From the Genetics of Development and Disease Branch,
NIDDK, National Institutes of Health, Bethesda, Maryland 20892, ** Section on Behavioral Neuropharmacology, Experimental Therapeutics
Branch, National Institute of Mental Health, Bethesda, Maryland 20892, and §§ Kekulé-Institut für Organische Chemie
und Biochemie der Universität Bonn, Gerhard- Domagk-Strasse 1, 53121 Bonn, Germany
Received for publication, November 30, 2000, and in revised form, December 26, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gangliosides are a family of
glycosphingolipids that contain sialic acid. Although they are abundant
on neuronal cell membranes, their precise functions and importance in
the central nervous system (CNS) remain largely undefined. We have
disrupted the gene encoding GD3 synthase (GD3S), a
sialyltransferase expressed in the CNS that is responsible for the
synthesis of b-series gangliosides. GD3S Gangliosides are glycosphingolipids that contain sialic acid
(reviewed in Ref. 1). They are found on the external leaflet of the
plasma membrane on eucaryotic cells and are most abundant in the
central nervous system (CNS)1
where they represent the major sialoglycoconjugate. Because of their
dramatic changes in expression during neuronal development and
differentiation (3-6), as well as their prominence in the mature CNS, gangliosides have long been assumed to have fundamental roles in CNS development and function.
In the ganglioside biosynthetic pathway (1) (see Fig. 2A),
lactosylceramide serves as the core structure. The first
ganglioside synthesized,
GM3,2 is produced by the
transfer of an We have now produced mice with a disrupted GD3 synthase
(GD3S) gene (12), of which expression is relatively high in
the CNS. Even though these mice lack the disialylated b-series
gangliosides in their CNS, they show no overt phenotype. By combining
the GalNAcT and GD3S mutant alleles, we have
established double knockout mice (DKO) that express only monosialylated
GM3 as their major CNS ganglioside. In contrast to each of the single
knockout mice, the DKO mice show a sudden death phenotype and a severe
CNS disturbance that is manifested by an exquisite susceptibility to
lethal, sound-induced seizures. These results provide compelling
evidence for the critical and essential role of gangliosides in CNS function.
Targeted Disruption of the Mouse GD3S Gene and Generation of
Mutant Mice--
To generate the GD3S knockout mice, a
genomic fragment was isolated from a 129/Sv strain library (Stratagene,
catalog no. 946306) containing the fifth exon of the 5-exon
GD3S gene (13). For knocking-in the LacZ reporter
gene and targeted inactivation of the GD3S gene, a
LacZ-neo (neomycin-resistant gene) cassette (14) was
inserted into the BamHII site of the fifth exon. The resulting targeting vector contained an ~11-kb GD3S 5'
fragment and a 2.2-kb GD3S 3' fragment separated by the
LacZ-neo cassette (Fig. 1A). The LacZ
coding region is preceded by an internal ribosomal entry sequence.
Therefore, targeted insertion generates a bicistronic transcription
unit in which the expression of the Glycosyltransferase Assays, Lipid Analysis, and MAG
Determination--
GD3 and GM3 synthase activities were determined as
previously described (16, 17).
The lipid analysis of mouse brain was done as described (18). The TLC
solvent system was CHCl3/MeOH/0.22% CaCl2
(60:35:8, v/v/v). Each lane was loaded with the equivalent of 10 mg wet weight of brain. The ganglioside bands were detected with a phosphoric acid/copper sulfate reagent (15.6 g of
CuSO4-(H2O)5 and 9.4 ml of
H3PO4 (85%, w/v) in 100 ml of water) (19).
Lipid-linked sialic acid determinations were done according to
Svennerholm (20) modified by the method of Miettinen and
Takki-Luukkainen (21). Determination of MAG levels was accomplished by
Western blotting.
GD3S Expression and Histology--
For visualization of
LacZ expression, timed matings were performed with
GD3S mutant mice. Embryos at embryonic day 11.5 (E11.5), E13.5, E15.5, and E17.5 were removed and immersed in fixative (1%
formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P-40 in PBS) at
4 °C for 2 h. Subsequently, the embryos were stained with
5-bromo-4-chloro-3-indolyl
For histological analysis, the mice were perfused with PBS and 10%
phosphate-buffered formalin. The brain, spinal cord, and visceral
organs were dissected out, fixed in 10% buffered formalin, and
processed for embedding in paraffin. The pathology of the organs were
surveyed on the sections stained with hematoxylin and eosin, and brain
and spinal cord were further examined with Luxol-Fast Blue and Nissl staining.
