Glycobiology Advance Access originally published online on June 2, 2004
Glycobiology 2004 14(9):851-857; doi:10.1093/glycob/cwh107
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Glycobiology vol. 14 no. 9 © Oxford University Press 2004; all rights reserved.
Myelin-associated glycoprotein (Siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides
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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Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin–axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid–binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased
60% and at 12 months of age 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface. Key words: Galgt1 / N-acetylgalactosaminyltransferase / GD1a / GT1b / MAG
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Introduction |
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Myelin-associated glycoprotein (MAG) is a minor myelin component, making up
1% of central nervous system and 0.1% of peripheral nervous system myelin proteins (Trapp, 1990
). It is preferentially expressed on the inner-most, periaxonal myelin membrane (Trapp et al., 1989), where it functions in myelin–axon interactions (Schachner and Bartsch, 2000). The axon is dependent on signals from myelin, specifically MAG, for its cytoarchitecture, structure, and long-term stability (Bjartmar et al., 1999). Genetic ablation of MAG results in reduced axon caliber, reduced axon neurofilament spacing and phosphorylation, and progressive axon degeneration (Fruttiger et al., 1995; Li et al., 1994; Montag et al., 1994; Yin et al., 1998). These observations led to the conclusion that MAG is an important signaling molecule in myelin–axon interactions and is required for optimal long-term axon stability (Schachner and Bartsch, 2000). In addition, MAG inhibits axon regeneration after injury (Li et al., 1996; McKerracher et al., 1994; Mukhopadhyay et al., 1994). Along with Nogo, oligodendrocyte myelin glycoprotein, and chondroitin sulfate proteoglycans, MAG contributes to a central nervous system environment that is highly inhibitory for nerve regeneration (Fournier and Strittmatter, 2001; Sandvig et al., 2004; Wang et al., 2002).
MAG, expressed on myelin, binds to complementary ligands on the apposing axon surface. MAG is a member of the Siglec family of sialic acid–binding Ig-family member lectins (Crocker and Varki, 2001; Crocker et al., 1996). Siglecs share significant domain and sequence similarity and bind to sialic acid–bearing 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 NeuAc1-3Galß1-3GalNAc 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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In prior studies we noted that MAG expression was decreased in mice lacking complex brain gangliosides (Sheikh et al., 1999
). The current work extends that observation. We report here that MAG expression in mice engineered to lack complex brain gangliosides is progressively and selectively decreased, even though the MAG gene is expressed equally in wild-type and Galgt1-null mice throughout life. These data support the conclusion that MAG interaction with complex brain gangliosides markedly affects its steady-state expression. |
Results |
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Ganglioside expression in Galgt1-null mice
Total brain weight and total brain myelin protein were not significantly different between wild-type, Galgt1-null, and Galgt1 heterozygote mice at any age tested (data not shown). Quantification of brain ganglioside- and glycoprotein-associated sialic acid in 13–15-month-old mice (Table I) demonstrated equivalent total expression of sialoglycoconjugates in wild-type and Galgt1 mutant mice. Analysis of gangliosides by thin-layer chromatography (TLC) (Table I) revealed that wild-type mice expressed four quantitatively major gangliosides (GM1a, GD1a, GD1b, GT1b) and lesser amounts of GQ1b and GD3. Galgt1 heterozygotes expressed a similar ganglioside pattern, except GD3 expression was increased and GQ1b expression was decreased. Galgt1-null mice lacked all complex gangliosides, instead expressing an equivalent total amount of GM3, GD3, and O-acetyl-GD3. These data are consistent with prior studies (Kawai et al., 2001
; Liu et al., 1999; Takamiya et al., 1996). Isolation of gangliosides under nonalkaline conditions to retain ester bonds revealed the relatively high expression of O-acetyl-GD3 in Galgt1-null mice, which was identified based on its relative migration and conversion to a species migrating with GD3 on alkali treatment (data not shown).
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MAG protein expression in Galgt1 mutant mice
MAG expression was quantified by immunoblotting equivalent amounts (based on protein) of myelin isolated from the brains of mice of different genotypes (Figures 2 and 3). At 1.5 months of age, MAG expression was the same in wild-type and Galgt1 mutant mice. However, a marked and significant decrease in MAG expression was observed in Galgt1-null mice compared to wild-type and Galgt1 heterozygote littermates by 6 months of age; this deficit was more striking at 12 months of age. When quantified and expressed relative to total myelin proteins, MAG expression was reduced 59% in 6-month-old and 70% in 12-month-old Galgt1-null mice compared to wild type. In contrast, expression of other myelin proteins (detected by Coomassie staining) was not changed during aging, and no significant difference in major myelin proteins among the three genotypes was observed (Figure 2 and data not shown).
