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Differential Expression of Three Sialidase Genes in Rat Development
Biochemical and Biophysical Research Communications 280, 726 –732 (2001) doi:10.1006/bbrc.2000.4186, available online at http://www.idealibrary.com on
Differential Expression of Three Sialidase Genes in Rat Development Takafumi Hasegawa,* ,† Carmen Feijoo Carnero,* Tadashi Wada,* Yasuto Itoyama,† and Taeko Miyagi* ,1 *Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, Natori, Miyagi 981-1293, Japan; and †Department of Neurology, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan
Received November 24, 2000
Mammalian sialidases have been reported to give a great influence on a number of cellular functions including cell differentiation and cell growth by removal of sialic acids from glycoproteins and gangliosides. To understand the roles of the sialidases during development, we investigated expression pattern of three types of sialidase in developing rat brain and liver. For this purpose we cloned a new membrane-associated sialidase cDNA from rat brain. The cDNA encodes 418 amino acids containing three ASP-boxes characteristic of sialidases and the major transcript of 3.5 kb is highly expressed in brain and cardiac muscle but low in liver. Competitive polymerase chain reaction methods were developed to evaluate the mRNA level together with activity assays in comparison with cytosolic and lysosomal sialidases previously obtained. The results indicate that the expression of individual sialidase genes is spatiotemporally controlled with distinct roles in determining the concentration and components of sialo-glycoconjugates during development. © 2001 Academic Press Key Words: sialidase; gangliosides; sialic acid; cDNA cloning; development; competitive PCR.
Sialic acids and their derivatives are ubiquitous at the terminal positions of oligosaccharides of glycoproteins and glycolipids in mammalian cells, and the roles have been implicated in various biological phenomena, such as cell proliferation, differentiation, signal transduction, cell surface interactions (1–5). Studying the developmental patterns of sialo-glycoconjugates in mammalian tissues is of interest, especially in the nervous system, because growth spurts such as neurogenesis, neuritogenesis, synaptogenesis and myelination 1 To whom correspondence should be addressed at Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, 47-1 Nodayama, Medeshima-shiode, Natori, Miyagi 981-1293, Japan. Fax: 81-22-381-1195. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
are characterized by striking alterations in concentration and composition of cell surface gangliosides (6 –9). In fact, during the second and third weeks of life, there is both an accumulation and a change in composition of gangliosides and sialoglycoproteins in mammalian brain (7, 8), and the turnover rate of rat brain gangliosides was found to be highest in this period (10). The contents of gangliosides and sialo-glycoproteins are mainly determined by the levels of sialyltransferase and sialidase and also by their proximity to and levels of substrate-oligosaccharides (11–15). Although not a few such enzymes have been identified and characterized so far, observations on the developmental pattern in neural and extraneural tissues remains scanty. To our knowledge, no information is available on the molecular basis of developmental changes of sialidase. In the light of recently obtained information on sialidase molecular cloning, we have now examined the expression patterns of three types of rat sialidase, including a newly cloned membrane sialidase. Mammalian sialidases have been classified based on their subcellular distribution and three types have recently been cloned: cytosolic, lysosomal and plasma membrane sialidases (16). In the present work, we have isolated cDNAs encoding rat membraneassociated sialidase and expression of this and two other rat sialidase genes was evaluated by the competitive reverse transcription-polymerase chain reaction (RT-PCR) method (17) as well as by activity assays in developing rat brain and liver. MATERIALS AND METHODS Animals and tissue preparation. Sprague-Dawley rats were purchased from Japan SLC (Shizuoka, Japan). Brain and liver tissues were obtained from the embryos at gestational days 17 and 19, postnatal (days 0, 2, 4, 6, 9, 11, 18, 30), and adult (day 50) rats. They were pooled from 8-9 embryonic and 3-4 postnatal rats, yielding about 0.4-0.5 g to avoid irregularity among the animals, and kept frozen at ⫺80°C until use.
