyl pyrophosphoryl oligosaccharides in their cerebral gray matter. Oligosaccharidescontaining 2 GlcNAc residues and 3 to 9 mannose residues were liberated by mild acid hydrolysis. The oligosaccharide profile given by brain tissue from 2 patients with infantile NCL was markedly different from that of late infantile and juvenile NCL brain, with Man,GlcNAc, as the most abundant component and decreasing amounts of Man,- Man,and MaQGlcNAc,. By contrast, Man,GlcNAc, was the most abundant oligosaccharide present in all juvenile NCL brain samples analyzed. Both the susceptibility of the isolated Man,GlcNAc, to endoglucosaminidase H digestion and permethylation analysis clearly indicated that it is not an intermediate in the biosynthesis of Glc,MmGlcNAc,-PP-dolichol but has undergone catabolism, probably either in the endoplasmic reticulum or in the Golgi apparatus. Treatment of cultured skin fibroblasts for 7 days with N-methyldeoxynojirimycin, a potent inhibitor of the endoplasmic reticulum processing enzymes glucosidase I and 11, resulted in an accumulation of the same Man,GlcNAc,-PP-dolichol species that was elevated in juvenile NCL brain. The level in untreated fibroblasts was undetectable, suggesting that inhibition of processing glu-
cosidases has interfered with the regulation and compartmentalization of lipid-linked oligosaccharides.
KEY WORDS: dolichyl pyrophosphoryl oligosaccharides, Batten disease, fibroblasts, brain, Nmethyldeoxynojirimycin, glucosidase-I, oligosaccharide processing
INTRODUCTION A significant elevation of dolichyl phosphate in brains from English setter dogs homozygous for NCL, and in brain from a late infantile (LINCL) and a juvenile (JNCL) patient, was first reported by Keller et al. [19841. Elevated dolichyl phosphate was subsequently confirmed in brain, liver, muscle, and pancreas from a number of patients with LINCL and JNCL by Hall and Patrick [1985, 19871. This dolichyl phosphate has now been shown to be produced from dolichyl pyrophosphoryl oligosaccharide (Dol-PP-OS) by the alkaline saponification step used as a part of the isolation procedure [Hall and Patrick, 19881. We have confirmed that there is a marked elevation in Dol-PP-OS species in human NCL brain tissue [Daniel e t al., 19891 and so has Pullarkat et al. [19881. Recently, Hall et al. [19891 have reported very high concentrations of Dol-PP-OS in storage cytosomes isolated from brain tissue of a sheep model of ceroid-lipofuscinosis. The regulation of the amount of Dol-PP-OS available for N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is imperfectly understood. Spiro and Spiro [19861 have shown that N-linked glycan synthesis in thyroid ER is controlled by membrane organiReceived for publication April 5,1991; revision received July 25, zation and by availability of dolichyl phosphate. Cacan 1991. et al. [19871 have demonstrated that there is further Address reprint requests to Dr. P. F. Daniel, Department of Biochemistry, E. K. Shriver Center, 200 "rapelo Road, Waltham, control at the level of the completed Dol-PP-OS, which is regulated by the activity of a Dol-PP-OS specific pyMA 02254. Rose-Mary N. Boustany is now at Department of Pediatric Neu- rophosphatase that produces dolichyl phosphate and phospho-oligosaccharide. The phospho-oligosaccharide rology, Duke University Medical Center, Durham, NC 27710. 0 1992 Wiley-Liss, Inc.
Abnormal Dolichol Metabolism in Batten Disease is rapidly dephosphorylated and deglucosylated and then undergoes endoglucosaminidase cleavage and stepwise removal of mannose residues, after translocation to a cytoplasmic compartment [Cacan et al., 19891. Normal levels of this pyrophosphatase have been demonstrated in LINCL and JNCL fibroblasts [Daniel et al., 19881, suggesting that it is unlikely that the elevated lipid-linked oligosaccharidesobserved in patients’ brain tissue is due to a genetic deficiency of this enzyme. Regardless of whether this elevation in brain from patients with NCL is due to a primary defect or is merely a secondary phenomenon, the increase is real and reproducible; it is found not only in human patients but also in sheep and dogs with NCL. Such lipid-linked oligosaccharides are thus worthy of study for any insight they may shed on the various forms of NCL in particular, and on factors regulating glycoprotein biosynthesis in general.