Audiogenic Seizure Induction--
The mice were 2-4
months old at the time of testing. An individual mouse was put in a
conventional cage with bedding and allowed to explore for at least 1 min. After the acclimation period, a key bundle was shaken steadily 15 cm above the cage by the experimenter for 5-10 s. The key bundle
consisted of five keys, and stimulus noise level was within the range
of 83 to 86 decibels. Background noise level in the room was 52 decibels. Observations were made for at least 60 s after the start
of key shaking.
Generation of GD3 Synthase Knockout Mice--
To disrupt the
GD3S gene in mice, we created a targeting vector that would
both render GD3 synthase inactive and allow monitoring of the
GD3S locus expression (Fig.
1A). The targeting vector was designed to introduce a LacZ reporter gene into the last
exon of the GD3S gene. This targeting strategy would cause
an early termination of the GD3 synthase protein, resulting in the
elimination of the highly conserved sialyl S motif, a critical portion
of the catalytic domain (22). Removal of the sialyl S motif from the
GD3 synthase in this manner renders the enzyme inactive (16, 23). After
electroporation of the targeting vector into embryonic stem
cells, we obtained clones with a homologously recombined GD3S allele. Targeted embryonic stem cells were used to
create chimeric mice and, subsequently, heterozygous mice.
Crossbreeding of the heterozygous mice resulted in the birth of
homozygous mutant mice (Fig. 1B) at the expected Mendelian
frequency, indicating an absence of embryonic lethality associated with
this mutation.
GD3 synthase activity was not detectable in extracts of
GD3S
We next determined the embryonic expression pattern of the
GD3S gene by whole mount staining of heterozygous and
homozygous mutant embryos with X-gal. At E11.5, very strong
GD3S expression was found in developing spinal cord. The
eyes and the top of the metencephalon were also positive (not shown).
Throughout later developmental stages, E13.5-E17.5, the targeted
GD3S gene expression continued to be relatively restricted
to the developing CNS (Fig. 1D).
As would be predicted from the location of GD3 synthase in the
biosynthetic pathway of gangliosides (Fig.
2A), the brain ganglioside pattern of GD3S Establishment of Mice That Express Only GM3 GalNAcT--
knockout
mice express predominantly GM3 and GD3 gangliosides in their CNS due to
the absence of
After their weaning we noted that the DKO mice had an extremely high
mortality rate. We followed the life span of a group of DKO mice and
found that by 13 weeks of age about 50% had died of unknown causes
(Fig. 3A). The overall
mortality was 92% during the observation period (36 weeks). We
searched for pathological changes in the DKO mice. We examined brain
sections by Nissl, and Luxol Fast Blue staining but could not discern
any gross pathological changes, including neuronal cell loss or
significant demyelination. Histological examination was done with mice
after timed sacrifice and after sudden death. It had been noted
previously that expression of MAG, which binds certain gangliosides,
was reduced in the GalNAcT mutant mice, presumably because
of a deficiency of ganglioside ligands for MAG (10). We found that the
single mutant GD3S mice had a level of MAG similar to
wild-type mice (not shown). This finding can be explained by the
presence of GD1a (Fig. 2, A and B), a relatively
high affinity MAG ligand (25). In the DKO mice, MAG expression was
similar to that of the single mutant GalNAcT mice (not
shown), presumably because of a comparable deficiency of MAG ligands in
the two types of mutant mice.
