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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
Sphingolipids are essential for cell functions as plasma membrane components and bioactive metabolites and defects in their lysosomal degradation cause lysosomal storage diseases, more specifically sphingolipidoses. The efficient in vivo degradation of sphingolipids in lysosome requires specific hydrolases and sphingolipid activator proteins, including the GM2 activator protein and four saposins (1,2) (Figure 1). Four saposins, A, B, C, D, are small hydrophobic glycoproteins produced by the sequential proteolytic cleavage of the precursor protein prosaposin (PSAP) in the lysosome. All saposins have a highly homologous structure with three disulfide bonds and at least one N-glycan. Instead of their structural similarities, their specificity for lysosomal hydrolases differs among saposins; saposin-A for galactosylceramide (GalCer)-β-galactosidase (GALC), saposin-B for arylsulfatase A (ARSA), saposin-C for glucosylceramide (GlcCer)-β-glucosidase (GBA1), and saposin-D for acid ceramidase (ASAH1). Mutations in the saposin-A, B, and C domains of the PSAP gene in human and mouse cause sphingolipidoses with characteristic sphingolipid accumulations, resembling Krabbe’s disease, metachromatic leukodystrophy, and Gaucher’s disease, respectively (1,3,4,5). Furthermore, mutations in the saposin-D domain of PSAP gene cause autosomal dominant form of hereditary Parkinson’s disease (6). Saposin-D-deficient mice show the accumulation of α-hydroxyl fatty acid-containing ceramide in the brain tissues and progressive motor decline accompanied by neuronal loss of cerebellar Purkinje cells and dopaminergic neurons in the substantia nigra (4,6). These findings highlight the critical role of saposins in sphingolipid degradation. Since saposins facilitate interactions between membrane-bound hydrophobic sphingolipids and water-soluble hydrolases, either by direct binding with their respective enzymes or as biological detergents that lift substrates out of the membrane (7), to determine the activator function for lysosomal hydrolase, it is ideal to use the labeled natural glycolipid substrates with minimal usage of detergents. However, due to their unavailability, the enzyme activities can be alternatively assayed with artificial fluorescent substrates embedded into the liposome mimicking the intra-lysosomal membrane.
Protocol
In this chapter, two kinds of protocols are described for determining saposin activity for sphingolipid hydrolase: 1) that using labeled natural glycolipid substrates and tissue homogenates and 2) that using fluorescent substrates embedded in the liposome and synthesized or recombinant saposin protein.
Materials
- 1.
Tissue or cell homogenates
- 2.
N-[14C] lauroyl-sphingenine ([14C] C12:0-ceramide) prepared from D-erythro-sphingenine (Biomol/Enzo Life Sciences, NY, USA) and [1-14C] lauric acid (GE Healthcare, NJ, USA) (8) (Note 1)
- 3.
Dithiothreitol (DTT) (GE Healthcare)
- 4.
Nonidet P-40 (Nacalai Tesque, Kyoto, Japan)
- 5.
Butylated hydroxyanisole (BHA) (Sigma-Aldrich, MO, USA)
- 6.
Precoated silica gel 60 F254 aluminum sheets for thin-layer chromatography (TLC) (20 × 20 cm, 0.2-mm thickness) (Merck, Darmstadt, Germany)
- 7.
Synthetic or recombinant saposin-C (9)
- 8.
Imiglucerase (Cerezyme®, Sanofi Genzyme, MA, USA)
- 9.
4-Nitrobenzo-2-oxa-1,3-diazole (NBD) labeled C12:0-glucosylceramide (GlcCer) (Avanti Polar Lipids, AL, USA)
- 10.
L-α-Phosphatidyl choline (Avanti Polar Lipids)
- 11.
Cholesterol (Sigma-Aldrich)
- 12.
Bis (monoacylglycerol) phosphate (BMP) (Avanti Polar Lipids)
- 13.
L-α-Phosphatidyl serine (Avanti Polar Lipids)
- 14.
A silica gel column (Inertsil SIL-150A, GL Science, Tokyo, Japan) of high-performance liquid chromatography (HPLC)
Instruments
- 1.
Physcotron homogenizer (Microtec, Funabashi, Japan)
- 2.
Image analyzer (Typhoon 9400, GE Healthcare)
- 3.
HPLC with a fluorescence detector (Prominence LC-20AT, CTO-20A, RF-20A, SHIMADZU, Kyoto, Japan)
Methods
- 1.
The determination of saposin-D activity for acid ceramidase activity using tissue homogenates (10)
- a.
The tissues (brains, kidneys, and livers) dissected from Saposin-D-/- mice and their wild-type littermates are cut into small pieces, homogenized in 9 times the volume (v/w) of 20 mM of Tris-HCl (pH 7.4)/0.32 M sucrose, and centrifuged at 800 g for 15 min at 4°C. The resultant supernatants are used as homogenates. Protein concentrations are determined by the Bradford method.
- b.
The 100 μg of protein from tissue homogenate is incubated with 100 μM of [14C] C12:0-ceramide (10,000 cpm, dissolved in 5 μL of ethanol) as a substrate in 100 μL of 100 mM of sodium acetate buffer (pH 4.5) containing 3 mM of DTT, 150 mM of NaCl, 0.05% bovine serum albumin (BSA), and 0.1% (w/v) Nonidet P-40 (Note 2) at 37°C for 30 min.
- c.
The reactions are terminated by adding 0.32 mL of a mixture of chloroform/methanol/1 M citric acid (8:4:1, by vol.) containing 5 mM of BHA. Centrifuge at 800 g for 5 min at 4°C.
- d.
The lower organic phase (100 μL) is subjected to TLC at 4°C for 15 min with a mixture of chloroform/methanol/acetic acid (94:1:5, by vol.) as a developing solvent.
- e.
Dry the TLC sheet, and expose the TLC sheet to imaging plate overnight.
- f.
The radioactivity of [14C] C12:0-ceramide as the substrate and [14C] free lauric acid as the product on the plate is quantified using an image analyzer.
- g.
For each TLC lane, the radioactivity of the product is divided by the total radioactivity, including the substrate and product, to obtain the conversion rate. The amount of product in each reaction is calculated by multiplying the amount of used substrate (10 nmol) by the conversion rate, and the enzyme activity is then obtained (Figures 2A and B).
- 2.
The determination of saposin-C activity for GlcCer-β-glucosidase (GBA1) activity using synthetic or recombinant protein (11).
- a.
Prepare liposomes containing 64% L-α-phosphatidyl choline, 23% cholesterol, 10% BMP, and 3% C12-NBD-GlcCer (Note 3).
- b.
Liposomes composed only of L-α-phosphatidyl serine are prepared as a control.
- c.
NBD-GlcCer containing liposomes (2.5 µM) are preincubated with 0–1 µM of chemically synthesized saposin-C (9,11) in 100 µL of 0.1 M sodium citrate-phosphate buffer (pH 5.5) at room temperature for 30 min.
- d.
1 nM of imiglucerase (Note 4) in 100 µL of 0.1 M sodium citrate-phosphate buffer (pH 5.5) with 1 µM of BSA is added to the reaction mixture, which is then incubated at 37°C for 30 min.
- e.
The reaction is stopped by adding 1 mL of chloroform/methanol (2:1, by vol.).
- f.
The sample is briefly vortexed and then centrifuged.
- g.
The organic phase is dried under nitrogen gas and dissolved in 500 µL of 2-propanol/n-hexane/water (55:44:1, by vol.).
- h.
The 50 µL aliquot of each sample is injected into a HPLC with a silica gel column (Inertsil SIL-150A) and eluted with 2-propanol/n-hexane/water (55:44:1, by vol.) at a flow rate of 1 mL/min (12).
- i.
Fluorescence under excitation and emission wave lengths of 470 and 530 nm, respectively, is detected using a fluorescence detector (Figure 3A).
- j.
GBA1 activity is calculated on the basis of the amount of NBD-Cer converted from NBD-GlcCer (Figure 3B).
Notes
- 1.
Purified saposin-D is reported to stimulate acid ceramidase activity when incubated with [14C] C12:0-ceramide (13).
- 2.
The use of Nonidet P-40 as the detergent might be important to observe the activity of saposins.
- 3.
NBD-GlcCer-containing liposomes, with higher content of characteristic anionic phospholipids, such as BMP and lower content of cholesterol, mimicking physiological intra-lysosomal membrane, are used as the substrate [1, 2].
- 4.
Cerezyme® (imiglucerase for injection) is a modified form of human GBA1 for enzyme replacement therapy for pediatric and adult patients with a confirmed diagnosis of Gaucher’s disease. It is produced by recombinant DNA technology using a mammalian Chinese Hamster Ovary cell culture and is tagged with mannose for targeting to macrophages.
References
- 1.
- Breiden B, Sandhoff K. Lysosomal Glycosphingolipid Storage Diseases. Annu Rev Biochem. 2019 Jun 20;88:461–485. [PubMed: 31220974] [CrossRef]
- 2.
- Sandhoff K. My journey into the world of sphingolipids and sphingolipidoses. Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(10):554–82. [PMC free article: PMC3552047] [PubMed: 23229750] [CrossRef]
- 3.
- Matsuda J, Vanier MT, Saito Y, Tohyama J, Suzuki K, Suzuki K. A mutation in the saposin A domain of the sphingolipid activator protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse. Hum Mol Genet. 2001 May 15;10(11):1191–9. [PubMed: 11371512] [CrossRef]
- 4.
- Matsuda J, Kido M, Tadano-Aritomi K, Ishizuka I, Tominaga K, Toida K, Takeda E, Suzuki K, Kuroda Y. Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in the mouse. Hum Mol Genet. 2004 Nov 1;13(21):2709–23. [PubMed: 15345707] [CrossRef]
- 5.
- Yoneshige A, Suzuki K, Matsuda J. A mutation in the saposin C domain of the sphingolipid activator protein (prosaposin) gene causes neurodegenerative disease in mice. J Neurosci Res. 2010 Aug 1;88(10):2118–34. [PubMed: 20175216] [CrossRef]
- 6.
- Oji Y, Hatano T, Ueno SI, Funayama M, Ishikawa KI, Okuzumi A, Noda S, Sato S, Satake W, Toda T, Li Y, Hino-Takai T, Kakuta S, Tsunemi T, Yoshino H, Nishioka K, Hattori T, Mizutani Y, Mutoh T, Yokochi F, Ichinose Y, Koh K, Shindo K, Takiyama Y, Hamaguchi T, Yamada M, Farrer MJ, Uchiyama Y, Akamatsu W, Wu YR, Matsuda J, Hattori N. Variants in saposin D domain of prosaposin gene linked to Parkinson’s disease. Brain. 2020 Apr 1;143(4):1190–1205. [PubMed: 32201884] [CrossRef]
- 7.
- Hill CH, Cook GM, Spratley SJ, Fawke S, Graham SC, Deane JE. The mechanism of glycosphingolipid degradation revealed by a GALC-SapA complex structure. Nat Commun. 2018 Jan 11;9(1):151. [PMC free article: PMC5764952] [PubMed: 29323104] [CrossRef]
- 8.
- Ueda N, Yamanaka K, Yamamoto S. Purification and characterization of an acid amidase selective for N-palmitoylethanolamine, a putative endogenous anti-inflammatory substance. J Biol Chem. 2001 Sep 21;276(38):35552–7. [PubMed: 11463796] [CrossRef]
- 9.
- Hojo H, Tanaka H, Hagiwara M, Asahina Y, Ueki A, Katayama H, Nakahara Y, Yoneshige A, Matsuda J, Ito Y, Nakahara Y. Chemoenzymatic synthesis of hydrophobic glycoprotein: Synthesis of saposin C carrying complex-type carbohydrate. J Org Chem. 2012 Nov 2;77(21):9437–46. [PubMed: 22800502] [CrossRef]
- 10.
- Tsuboi K, Tai T, Yamashita R, Ali H, Watanabe T, Uyama T, Okamoto Y, Kitakaze K, Takenouchi Y, Go S, Rahman I.A.S, Houchi H, Tanaka T, Okamoto Y, Tokumura A, Matsuda J, Ueda N. Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Sep;1866(9):158972. [PubMed: 34033896] [CrossRef]
- 11.
- Yoneshige A, Muto M, Watanabe T, Hojo H, Matsuda J. The effects of chemically synthesized saposin C on glucosylceramide-β-glucosidase. Clin Biochem. 2015 Nov;48(16-17):1177–80. [PubMed: 26068040] [CrossRef]
- 12.
- Hayashi Y, Zama K, Abe E, Okino N, Inoue T, Ohno K. Makoto Ito. A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidases. Anal Biochem. 2008 Dec 1;383(1):122–9. [PubMed: 18708024] [CrossRef]
- 13.
- Azuma N, O’Brien JS, Moser HW, Kishimoto Y. Stimulation of acid ceramidase activity by saposin D. Arch Biochem Biophys. 1994 Jun;311(2):354–7. [PubMed: 8203897] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
Figures
Figure 1:
Pathway of glycosphingolipid degradation in lysosome.
Lysosomal glycosphingolipids are degraded in a stepwise manner. (Red) sphindolipidosis, (Blue) hydrolase, and (Black) sphingolipid activator protein. Sap, saposin; LacCer, lactosylceramide.
Figure 2:
Hydrolysis of ceramide using acid ceramidase in the tissue homogenates from wild-type (WT) and Saposin-D-/- (KO) mice.
B: The C12:0-ceramide-hydrolyzing activities of the tissue homogenates from KO mice were significantly lower than those from WT mice. Bars represent mean values ± S.D. (n = 3). *, p < 0.01 versus WT mice (Student’s t-test).
A: Representative thin-layer chromatography (TLC) to show the produced 14C labeled free fatty acids (FFA).
Figure 3:
GBA1 activity in the presence of various concentrations of synthetic saposin-C
B: Imiglucerase (1 nM) was incubated with 2.5 μM of NBD-GlcCer-containing liposomes and the indicated concentrations of saposin-C. GBA1 activity (pmol/h/ng GBA1) was defined on the basis of the amount of NBD-Cer converted from NBD-GlcCer. The averages and standard errors of three to five experiments are plotted.
A: Representative high-performance liquid chromatography (HPLC) profiles show the amount of NBD-Cer converted from NBD-GlcCer lipids. Standard mixture containing 20 pmol of NBD-Cer and NBD-GlcCer was incubated: in the absence of saposin-C and in the presence of 2 µM of saposin-C. The addition of saposin-C resulted in the highest hydrolytic conversion of NBD-GlcCer to NBD-Cer.