587
Labelled oligosaccharides were analyzed on an amino column (4.6 x 250 mm; Supelco, Inc., Bellefonte, PA) in acetonitrile/water at a flow rate of 1 ml/min. Oligosaccharides were eluted with a linear gradient of acetonitrile/water changing from 65 :35 to 45 :55 over 65 min. Separation of oligosaccharides was monitored by continuous flow detection using a Flo-One f3 radioactivity flow detector (Radiomatic Instruments, Tampa, FL). Permethylation Analysis Man,GlcNAc was isolated by semi-preparative HPLC on a 5 p,m C8 column (10 x 250 mm; Rainin Instruments) at a flow rate of 4 ml/min. Because preparative columns do not possess as great a resolving power as 3 p,m analytical columns, a second, isocratic separation on the preparative column was necessary to achieve sufficient purity for structural characterization (>95% pure). Purified Man,GlcNAc was permethylated by the use of solid sodium hydroxide in dimethyl sulphoxide by the method of Gunnarson 119871, purified by partition chromatography on a C18 Bond Elut cartridge (Analytichem International, Harbor City, CA), and then hydrolysed in 2 M aqueous trifluoroacetic acid and converted to permethylated alditol acetates [Waeghe et al., 19831. Permethylated alditol acetates were analyzed by capillary GC-MS with ammonia chemical ionization to increase sensitivity and specificity [Geyer et al., 19821.
MATERIALS AND METHODS Isolation of Dolichyl Oligosaccharides From Brain Dol-PP-OSwere isolated from up to 1 g of brain tissue by the procedure of Hall and Patrick [19881. Dissected cerebral cortex grey matter was homogenized in distilled water (0.5 g tissue/ml) using a Polytron homogenizer. The homogenate was first delipidated by extraction twice with 2 volumes of butanol/diisopropyl ether Metabolic Labelling Studies (2 :3 vlv). Chloroform (3 vol) and methanol (1.5vol) were then added to the lower phase yielding two phases after Cultured fibroblasts from JNCL and LINCL patients centrifugation. The upper phase was discarded, and the and controls (1-4 x lo6 cells/ml) were metabolically lower phase was washed twice with methanollwater labelled with 3.7 MBq/ml of [2-3Hlmannose(NEN Re(1 : 1 v/v; 1.5 vol) and then mixed with methanol (1.3vol) search Products, Boston, MA) for 1 h in glucose-free and water (0.3 vol) to produce a single phase solvent of DMEM medium, as described by Cacan et al. [19871. compositionchlorofordmethanol/water (10 : 10 : 3). Two Cells were pre-treated with either N-methyldeoxynofurther extracts of the tissue residue with 2.5 vol of jirimycin or mannojirimycin in complete DMEM me10 : 10 :3were combined with the initial 10 :10 :3 extract dium containing 15%fetal calf serum for either 1h or for and concentrated by nitrogen evaporation. 7 days. Inhibitors were purchased from Genzyme Corporation (Boston, MA). Analysis by HPLC After incubation, the cells were pelleted and sequenOligosaccharides were quantitatively released from tial lipid extraction was performed as described by Cathe 10 : 10 :3 extract by mild acid hydrolysis as described can et al. [19871. The soluble oligosaccharides released by Chalifour and Spiro [ 19841. Water-soluble material into the aqueous phase were separated from precursor was deionized on coupled columns of Dowex 50 (H + 1and mannose by gel filtration on Bio-Gel P-2. Radioactivity Dowex 1 (acetate) in a pasteur pipette and then digested in the chlorofodmethanol fraction (Man-P-Dol),in the with endoglucosaminidase H (Endo H; Genzyme Corpo- chlorofordmethanol/water 10: 10 :3 fraction (Dol-PPration, Boston, MA). The Endo H digested oligosac- OS), and in the glycoprotein pellet was determined by charides were desalted on a column of Bio-Gel P-2, re- liquid scintillation counting. Analysis of the oligosacduced with sodium borohydride, perbenzoylated, and charide moiety from Dol-PP-OS was performed after analyzed by reversed phase HPLC on a 3 micron OC- mild acid hydrolysis (0.01N HC1, 100°C, 15 min) of the tylsilica column (3.2 x 100 mm; Rainin Instruments, chloroform/methanol/water fraction, followedby Endo H Woburn, MA), as described by Daniel [1987]. Oligosac- treatment overnight in 50 mM citrate buffer, pH 5.5, charides were eluted at a flow rate of 1 ml/min with a and gel filtration on Bio-Gel P-2. gradient of acetonitrile-water (80 :20) changing to pure RESULTS acetonitrile over 20 min. Oligosaccharideswere detected Quantification of Dolichyl Oligosaccharides in by their UV absorbance at 230 nm using an Applied Brain Biosystems model 757 variable wavelength detector and Patients with JNCL or LINCL have greatly increased chromatograms were plotted on a Spectra Physics SP4270 integratorlplotter. Oligosaccharides isolated levels (5-fold to 20-fold) of Dol-PP-OS in their cerebral from a sheep fed “locoweed” (Astragalus lentiginosus) gray matter (Table I). Diseased controls consisted of 2 Hurler, a Tay-Sachs, and a Fabry patient. Oligosacwere used as standards [Warren et al., 19881.
588
Daniel et al.
TABLE I. Measurement of Dolichyl-PP-Oligosaccharidesin Brain
Normal controls (N = 14) Diseased controls (N = 4) Infantile NCL ( N = 2 ) Late-infantile NCL (N = 4) Juvenile NCL (N = 7)
nmol/g tissue f S.D. 2.1 f 1.9 4.2 ? 3.9 13.7 10.8 f 5.4 41.8 13.2
*
9
A
Range