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Cloning of a membrane-associated sialidase from rat brain. A rat membrane-associated sialidase cDNA was obtained by homologybased RT-PCR using rat brain poly (A) ⫹ RNA prepared by acid guanidium-phenol-chloroform extraction methods (18) followed by oligo (dT) column chromatography. The primers 7S (5⬘-GGACACCGGACCATGAACCCCTGTCCT-3⬘) and 9A (5⬘-CCTGGCCC CACAGCAAAAGTGGCCCA-3⬘) were designed from sequences in regions in which the amino acids of bovine membrane sialidase (19) are identical to those of rat cytosolic sialidase (20). They were used for PCR with the template cDNA of rat brain under the following conditions; initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min and extension at 72°C for 2 min with final elongation at 72°C for 10 min. The PCR products were subcloned into pBluescript and sequenced with an ALF DNA autosequencer (Amersham Pharmacia) using a Thermo Sequenase Cycle Sequencing Kit (Amersham Pharmacia). The 245 bp fragment (RBI) thus obtained was used as a probe for library screening. RBI was radiolabeled with [␣- 32P]-dCTP using a Random Primer DNA Labeling Kit (Takara, Japan) for screening of a 5⬘-stretch gt10 cDNA rat brain library (Clontech). Hybridization with the radiolabeled RBI probe was carried out on Hybond-N ⫹ membranes (Amersham Pharmacia) at 65°C overnight. The filters were washed and used to expose to an X-ray films at ⫺80°C overnight. Positive clones were isolated, subcloned, and sequenced as described previously (21). Expression of rat membrane-associated sialidase cDNA in COS-1 cells. The entire open reading frame (ORF ) of rat membrane sialidase cDNA was PCR-amplified using primers, 1S (5⬘-CCCGAATTCGTCATGGAAGAAGTTT CATCC-3⬘) and 2A (5⬘-CCCGAATTCTTAGTTGCTACTAGGGCTGGTA-3⬘) and subcloned into a pME18S expression vector. COS-1 cells (Riken Cell Bank, Japan) were transfected by electroporation (at 240 V, 975 F) with the empty vector and the vector containing entire ORF (pMErmSD) of the membrane sialidase cDNA using Bio-Rad Genepulser. After 48 h, cells were pelleted, lysed in 9 volumes of ice-cold PBS by sonication and centrifuged at 1,000 ⫻ g, 4°C for 10 min. The supernatant was assayed for sialidase activity with various substrates, including gangliosides. Sialidase assay. Tissue homogenates (1:8, w/v) were prepared in ice-cold sucrose solution containing 0.25 mM sucrose, 10 mM phosphate buffer (pH 7.0), 1 mM EDTA and 0.5 mM phenylmethanesulfonyl fluoride using a glass homogenizer at 1000 rev./min with 7-strokes. After centrifugation at 1000 ⫻ g, 4°C for 10 min, the crude homogenates were further centrifuged at 100000 ⫻ g, 4°C for 1 h. The resultant supernatant was used as the enzyme fraction for cytosolic sialidase, and the sediment homogenized with 0.25 M sucrose solution was applied as the enzyme source for membrane and lysosomal sialidases. The reaction mixture (200 l) for sialidase assay contained 30 nmol of substrate as bound sialic acid, 0.2 mg of BSA, 10 mol of sodium acetate buffer (pH 4.6 or 5.5). The membrane sialidase activity was assayed by incubating the particulate fraction in pH 4.6 buffer with (for total activity) or without (for endogeneous activity) bovine brain mixed gangliosides (Sigma) in the presence of 0.1% Triton X-100, whereas for the assays of cytosolic and lysosomal sialidases the cytosolic and particulate fractions were incubated at pH 5.5 and 4.6, respectively, with synthetic substrate 4-methylumbellyferone-neuraminic acid (4MU-NeuAc). After incubation at 37°C for 2 h, released sialic acids were determined by the thiobarbituric acid method after passing through an AG1X-2 minicolumn and 4MU by fluorometric measurement, respectively, as described elsewhere (22). Quantitative PCR methods. A rat lysosomal sialidase cDNA (Accession No. AB035722) was obtained with homology-based hybridization by screening a rat liver cDNA library using human lysosomal sialidase cDNA as a probe, which was generously provided by Dr. Milner (23), and was confirmed to encode an acidic sialidase for
4MU-NeuAc in the same manner described above. The cDNA competitors were prepared by single- or double-digestion of the Bluescript vectors containing entire ORFs of each sialidase cDNAs with BstPI and BglII for membrane sialidase, XcmI for cytosolic (20), and BbsI for lysosomal sialidase (Accession No. AB035722), respectively. From 5 g of total RNA, cDNA was synthesized using 200 units of Moloney murine leukemia virus-reverse transcriptase and oligo (dT) primers in a final volume of 20-l. The 25-fold diluted cDNA (10 l )was subjected to quantitative PCR analysis with the competitors adjusted to the following concentrations: 40 fg (brain) and 6 fg (liver) for membrane, 8 fg (brain) and 0.4 fg (liver) for cytosolic, 50 fg (brain) and 25 fg (liver) for lysosomal sialidase. PCR was performed with primers rm4S (5⬘-CCTACTTGATATCATGCTGG-3⬘) and rm4A (5⬘TTAGTTGCTACTAGGGCTGG-3⬘) for membrane (target: 718 bp, competitor: 484 bp), rc3S (5⬘-CCGTCCAGGACCTCACAGAG-3⬘) and rc3A (5⬘-TCACTGAGCACCATGTACTG-3⬘) for cytosolic (target: 727 bp, competitor: 387 bp), rlSD12S (5⬘-TTGGAGTAAG GATGACGGCC-3⬘) and rlSD10A (5⬘-TCAAAGCGTGCCGTAGACGC-3⬘) for lysosomal sialidases (target: 707 bp, competitor: 401 bp), respectively. PCRs were conducted under the conditions described previously (21). The annealing temperatures and PCR cycles were 52, 55, and 57°C and 40, 36 and 38 cycles for membrane sialidase, cytosolic sialidase, and lysosomal sialidase, respectively. To normalize for sample variation, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (24) was also measured as an internal control using a GAPDH competitor, prepared by digestion of the 0.5 kb cDNA (nucleotides 566 to 1017) with StyI and BspMI. After amplification, 10-l aliquots were electrophoresed in 1.5% agarose gels. Gel photos were scanned and densitometric analyses were performed using the NIH-image program, version 1.61.
RESULTS AND DISCUSSION Isolation of a cDNA encoding rat membraneassociated sialidase. cDNA from rat brain poly(A) ