5 Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205; 6 The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205; 7 Department of Neurology, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205; and 8 Department of Neuroscience, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205
Received on April 8, 2004; revised on May 27, 2004; accepted on May 28, 2004
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Abstract |
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Key words: Galgt1 / N-acetylgalactosaminyltransferase / GD1a / GT1b / MAG
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Introduction |
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MAG, expressed on myelin, binds to complementary ligands on the apposing axon surface. MAG is a member of the Siglec family of sialic acidbinding Ig-family member lectins (Crocker and Varki, 2001; Crocker et al., 1996
). Siglecs share significant domain and sequence similarity and bind to sialic acidbearing glycoconjugates with varying specificities for the sialic acid linkage and penultimate saccharides. Gangliosides are the major sialoglycoconjugates in the brain (Schnaar, 2000
) (Figure 1). MAG binds with high affinity and specificity to two major brain gangliosides, GD1a and GT1b, that are expressed prominently on axons (DeVries and Zmachinski, 1980
) and that bear the MAG-binding terminal sequence NeuAc
1-3Galß1-3GalNAc (Collins et al., 1997
; Yang et al., 1996
). Mice engineered to lack a key enzyme in ganglioside biosynthesis, UDP-N-acetyl-D-galactosamine:GM3/GD3 N-acetyl-D-galactosaminyltransferase (EC 2.4.1.92), do not express the NeuAc
1-3Galß1-3GalNAc terminus, and display axon degeneration and dysmyelination similar to Mag-null mice (Sheikh et al., 1999
), as well as progressive motor behavioral deficits (Chiavegatto et al., 2000
). Furthermore, nerve cells from these mice are less responsive to MAG as an inhibitor of neurite outgrowth (Vyas et al., 2002
). These and other data (Vinson et al., 2001
; Yamashita et al., 2002
) implicate complex brain gangliosides, particularly GD1a and GT1b, as functional MAG ligands.
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Results |
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Discussion |
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One hypothesis that fits the data is that the stability of MAG in the periaxonal myelin membrane depends on its productive engagement with its ganglioside ligands, GD1a and GT1b, on the axon surface. Although the observed lack of MAG depletion and neuropathy in Siat8a-null mice appears to dismiss a role for GT1b, it remains possible that GD1a and GT1b, which are equivalently potent MAG ligands (Collins et al., 1997), act redundantly. Another consistent hypothesis is that MAG stability depends on the presence of GM1, the major ganglioside of rat myelin (DeVries and Zmachinski, 1980
). Resolving the roles of GM1, GD1a, and GT1b will require mice engineered to lack the sialyltransferase(s) that generate GD1a and GT1b from GM1 and GD1b, respectively. To date, the genes responsible for expression of these sialyltransferases have not been definitively identified.
The progressive and selective decrease in MAG expression documented in the current study is not likely to precede the axonal degeneration and motor behavioral deficits we previously documented in Galgt1-null mice (Chiavegatto et al., 2000; Sheikh et al., 1999
). Mutant mice heterozygous for the Mag gene express only half the level of normal MAG from birth yet are without documented behavioral or neurohistological pathology (Montag et al., 1994
). In contrast, 3-month-old Galgt1-null mice already show marked axon degeneration (Sheikh et al., 1999
). Based on these data, we favor the hypothesis that the deficiency of MAG ligands GD1a and GT1b results in decreased function of all MAG molecules, thereby initiating axonal degeneration and motor behavioral defects. Nevertheless, at older ages, as MAG expression drops below 50% of normal, loss of MAG may contribute to the ongoing nervous system deficits documented in aging Galgt1-null mice. A comparison of nervous system pathologies in Galgt1-null, Mag-null and Galgt1/Mag double-null mice may help resolve the relative roles of complex gangliosides and MAG in stabilizing myelinaxon interactions.
The progressive disappearance of MAG in Galgt1-null mice was not due to a decrease in MAG mRNA so must be due to a change in the translation, cell surface expression, and/or degradation rates of MAG, any one of which may be affected by the absence of complex gangliosides. Little prior data address the long-term metabolism of MAG in vivo. In one study (Toews et al., 1988), young rats were injected with [14C]glycine, and [14C]MAG levels were determined for up to 1 month thereafter. The degradation of MAG was biphasic, with a significant portion remaining stable for the month-long experimental observation period. Therefore even a modest decrease in MAG stability might result in its selective loss from myelin. The observation that MAG levels are normal in young (1.5-month) Galgt1-null mice but are notably decreased after 3 months (Sheikh et al., 1999
) and markedly decreased in older Galgt1-null mice (this study) is consistent with an increase in the degradation rate of an otherwise very stable protein. The present study demonstrates a progressive and selective decrease in MAG protein level in Galgt1-null mice. These data are consistent with a direct role for complex brain gangliosides in MAG metabolism, perhaps due to GD1a and GT1b acting as complementary MAG ligands and thereby stabilizing MAG in vivo.