Again, as had been noted in the GalNAcT Double Mutant Mice Are Sensitive to Sound-induced Seizures--
We
noticed that some of the DKO mice showed apparently spontaneous and/or
handling-induced seizures. In one case, a mouse died after such a
handling-induced seizure attack. Therefore, we hypothesized that the
reason for sudden death in the DKO mice may be enhanced susceptibility
to lethal seizure. Sound is a classic means of inducing seizures in
certain susceptible strains of mice (27). We used a relatively mild
sound stimulus, key jangling, to test the DKO and single mutant mice
for sensitivity to audiogenic seizure. We found that the double mutant
mice were exquisitely sensitive to seizure induction by this method
(Fig. 3B). The typical seizure began with a brief wild
running phase, followed by a clonic-tonic seizure. In most cases the
seizure resulted in death. All DKO mice tested were induced to have a
clonic-tonic seizure. Ultimately, 9 of 10 DKO mice died during tonic
phase, apparently because of respiratory arrest. None of the wild-type
or GD3S Why Are Mice That Express Only Monosialoganglioside GM3 Exquisitely
Susceptible to Lethal Seizures?--
Seizure susceptibility in mice
has been shown to result from mutations in a large number of genes with
diverse functions, including a variety of proteins involved in neuronal
cell signaling (28). Gangliosides have been proposed to act as
modulators of transmembrane signaling (29, 30). Recently, Kasahara
et al. (31, 32) have shown that GD3 ganglioside is
associated with the Src family kinase, Lyn, and with the
glycosylphosphatidylinositol-anchored signaling protein, TAG-1,
in glycosphingolipid-enriched microdomains. It is also of interest that
mice deficient in Fyn kinase, another Src family kinase, are
susceptible to audiogenic seizures (33). In the DKO mice, the
undersialylated ganglioside complement could alter the properties of
these glycosphingolipid-enriched membrane microdomains, resulting in
impaired neuronal cell signaling and seizure susceptibility.
A role for gangliosides in the development of the CNS has been
suggested based on the striking changes in ganglioside composition during the differentiation of neurons (3-6). Thus, it is also possible
that the seizure susceptibility in DKO mice may be a result of abnormal
CNS development. Although histologic examination of DKO mutant brains
revealed no obvious abnormalities, subtle defects cannot be ruled out.
Finally, our findings may have relevance for human epilepsy, a disorder
that burdens about 1% of the population (28). Our results raise the
possibility that mutations altering the ganglioside synthesis pathways
may be factors in contributing to some forms of this disorder.
/
mice, even
with an absence of b-series gangliosides, appear to undergo normal
development and have a normal life span. To further restrict the
expression of gangliosides, the GD3S mutant mice were
crossbred with mice carrying a disrupted GalNAcT gene encoding
1,4-N-acetylgalactosaminyltransferase.
These double mutant mice expressed GM3 as their major ganglioside. In
contrast to the single mutant mice, the double mutants displayed a
sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate
unequivocally that gangliosides play an essential role in the proper
functioning of the CNS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2,3-linked sialic acid residue to lactosylceramide.
Subsequently, GM3 can be modified by the action of
1,4-N-acetylgalactosaminyltransferase (GalNAcT, EC 2.4.1.92) to produce GM2 and other complex gangliosides. Alternatively, GM3 can be modified by the action of GD3 synthase (CMP-sialic acid:GM3
-2,8-sialyltransferase, EC 2.4.99.8) to produce the disialoganglioside GD3, which diverts the pathway to the synthesis of
b- and c-series gangliosides. Gene targeting in mice has been a
particularly fertile approach for uncovering the functions of gangliosides in the CNS. Disruption of the GalNAcT gene (7) blocks the synthesis of complex gangliosides and results in the expression of only the simple gangliosides GM3 and GD3. Surprisingly, these mutant mice are viable, with a normal life span and a CNS that is
largely intact both morphologically and functionally (8, 9). These mice
do, however, exhibit an age-related dysmyelination process that is
associated with axonal degeneration (10). The mechanism for
dysmyelination may be the absence of neuronal ganglioside ligands for
myelin-associated glycoprotein (MAG) resulting in myelin instability.
Ultimately, motor defects are observed in aged, 12-month-old
GalNAcT
/
mice, suggesting a role for complex gangliosides in long-term CNS maintenance (11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase reporter protein
is under the control of GD3S transcriptional regulatory
elements. The herpes simplex virus thymidine kinase (TK) gene
was positioned outside the homologous sequence to deter random
integration events. Targeting of TC1 embryonic stem cells and
establishment of chimeric mice were performed as in past experiments (8, 15). Male GD3S
/
mice were crossed to female
GalNAcT
/
mice (8) to generate GD3S+/
GalNAcT +/
double heterozygotes. These double heterozygote
mice were intercrossed to generate GalNAcT
/
GD3S
/
double mutant mice. Genotyping was
accomplished by polymerase chain reaction and Southern analysis
of genomic DNA isolated from tail biopsies.