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Two possibilities to explain these findings were considered. Either MAG is selectively lost in Galgt1-null mice, or MAG is expressed equally but is not recovered with the purified myelin fraction. To distinguish between these possibilities, total brain homogenates from 1-year-old wild-type and mutant mice were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted to determine the total brain expression levels of MAG and, as a quantitative marker for myelin, myelin basic protein (MBP). The results (Figures 4 and 5) support the conclusion that MAG expression is decreased in the brains of Galgt1-null mice, whereas there is no difference in MBP expression between the wild type and mutants. Based on equivalent brain protein loaded, total MAG expression was decreased 68% in the brains of Galgt1-null mice compared to wild-type littermates (data not shown). When expressed relative to MBP in the same extracts (Figure 5) MAG expression in Galgt1-null mice was reduced 77% compared to wild-type mice. MAG expression was reduced modestly in the heterozygotes compared to the wild-type mice, but this reduction did not reach statistical significance.
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To further determine the expression and distribution of MAG in wild-type and Galgt1 mutant mouse brains, immunohistochemistry was performed on parasagittal brain sections of 10-month-old animals (Figure 6). Anti-MBP antibody–stained white matter tracts equivalently throughout the brains of wild-type, Galgt1 heterozygote, and Galgt1-null mice. Anti-MAG antibody also strongly stained white matter tracts of wild-type and Galgt1 heterozygote mouse brains (note corpus callosum, white matter tracts of the cerebellum, and midbrain). However, consistent with biochemical analyses, immunohistochemical staining of Galgt1-null mice revealed markedly diminished staining of the same white matter tracts with anti-MAG antibody. We conclude that in Galgt1-null mice, MAG is still concentrated in myelin, although its expression level is markedly and selectively reduced.
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MAG gene expression in Galgt1 mutant mice
The progressive decrease in expression of MAG in the brains of Galgt1-null mice may occur at the level of gene expression or at subsequent steps, such as relative translation and/or degradation rates. To test the former possibility, MAG mRNA levels were determined by northern analysis using total RNA recovered from 1-year-old wild-type and Galgt1 mutant mouse brains. In contrast to protein levels, mRNA levels were indistinguishable among the three genotypes (Figures 7 and 8). When quantified as MAG mRNA relative to 28S RNA (Figure 8), MAG gene expression in Galgt1 mutant mice was equivalent to that in wild-type mice, even though MAG protein expression was reduced >70% in Galgt1-null mice compared to wild-type mice.
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Discussion |
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MAG expression is progressively and selectively decreased in the brains of mice lacking complex brain gangliosides. Evidence that MAG expression was ganglioside-dependent was first reported in our earlier publication, in which a decrease in MAG expression was observed in brains of 3-month-old Galgt1-null mice (Sheikh et al., 1999
). This finding was confirmed, and it was further found that mice lacking GD3 synthase (Siat8a-null mice), which express GM1 and GD1a but not GD3, GD1b, or GT1b, expressed normal levels of MAG (Kawai et al., 2001). Siat8a/Galgt1 double-null mice, which express only monosialoganglioside GM3, expressed MAG levels similar to those in Galgt1-null mice (Kawai et al., 2001). We conclude from these combined observations that the decrease in MAG expression in Galgt1-null mice is neither due to increased GD3 expression nor to decreased GD1b or GT1b per se.
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 myelin–axon 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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Mice
Mice with a disrupted Galgt1 gene were engineered by homologous recombination of exons 6 and 7 and part of exon 8 with a selectable cassette as described (Liu et al., 1999
). Founding breeders stably carrying the mutant gene were generously provided by Dr. Richard Proia (National Institutes of Health, Bethesda, MD). Offspring of heterozygote crosses were genotyped using polymerase chain reaction analysis of DNA isolated from mouse tails with the forward primer TACCAGGCCAACACAGCA from exon 5 and the reverse primer CAGGTCCAGGGGCGTCTT from exon 8, resulting in a 2.9-kb product from the wild-type allele and a 2.6-kb product from the null allele. Tissues from age-matched wild-type, heterozygote, and mutant littermates were compared in all experiments.
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 (1–10 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 SDS–PAGE (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 steptavidin–alkaline 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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We thank Dr. Richard Proia for providing founder mice for establishing the Galgt1 mutant mouse colony, Dr. Norman Latov for providing the anti-MAG monoclonal antibody Gen-S3, and Susan Fromholt for maintenance and genotyping of the mouse colony.
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Footnotes |
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4 To whom correspondence should be addressed; e-mail: schnaar{at}jhu.edu
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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ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; PVDF, polyvinylidene difluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TLC, thin-layer chromatography
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