©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Cytidine Monophospho-N-acetylneuraminic Acid Hydroxylase
REGULATION OF SPECIES- AND TISSUE-SPECIFIC EXPRESSION OF N-GLYCOLYLNEURAMINIC ACID (*)

Takehiro Kawano (1)(§), Susumu Koyama (3), Hiromu Takematsu (3), Yasunori Kozutsumi (3), Hiroshi Kawasaki (2), Seiichi Kawashima (2), Toshisuke Kawasaki (3), Akemi Suzuki (1)(¶)

From the (1)Department of Membrane Biochemistry, and (2)Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113 and the (3)Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase, which is the key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), has been purified from the cytosolic fraction of mouse liver, as described in our previous paper. The amino acid sequences of the purified CMP-NeuAc hydroxylase, and peptides obtained by lysylendopeptidase digestion, were used to synthesize specific oligonucleotide primers. A mouse cDNA clone of the enzyme was obtained by a combination of the polymerase chain reaction and rapid amplification of cDNA ends. The sequence of the clone contained an open reading frame coding for a protein of 577 amino acids with a predicted molecular mass of 66 kDa. The deduced sequence included the amino acid sequences obtained for the purified enzyme and peptides, and a complete match was obtained for 159 residues. The enzyme has neither a signal peptide sequence nor a membrane spanning domain, which is consistent with localization of the enzyme in the cytosol. Transfection of a cDNA construct to COS-1 cells increased the enzyme activity and the amount of NeuGc. Comparison of the sequence with GenBank data indicated that no similar sequence has been reported so far. Northern blot analysis of various mouse tissues with the enzyme cDNA as a probe indicated that expression of NeuGc is related to the level of CMP-NeuAc hydroxylase mRNA. On Southern blot analysis with the same probe, cross-hybridizing bands were detected in the human and fish genomes.


INTRODUCTION

Cell membrane sialic acid is involved in cell-cell (1) and cell-pathogen interactions (2, 3) and in binding of cells to extra cellular matrix(4) . Sialic acid is a generic designation used for N-acylneuraminic acids and their many derivatives(5, 6) . N-Acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc)()are two of the most abundant derivatives, and both may be further modified by O-acetylation. Some reports suggested that NeuGc was produced from NeuAc through enzymatic hydroxylation of the N-acetyl residue of free NeuAc, CMP-NeuAc, or glycoconjugate-linked NeuAc(7, 8) , but recent results have indicated that the major mechanism for biosynthesis of NeuGc is hydroxylation of CMP-NeuAc(9, 10, 11) . In addition, the hydroxylation is carried out by an electron transport system, which includes NADH-dependent cytochrome b reductase, cytochrome b, and CMP-NeuAc hydroxylase. The hydroxylase accepts electrons from cytochrome b and catalyzes the terminal reaction(12, 13, 14) . We have purified CMP-NeuAc hydroxylase from the cytosolic fraction of mouse liver and demonstrated that the enzyme is highly specific to CMP-NeuAc and does not use free NeuAc or NeuAc-containing GM3 as a substrate(15) .

The ratio of NeuGc to NeuAc in glycoconjugates varies among animal species (16, 17) and among tissues of a single species(18, 19, 20) . The regulation of NeuGc and CMP-NeuGc expressions in mouse and rat tissues was suggested to be dependent on the level of CMP-NeuAc hydroxylase activity(14, 19) . NeuGc is barely detectable in the brain of most mammals, although the sialic acid content of brain is quite high(21) . Polysialic acids of N-CAM and gangliosides, which are regarded to be functionally important in neural tissues, contain NeuAc and not NeuGc. Thus, brain-specific suppression of NeuGc is evident but its functional role is unknown. Normal chicken and human tissues do not contain NeuGc. The occurrence of NeuGc in human T cells activated in vitro (22) and various types of human cancer tissues (23, 24, 25, 26, 27) was demonstrated by immunological methods. Although the occurrence of NeuGc remains to be confirmed by chemical methods, it is possible that NeuGc would be an excellent cancer-associated antigen or tumor marker.