-D-galactopyranoside (X-gal)
solution (0.1 M sodium phosphate at pH 7.3, 2 mM MgCl2, 5 mM K3
Fe(CN)6, 5 mM
K4Fe(CN)6, 0.01% sodium deoxycholate, 0.02%
Nonidet P-40, 20 mM Tris-HCl, 1 mg/ml X-gal) at room
temperature for 16 h. After the incubation the embryos were rinsed
with PBS.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 1.
Targeted disruption and expression of
the GD3S gene. A, schematic
representation of the GD3S targeting strategy. The structure
of the 3' end of the mouse GD3S locus is shown at the
top, the structure of the targeting vector in the
middle, and the predicted structure of the homologous
recombined locus is on the bottom. B,
BamHI; H, HindIII; probe,
probe for Southern blot; WT, wild type;
RV, EcoRV. B, identification
of mice homozygous for the GD3S locus. Southern analysis
yielded a 4.5-kb HindIII band corresponding to the wild-type
GD3S allele in wild-type DNA using the external probe shown
in A. DNA from mice homozygous for the disrupted
GD3S allele yielded a 7-kb band. C,
GD3S /
mice are devoid of GD3 synthase activity. As
demonstrated by TLC, GD3 synthase activity was not detectable in the
extract from the GD3S
/
mouse brain but was readily
apparent in the extract from the wild-type mouse brain. Both extracts
contained GM3 synthase activity as indicated by the synthesis of
radiolabeled GM3. The TLC migration positions of labeled GD3 and GM3
are indicated by arrows. D, GD3S
/
,
embryos were obtained and were incubated with X-gal to visualize
expression from the GD3S locus. Embryos were sectioned along
the saggital plane prior to staining to expose internal structures.
Note the high level of expression in the CNS.
/
brain (Fig. 1C). However,
GM3 synthase, the sialyltransferase that catalyzes the preceding step
of ganglioside biosynthesis, was readily detectable in the same
extracts (Fig. 1C).
/
mice was devoid of the
b-series structures, GD1b and GT1b, and was dominated by the a-series
gangliosides, GM1 and GD1a (Fig. 2B). The absence of
b-series gangliosides further documents the disruption of the
GD3S gene and validates the ganglioside synthesis scheme
proposed by Sandhoff and colleagues (1, 24). The
GD3S
/
mice were viable and fertile, had
normal growth, and were without gross behavioral abnormalities.
Histologic examination of brains from GD3S
/
mice did not reveal any obvious abnormalities. The GD3S
/
mice did not show observable demyelination as
determined by Luxol Fast Blue staining of brain sections.
View larger version (60K):
[in a new window]
Fig. 2.
Ganglioside and MAG expression in ganglioside
mutant mice. A, the partial biosynthetic pathway of
gangliosides with blocks in the pathway predicted in the single and
double mutant mice. B, brain ganglioside patterns from
wild-type (WT) and ganglioside mutant mice. The positions of
major brain gangliosides are shown on the sides of the
panel. C, lipid-linked sialic acid levels from brains
(n = 3) of wild-type and ganglioside mutant mice. An
estimation of the total brain ganglioside concentration (nmol/10 mg wet
weight) based on scanning the TLC plates and the sialic acid
determination is as follows: wild-type, 7-10;
GD3S /
, 11-16; GalNAcT
/
,
12-14; GD3S/GalNAcT
/
, 13-14.
1,4-N-acetylgalactosaminyltransferase, yet
they have fundamentally normal nervous system development and a normal
life span (8, 9). We further narrowed the expression of ganglioside
structures by crossing the two types of ganglioside mutant mice. Mice
doubly mutant for the GalNAcT and GD3S genes were
born at the expected Mendelian frequency. As predicted from the
biosynthetic pathway of gangliosides (Fig. 2A), elimination of both transferases in the DKO mice resulted in the expression of GM3 as their major CNS ganglioside (Fig. 2B). Although
roughly similar amounts of total brain ganglioside were present in
wild-type and mutant mice, DKO mice contained markedly reduced levels
of lipid-linked sialic acid in brain compared with wild-type and single
mutant mice (Fig. 2C).
View larger version (27K):
[in a new window]
Fig. 3.
Life span and audiogenic seizures of
ganglioside mutant mice. A, groups of mice
(n = 9) of each of the genotypes were monitored for
survival from an age of 4 weeks until 35 weeks. B, mice from
each genotype (wild-type (WT), n = 5;
GalNAcT /
, n = 9; GD3S
/
,
n = 5; GD3S/GalNAcT
/
, n = 10) were
exposed individually to a sound stimulus created by jangling a set of
keys for ~5 s. The behavior of the mice was observed for the
following: wild running, clonic seizure, tonic seizure, and death. *,
one GalNAcT
/
mouse responded to the sound stimulus with
wild running and a clonic seizure but not a tonic seizure or death. **,
p < 0.001;
2 test.
/
mice, the male
DKO mice displayed a defect in sperm maturation resulting in sterility (not shown) (8, 26). No pathologic abnormalities were found in visceral
organs in the DKO mice.
/
mice showed any seizure response to the sound
stimulus. One of the nine GalNAcT
/
mice responded with
wild running activity and a clonic seizure, but the episode did not
progress further to lethality.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank April Howard for expert help in preparing the figures and for maintaining the mouse colony and Bettina Kircharz for excellent help in preparing the thin layer chromatography.
![]() |
FOOTNOTES |
---|
* This work was supported in part by funding from the Deutsche Forschungsgemeinschaft (SFB 284 to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Third Dept. of Medicine, Shiga University of Medical Science, Otsu 520-2192, Japan.
¶ Present address: Dept. of Pathology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Present address: Dept. of Developmental Morphology, Tokyo
Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan.
Present address: Dept. of Pharmacology, Vanderbilt University
Medical Center, Nashville, TN 37232-6600.
¶¶ Present address: Biacore AB, Königsheide 28, D-44536 Lünen, Germany.
To whom correspondence should be addressed:
Bldg. 10, Rm. 9N-314, National Institutes of Health, Bethesda, MD
20892-1821.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.C000847200
2 The nomenclature for gangliosides follows the system of Svennerholm (2).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CNS, central nervous
system;
DKO, double knockout mice;
MAG, myelin-associated glycoprotein;
GD3S, the gene encoding GD3 synthase;
GalNAcT, the gene encoding 1,4-N-acetylgalactosaminyltransferase;
TLC, thin layer chromatography;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
kb, kilobase pair;
PBS, phosphate-buffered saline;
E, embryonic day (e.g.
E11.5).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Huwiler, A., Kolter, T., Pfeilschifter, J., and Sandhoff, K. (2000) Biochim. Biophys. Acta 1485, 63-99[Medline] [Order article via Infotrieve] |
2. | Svennerholm, L. (1994) Prog. Brain Res. 101, x-xiv |
3. | Yu, R. K. (1994) Prog. Brain Res. 101, 31-44[Medline] [Order article via Infotrieve] |
4. |
Ledeen, R. W.,
Wu, G.,
Lu, Z. H.,
Kozireski-Chuback, D.,
and Fang, Y.
(1998)
Ann. N. Y. Acad. Sci.
845,
161-175 |
5. |
Walkley, S. U.,
Zervas, M.,
and Wiseman, S.
(2000)
Cereb. Cortex
10,
1028-1037 |
6. | Futerman, A. H. (1998) Acta Biochim. Pol 45, 469-478[Medline] [Order article via Infotrieve] |
7. | Sango, K., Johnson, O. N., Kozak, C. A., and Proia, R. L. (1995) Genomics 27, 362-365[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Liu, Y.,
Wada, R.,
Kawai, H.,
Sango, K.,
Deng, C.,
Tai, T.,
McDonald, M. P.,
Araujo, K.,
Crawley, J. N.,
Bierfreund, U.,
Sandhoff, K.,
Suzuki, K.,
and Proia, R. L.
(1999)