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Materials and methods |
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Sialoglycoconjugate analyses
Mice were euthanized; their brains were removed and homogenized in 3 volumes of ice-cold water. Methanol was added with vigorous stirring to give a methanol-aqueous ratio of 8:3. The suspension was brought to ambient temperature, and chloroform was added to give a final chloroform-methanol-water ratio of 4:8:3 (Schnaar, 1994). Precipitated protein was removed by centrifugation (the resulting protein pellet was solubilized in 0.1 M aqueous sodium hydroxide and retained for subsequent sialic acid determination). The supernatant was collected and water added to generate a biphasic mixture of chloroform-methanol-water (4:8:5.6). After thorough mixing, the resulting upper phase (containing gangliosides) was reextracted with theoretical lower phase, then the upper phase was loaded on a reverse phase chromatography cartridge (Sep-Pak tC18, Waters, Milford, MA) to remove nonlipid contaminants (Schnaar, 1994
). Gangliosides were eluted with methanol, analyzed for sialic acid content, then subjected to silicic acid TLC using chloroform-methanol-0.25% aqueous potassium chloride (60:35:8) as developing solvent (Schnaar and Needham, 1994
). TLC-resolved gangliosides were detected using a resorcinol reagent (Schnaar and Needham, 1994
). The resulting image was captured using a CCD camera, and ganglioside concentrations were determined by quantitative densitometry (Scion Image, Scion, Frederick, MD).
To determine sialic acid content, an aliquot of the ganglioside fraction (or of the resolubilized protein) was added to a 500-µl polypropylene tube and evaporated to dryness; 20 µl of a solution containing 0.1 M HCl and 0.25 M NaCl were added. The sample was hydrolyzed for 3 h at 80°C. Released sialic acid was analyzed by injecting an aliquot (110 µl) onto a Dionex (Sunnyvale, CA) high-pressure liquid chromatography system using a HPIC AS6 column and a pulsed amperometric detector as described (Manzi et al., 1990). NeuAc was identified by its elution time and quantified by comparison with known standards.
Myelin
Myelin was purified from freshly collected mouse brains as described (Norton and Poduslo, 1973). A protease inhibitor cocktail (P8340, Sigma, St. Louis, MO) was included in all isolation steps. Purified myelin was collected from the final sucrose density centrifugation, washed with 0.32 M sucrose without protease inhibitors, and resuspended in 0.32 M sucrose. Myelin protein was quantified using a bicinchoninic acid assay (Pierce, Rockford, IL). The resulting myelin suspension was used immediately or was stored frozen in small aliquots at 70°C.
Immunoblotting
Anti-MAG monoclonal antibody (Gen-S3; Nobile-Orazio et al., 1984) was kindly provided by Dr. Norman Latov (Cornell University, Ithaca, NY). Anti-myelin basic protein was from QED Bioscience (San Diego, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) was from Jackson ImmunoResearch Laboratories (West Grove, PA). Enhanced chemiluminescence (ECL) reagents were from Santa Cruz Biotechnology (Santa Cruz, CA), and ECL Hyperfilm was from Amersham Biosciences (Piscataway, NJ).
Equal protein amounts (myelin or total tissue homogenate) were subjected to SDSPAGE (Laemmli, 1970). Resolved proteins were either stained with Coomassie brilliant blue or transferred to polyvinylidene difluoride (PVDF) membranes using a semi-dry blotting apparatus for immunoblotting overnight at 4°C using concentrations of each primary antibody optimized for quantitative detection of its antigen. The blots were then washed three times with 0.2% Tween 20 in Tris-buffered saline (TBS) and incubated with 50 ng/ml HRP-conjugated goat anti-mouse IgG for 1 h. The blots were then washed twice with Tween-containing TBS, once with TBS, then were developed using ECL reagents according to the manufacturer's instructions. Gels and blots were digitally scanned and band densities quantified using Scion Image software.
Northern blot analyses
Total RNA was extracted from fresh mouse brains using TRIzol Reagent (Invitrogen, Carlsbad, CA). Ten micrograms of each RNA preparation were separated on a 1.2% agarose formaldehyde gel and the 28S and 18S RNA bands were stained with ethidium bromide and detected by UV transillumination. The RNA was then transferred to Nytran (Schleicher & Schuell, Keene, NH) for hybridization with a 32P-labeled cDNA probe consisting of the rat L-MAG full coding sequence (95% identical to the mouse sequence). The probe was labeled using Ready-To-Go DNA labeling beads (Amersham) using the manufacturer's instructions. Northern blot was done using ULTRAhyb buffer from Ambion (Austin, TX). Bound probe was detected using a Fuji BAS phosphorimaging system (Fujifilm Medical Systems, Stamford, CT).
Immunohistochemistry
Mice were anesthetized and perfused transcardially with 4% paraformaldehyde. The brain was removed and postfixed 24 h in the same fixative, then cryopreserved by incubation for 24 h in 10% sucrose. Parasagittal 30-µm frozen sections were collected into TBS. Immunostaining of free-floating sections was performed at 4°C. Sections were preblocked for 2 h in TBS containing 1% bovine serum albumin, 5% goat serum, and 1% Triton X-100, then were incubated for 16 h in the same solution containing 4 µg/ml anti-MAG monoclonal antibody 513 (Chemicon, Temecula, CA) or anti-MBP (QED Bioscience). Sections were washed with TBS containing 1% Triton X-100, then incubated for 6 h in the same solution containing 2 µg/ml biotin-conjugated goat anti-mouse IgG (Fc specific, Jackson ImmunoResearch). Sections were washed as before and incubated with steptavidinalkaline phosphatase conjugate for 2 h, then developed with Vector Red alkaline phosphatase substrate using the manufacturer's protocols (Vector Laboratories, Burlingame, CA).
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
2 Present address: Department of Chemistry, Shiga University, Shiga 520-0862, Japan
3 Present address: Faculty of Medicine, J. J. Strossmayer University of Osijek, Osijek, Croatia
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Abbreviations |
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References |
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