The specialized physiological functions of NeuAc and NeuGc are not clear at present. Influenza virus hemeagglutinins bind glycoconjugates that contain NeuAc and NeuGc with different affinities, and the neuraminic acids may play a role in determining susceptibility to viral infection(28) . A cell adhesion molecule on marginal zone macrophages, sialoadhesin, preferentially recognizes NeuAc-Gal-GalNAc structure but not NeuGc-Gal-GalNAc(29) . These examples suggest that sialic acid difference is biologically important in recognition events mediated by carbohydrates.

To elucidate the mechanisms for species- and tissue-specific and cancer associated regulations of NeuGc expression, information about the structure of CMP-NeuAc hydroxylase is required. Here we report the cDNA cloning of CMP-NeuAc hydroxylase, analysis of its mRNA expression in mouse tissues, and detection of the similar sequences in several animal species.


EXPERIMENTAL PROCEDURES

Protein Sequencing

Purification of CMP-NeuAc hydroxylase was described previously(15) . An aliquot (37 µg) of the finally purified enzyme was lyophilized and then dissolved in 40 µl of 8 M urea. After dilution with 120 µl of 0.1 M Tris-HCl buffer, pH 9.0, 0.4 µg of lysylendopeptidase (Wako Pure Chemicals) was added, followed by incubation at 37 °C overnight. The resulting peptide fragments were separated by HPLC on a Wakosil 5C18 column (4 150 mm; Wako Pure Chemicals). Elution was performed with a linear concentration gradient of acetonitrile, 0-60%, in 0.1% trifluoroacetic acid, in 60 min. Twelve major peaks were recovered and subjected to protein sequencing using an Applied Biosystems 470A protein sequencer. NH-terminal amino acid sequencing was also performed using 6 µg of the purified hydroxylase.

Polymerase Chain Reaction (PCR) with Degenerate Oligonucleotides

Degenerate oligonucleotides designed on the basis of the amino acid sequences of the lysylendopeptidase-digested peptides and the intact enzyme, as shown in Fig. 1A, were synthesized with an Applied Biosystems 391 DNA synthesizer. Each oligonucleotide primer contained either an EcoRI or HindIII recognition sequence at the 5`-end. A pool of cDNA was prepared by reverse transcription with random primers using mouse liver total RNA as a template. PCR was performed using sense and antisense primers (125 pmol each), and 1.3 units of Taq DNA polymerase (Promega) in a reaction mixture (25 µl) containing 1 µg of cDNA(30) . Amplification was carried out by 30 cycles of 94 °C for 0.5 min, 37 °C for 1 min, and 72 °C for 1.5 min, using a Zymoreactor II thermal cycler (Atto Corp.). The PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide.


Figure 1: A, NH-terminal amino acid sequence and partial amino acid sequences of lysylendopeptidase-digested peptides a and b. The sequences of the degenerate oligonucleotide primers (pr. 1, pr. 2, and a-pr. 1) used for the PCR experiments are shown under the corresponding amino acid sequences (underlined). Boxes show the linker sequences (GAATTC, EcoRI; and AAGCTT, HindIII). B, amplification of cDNA fragments by PCR using the above mentioned primers and newly synthesized completely matched primers (pr. 3, a-pr. 2, a-pr. 3, and a-pr. 4). Hooked arrows indicate the positions and directions of these primers. The sequences of these primers are shown in Fig. 2. pr. 3 and a-pr. 2 have linker sequences TCTAGA (XbaI) and CTCGAG (XhoI), respectively.



Isolation and Characterization of a cDNA Clone

After being digested with appropriate restriction enzymes, the PCR amplified fragments were subjected to electrophoresis, excised from the gel, purified with a QIAEX gel extraction kit (Qiagen), and then subcloned into Bluescript II KS (Stratagene). The constructs were transfected into XL1-Blue strain. Clones with the inserts were identified by color selection with isopropyl-1-thio--D-galactopyranoside and 5-bromo-4-chloro-3-indolyl--D-galactoside, and plasmid DNAs were isolated from Escherichia coli grown in small scale cultures(31) . The sequencing reaction with a thermal cycler was performed with Dye Deoxy terminators (Applied Biosystems) and Ampli Taq DNA polymerase (Applied Biosystems). The M13 universal primer or several synthetic oligonucleotide primers were used. Gel electrophoresis and analysis of data were performed with a 373A DNA sequencer (Applied Biosystems). Three clones were sequenced to compensate for misreading by Taq polymerase. In the case of the 5`-non-coding sequence, the PCR amplified fragments were directly sequenced, without subcloning, using specific oligonucleotides as primers.

Hydropathy indexes by Kyte-Doolittle (32) were estimated with a sliding window of nine amino acids.

Expression of the cDNA in COS-1 Cells

To amplify the entire coding region of CMP-NeuAc hydroxylase, two primers, each of which contains either the 5`- or 3`-end of coding region and BamHI recognition sequence at the 5`-end, were synthesized. Amplified fragments with mouse liver cDNA were digested with BamHI (Takara Shuzo Corp.) and inserted into a Bluescript II vector. An insert, the sequence of which was confirmed to be the same as the cloned sequence, was ligated into BamHI site of the expression vector, pdKCR(33) . A construct of the correct direction was amplified in a large scale, and purified by two cycles of CsCl gradient centrifugation(31) . COS-1 cells obtained from the Japanese Cancer Research Bank were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co. Ltd.) with 10% fetal calf serum (Life Technologies, Inc.) and kanamycin. Six dishes of COS-1 cells (5 10 cells/10 cm dish) were transfected with the purified plasmid (20 µg/dish) by calcium phosphate precipitation methods(34) . At 60 h of incubation, the cells were harvested, washed, and collected by centrifugation. One-fifth of the pelleted cells was subjected to the determination of sialic acid species according to Hara et al.(35) . Briefly, the pelleted cells were hydrolyzed in 0.9 ml of 0.05 N HSO at 80 °C for 3 h, and released sialic acids were treated with 3.6 ml of 3. 5 mM of 1,2-diamino-4,5-methylenedioxybenzene in 1.7 M acetic acid at 50 °C for 2.5 h. NeuAc and NeuGc were determined by HPLC with a TSK-gel ODS-80TM column (Tosoh). Four-fifths of the pelleted cells were homogenized, and the cytosolic fraction was used for the determination of protein concentration and CMP-NeuAc hydroxylase activity as previously reported (14).

Northern Blot Analysis

Poly(A) RNA from mouse tissues was prepared using an ISOGEN acid phenol procedure kit (Wako Pure Chemicals) and Oligotex dT (Takara Shuzo Corp.), following the manufacturers' protocols. Approximately 3 µg of poly(A) RNA samples was subjected to electrophoresis in a 1% agarose gel containing formaldehyde and then transferred to a MagnaGraph nylon membrane filter (Micron Separations Inc.). A radiolabeled probe (3 10 counts/min/µg) was prepared with a gel-purified cDNA containing the whole coding region of CMP-NeuAc hydroxylase or 2.0-kb human -actin cDNA (Clontech) using a random primer DNA labeling kit (Takara Shuzo Corp.). Hybridization was performed overnight at 42 °C in 50% formamide, 5 SSC (1 SSC, 150 mM NaCl, and 15 mM sodium citrate, pH 7.0), 50 mM sodium phosphate buffer, pH 7.0, 0.5% skim milk, 1.5% SDS, and 100 µg/ml yeast RNA. The blot was washed twice in 1 SSC and 0.1% SDS at 65 °C for 10 min, and then three times in 0.1 SSC and 0.1% SDS at 65 °C for 20 min. The bands were visualized and quantified with a Fujix BAS 2000 Bio-imaging Analyzer (Fuji Photo Film).

Southern Blot Analysis

A BIOS EVO Blot digested with EcoRI was purchased from BIOS Laboratories. The amount of DNA blotted is adjusted to assure the same copy number of the genome available among seven species. Preparation of a radiolabeled probe was performed in the same way as described for Northern blot analysis. Hybridization was performed essentially as for Northern blot analysis except that the proportion of formamide was decreased to 37.5%. After overnight hybridization, the blot was washed with 1 SSC and 0.1% SDS at room temperature, 50 °C, and finally 65 °C. The radioactivity was monitored with a Fujix BAS 2000 Bio-imaging Analyzer. Discrete bands were visible only after the washing at 65 °C.


RESULTS

Protein Sequencing and Amplification of cDNA Fragments by PCR

The amino acid sequences of the two major peptides and the NH terminus are shown in Fig. 1A, and these sequences were used for designing mixed oligonucleotide primers for PCR. First, a fragment of 158 bp was amplified from mouse liver cDNA with sets of degenerated primers designed for peptides a and b (pr. 2 and a-pr. 1, respectively, in Fig. 1A). This fragment could not be amplified with the cDNA prepared from mouse brain, which was used as a negative control because it does not express CMP-NeuAc hydroxylase(14) . The amplified fragment was subcloned into Bluescript II KS and then sequenced. Based on the sequence, completely matched oligonucleotide primers (a-pr. 2 and pr. 3, see Fig. 1B and 2) were synthesized for further experiments. The next PCR was performed between pr. 1, designed for the NH-terminal amino acid sequence, and a-pr. 1. On this PCR, no specific band was visibly amplified, but when an aliquot of the reaction product was diluted and subjected to a second PCR (30) with a primer corresponding exactly to an inner sequence (a-pr. 2) and pr. 1, a fragment of 1.5 kb pairs was amplified. This fragment seemed to cover the whole coding region of the enzyme except for the 5`- and 3`-ends, as judged on comparison of its size with the molecular mass of the hydroxylase(15) . Since the fragment had an inner EcoRI site, the PCR product was digested with EcoRI, ligated into the EcoRI site of Bluescript II KS, and then sequenced. Antisense primers (a-pr. 3 and a-pr. 4) were synthesized for the following experiments (see Fig. 1B and 2).

Rapid Amplification of the 5`- and 3`-cDNA Ends

For amplification of the 5`-end of the cDNA, a template was prepared by reverse transcription with a-pr. 3 and polyadenylation of the 5`-end (30). A 650-bp fragment was amplified by PCR with oligo(dT) and a-pr. 4, as shown in Fig. 1B, and a part of the fragment was sequenced. The 3`-end of the cDNA was also amplified with pr. 3 and oligo(dT), using a cDNA prepared by reverse transcription with oligo(dT) as a primer. Approximately 250- and 700-bp fragments were amplified. The 250-bp fragment and a part of the 700-bp fragment were sequenced, and the sequences indicated that the 700-bp fragment included the 250-bp fragment and both contained an 89-bp coding sequence.

Nucleotide Sequence and Expression of CMP-NeuAc Hydroxylase

The merged sequence of the overlapping cDNA fragments indicated a single open reading frame, and about 100-bp 5`- and 3`-untranslated regions. The nucleotide sequence (1731 bp) and the deduced amino acid sequence (577 amino acids) for the open reading frame are shown in Fig. 2. Although the 5`-untranslated region has not been completely sequenced, stop codons were emerged at -93, -59, and -46 in each of three frames. There are adjoining methionines at the potential site for translation initiation. The upper methionine was tentatively taken as an initiation site. The first four amino acids at the NH terminus could not be detected on sequencing of the purified hydroxylase, and thus these amino acids were cleaved, possibly through post-translational processing or artificial cleavage during the purification. All the amino acid sequences of the 12 lysylendopeptidase-digested peptides and NH terminus of the purified enzyme, 159 amino acid residues long in total, were found in the open reading frame of the deduced amino acid sequence (underlined in Fig. 2). The molecular mass calculated from the deduced amino acid sequence is 66 kDa, which is in good accordance with that determined by SDS-polyacrylamide gel electrophoresis under reducing conditions (64 kDa)(15) . A search of GenBank data revealed no similar sequence reported so far. A hydropathy plot (32) of the enzyme did not identify a potential transmembrane region, suggesting that the enzyme is translated on free ribosomes as a cytosolic protein.


Figure 2: Nucleotide sequence and deduced amino acid sequence of CMP-NeuAc hydroxylase. The sequences corresponding to the NH terminus and the lysylendopeptidase-digested peptides of the enzyme are indicated by underlines. There are two potential sites for translation initiation. The upper Met is taken as site 1 here. The sequences corresponding to the synthesized primers are boxed.



As shown in , the COS-1 cells transfected with the cloned cDNA contained an increased amount of NeuGc, compared with a mock transfectant. This change is confirmed to be due to the increase of CMP-NeuAc hydroxylase activity. The results all togehter indicate that the cloned cDNA encodes for mouse CMP-NeuAc hydroxylase.

Northern Blot Analysis

To test the possibility that the expression of CMP-NeuAc hydroxylase regulates the tissue-specific expression of NeuGc-containing glycoconjugates, mRNA isolated from mouse brain, thymus, liver, kidney, and spleen was probed at high stringency with a radiolabeled cDNA fragment. It was reported that NeuGc was hardly detected in the brain gangliosides, whereas both NeuAc and NeuGc were expressed in the other tissues(18) . Consistent with this observation, 3.6 and 10 kb mRNA bands were detected for all tissues except brain (Fig. 3). The intensity ratio of 3.6 and 10 kb mRNA bands is almost the same among tissues expressing NeuGc, and both bands are not detected in the brain. The 10-kb transcript, which is much longer than the cDNA we obtained, would be a product of alternative splicing or use of alternative polyadenylation signals in a 3`-untranslated region.


Figure 3: Northern blot analysis of CMP-NeuAc hydroxylase. Approximately 3 µg of poly(A) RNA obtained from mouse liver (l), brain (b), thymus (t), spleen (s), and kidney (k) was subjected to electrophoresis and probed at high stringency with a radiolabeled cDNA containing the whole coding region of CMP-NeuAc hydroxylase or 2.0-kb human -actin cDNA. The positions and sizes of marker RNAs are indicated at the right.



Southern Blot Analysis

As shown in Fig. 4, human and fish genomic DNAs contain cross-hybridizing sequences, whereas chicken, frog, lobster, and mussel DNAs were negative. These data are quite interesting because expression of NeuGc was detected in several human cancerous tissues (23, 24, 25, 26, 27) and in polysialoglycoproteins of several kinds of fish (36) but not in chickens or frogs(37) . Sialic acid have not been detected in bivalves to date(38) .


Figure 4: Genomic Southern blot analysis of various animal species. Blots containing EcoRI digested genomic DNA of man, mouse, chicken (Gallus domesticus), frog (Xenopus laevis), lobster (Homarus americanus), mussel (Mytilus edulis), and a fish (Tautoga onitis) were probed at low stringency with a radiolabeled CMP-NeuAc hydroxylase cDNA. The positions and sizes of marker DNA (-HindIII digest) are indicated at the left.




DISCUSSION

Knowledge of the amino acid sequence of CMP-NeuAc hydroxylase enabled us to design oligonucleotide primers and to generate cDNA that encoded the entire protein and approximately 100 bp of both 5`- and 3`-untranslated regions. The structure of this enzyme is not similar to other known hydroxylases, which is consistent with its unique substrate specificity for CMP-NeuAc. Several sialyltransferases contain a conserved amino acid sequence known as the ``sialylmotif,'' which may be a site that recognizes sialic acid(39) , but this motif was not present in the CMP-NeuAc hydroxylase.

The enzyme was purified from the cytoplasm(15) , but electron transport systems are usually located in microsomal membranes. Alternatively, one might suppose that the hydroxylase is a transmembrane protein or contains a phosphatidylinositol-anchor and that it is artificially solubilized during the purification by the action of proteinases or phospholipase C. However, the nucleotide sequence of the hydroxylase obtained in this study did not contain transmembrane domain and signal peptides for membrane insertion, confirming the cytosolic origin of the enzyme. We suggest the following models to explain how the soluble enzyme interacts with the electron transport system. 1) soluble CMP-NeuAc hydroxylase interacts for a short time with cytochrome b on microsomes, when the transfer of electrons occurs; or 2) soluble forms of cytochrome b and NADH-cytochrome b reductase are involved in the reaction. At present, the latter idea is unlikely because mRNA encoding only the microsomal form of cytochrome b was observed in mouse liver(40) . To clarify this point, reconstitution experiments mimicking the in vivo interaction between soluble CMP-NeuAc hydroxylase and the membrane-bound electron transport system are required.

The relationship between the level of mRNA of CMP-NeuAc hydroxylase and the expression of NeuGc in glycoconjugates was confirmed in several tissues. The inability of the brain to produce NeuGc-containing glycoconjugates (18, 21) was evidenced by the absence of the CMP-NeuAc hydroxylase mRNA (Fig. 3). Sialic acid-containing glycoconjugates in the brain are considered to be functionally important. For example, polysialylation of N-CAM reduces homophilic interaction and cellular adhesion(1) , tetrasialoganglioside GQ1b potentiates neuritogenesis(41) , and gangliosides exhibit cell type-specific distribution(42, 43) . Interestingly, the sialic acids of these functionally important sialoglycoconjugates are all NeuAc. It is unclear at present why the expression of NeuGc should be suppressed in the brain. Overexpression of the hydroxylase in the brain of experimental animals could provide insight into this issue.

Although the expression of NeuGc-containing glycoconjugates was detected in some cancerous tissues(23, 24, 25, 26, 27) , the existence of human CMP-NeuAc hydroxylase has not been documented previously. The detection of a cross-hybridizing sequence in the human genome suggests participation of CMP-NeuAc hydroxylase activation in the tumor-associated expression of NeuGc-glycoconjugates. It will be interesting to determine if hydroxylase mRNA can be detected in cancerous tissues. A related fragment was also detected in a fish genome. In some salmonid fish, the species-specific expression of poly-NeuGc-glycoproteins in the eggs was reported(36) . Therefore, investigation of the expression of the CMP-NeuAc hydroxylase gene in salmonid fish eggs is also of interest.

CMP-NeuAc hydroxylase, which regulates the overall velocity of CMP-NeuAc hydroxylation, may be responsible for the species- and tissue-specific expression of NeuGc-containing glycoconjugates(14) . Since the synthesis of oligosaccharide structures is dependent upon not only the level of glycosyltransferases, but also the amounts of the acceptor oligosaccharides and donor nucleotide sugars, the activities of glycosyltransferases do not always correlate well with the abundance of oligosaccharide structures expressed in the cells. On the other hand, CMP-NeuAc hydroxylase modifies the structure of the donor and may directly regulate the expression of NeuGc in glycoconjugates. The expression of NeuGc-glycoconjugates may also be regulated by factors other than the hydroxylase itself, e.g. post-translational modification of the enzyme, regulation of the accessibility of cytochrome b and cytochrome b reductases, reutilization of incorporated NeuGc or NeuGc-glycoconjugates as a source of CMP-sialic acids(44) , and involvement of sialyltransferases and CMP-sialic acid transporters specific to either CMP-NeuAc or CMP-NeuGc, although there are no data to support such specificity at present(45, 46) . Modification of sialic acids from NeuAc to NeuGc by artificial expression of CMP-NeuAc hydroxylase in cultured cells or animals is now possible because of availability of cDNA. Further studies of the enzyme, using the hydroxylase cDNA, will facilitate elucidation of functions of NeuGc-containing glycoconjugates and the mechanisms of NeuGc expression in ontogeny and phylogeny.

  
Table: Expression of cloned cDNA in COS-1 cells

After 60 h, the transfectant and the mock transfectant were harvested and subjected to the sialic acid analysis and the CMP-NeuAc hydroxylase assay. CMP-NeuAc hydroxylase activities in the cytosolic fractions were measured.



FOOTNOTES

*
This work was partly supported by Grants-in-Aid 05274107 and 05858084 for Scientific Research in Priority Areas, from the Ministry of Education, Science and Culture of Japan, and a grant from the Human Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) D21826.

§
Present address: Dept. of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan.

To whom correspondence should be addressed: Dept. of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3823-2101 (ext. 5483); Fax: 81-3-5685-6607; E-mail: asuzuki@rinshoken.or.jp.

The abbreviations used are: NeuGc, N-glycolylneuraminic acid; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s). The abbreviations for gangliosides follow Svennerholm's nomenclature system (47).


ACKNOWLEDGEMENTS

We are grateful to Drs. Kiyomitsu Nara, Hideo Kubo, and Yoko Nadaoka of the Tokyo Metropolitan Institute of Medical Science for the suggestions and technical support, and Dr. Hiroshi Kurata, Yukiko Tamagawa, and Mitsuyo Ohkawa of Kyoto University for their technical advice and support. The authors also wish to thank Prof. Donald M. Marcus for revising the manuscript and Prof. Tamio Yamakawa for his encouragement.


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