J. Clin. Invest.
103,
497-505 |
9. |
Takamiya, K.,
Yamamoto, A.,
Furukawa, K.,
Yamashiro, S.,
Shin, M.,
Okada, M.,
Fukumoto, S.,
Haraguchi, M.,
Takeda, N.,
Fujimura, K.,
Sakae, M.,
Kishikawa, M.,
Shiku, H.,
Furukawa, K.,
and Aizawa, S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10662-10667 |
10. |
Sheikh, K. A.,
Sun, J.,
Liu, Y.,
Kawai, H.,
Crawford, T. O.,
Proia, R. L.,
Griffin, J. W.,
and Schnaar, R. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7532-7537 |
11. | Chiavegatto, S., Sun, J., Nelson, R. J., and Schnaar, R. L. (2000) Exp. Neurol. 166, 227-234[CrossRef][Medline] [Order article via Infotrieve] |
12. | Matsuda, Y., Nara, K., Watanabe, Y., Saito, T., and Sanai, Y. (1996) Genomics 32, 137-139[CrossRef][Medline] [Order article via Infotrieve] |
13. | Takashima, S., Kono, M., Kurosawa, N., Yoshida, Y., Tachida, Y., Inoue, M., Kanematsu, T., and Tsuji, S. (2000) J. Biochem. (Tokyo) 128, 1033-1043[Abstract] |
14. | Nehls, M., Kyewski, B., Messerle, M., Waldschutz, R., Schuddekopf, K., Smith, A. J., and Boehm, T. (1996) Science 272, 886-889[Abstract] |
15. |
Yamashita, T.,
Wada, R.,
Sasaki, T.,
Deng, C.,
Bierfreund, U.,
Sandhoff, K.,
and Proia, R. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9142-9147 |
16. |
Kawai, H.,
Sango, K.,
Mullin, K. A.,
and Proia, R. L.
(1998)
J. Biol. Chem.
273,
19634-19638 |
17. | Kono, M., Takashima, S., Liu, H., Inoue, M., Kojima, N., Lee, Y. C., Hamamoto, T., and Tsuji, S. (1998) Biochem. Biophys. Res. Commun. 253, 170-175[CrossRef][Medline] [Order article via Infotrieve] |
18. | Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C. J., Skop, E., Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L. (1996) Nat. Genet. 14, 348-352[Medline] [Order article via Infotrieve] |
19. | Yao, J. K., and Rastetter, G. M. (1985) Anal. Biochem. 150, 111-116[Medline] [Order article via Infotrieve] |
20. | Svennerholm, L. (1957) Biochim. Biophys. Acta 24, 604-611[CrossRef] |
21. | Miettinen, T., and Takki-Luukkainen, I. T. (1959) Acta Chem. Scand. 13, 856-858 |
22. |
Datta, A. K.,
Sinha, A.,
and Paulson, J. C.
(1998)
J. Biol. Chem.
273,
9608-9614 |
23. |
De Maria, R.,
Lenti, L.,
Malisan, F.,
d'Agostino, F.,
Tomassini, B.,
Zeuner, A.,
Rippo, M. R.,
and Testi, R.
(1997)
Science
277,
1652-1655 |
24. | Iber, H., Zacharias, C., and Sandhoff, K. (1992) Glycobiology 2, 137-142[Abstract] |
25. |
Schnaar, R. L.,
Collins, B. E.,
Wright, L. P.,
Kiso, M.,
Tropak, M. B.,
Roder, J. C.,
and Crocker, P. R.
(1998)
Ann. N. Y. Acad. Sci.
845,
92-105 |
26. |
Takamiya, K.,
Yamamoto, A.,
Furukawa, K.,
Zhao, J.,
Fukumoto, S.,
Yamashiro, S.,
Okada, M.,
Haraguchi, M.,
Shin, M.,
Kishikawa, M.,
Shiku, H.,
and Aizawa, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12147-12152 |
27. | Seyfried, T. N., Todorova, M. T., and Poderycki, M. J. (1999) Adv. Neurol. 79, 279-290[Medline] [Order article via Infotrieve] |
28. | Puranam, R. S., and McNamara, J. O. (1999) Curr. Opin. Neurobiol. 9, 281-287[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Hakomori, S.
(1990)
J. Biol. Chem.
265,
18713-18716 |
30. | Hakomori, S., Handa, K., Iwabuchi, K., Yamamura, S., and Prinetti, A. (1998) Glycobiology 8, xi-xix[Medline] [Order article via Infotrieve] |
31. |
Kasahara, K.,
Watanabe, Y.,
Yamamoto, T.,
and Sanai, Y.
(1997)
J. Biol. Chem.
272,
29947-29953 |
32. |
Kasahara, K.,
Watanabe, K.,
Takeuchi, K.,
Kaneko, H.,
Oohira, A.,
Yamamoto, T.,
and Sanai, Y.
(2000)
J. Biol. Chem.
275,
34701-34709 |
33. | Miyakawa, T., Yagi, T., Taniguchi, M., Matsuura, H., Tateishi, K., and Niki, H. (1995) Brain Res. Mol. Brain Res. 28, 349-352[CrossRef][Medline] [Order article via Infotrieve] |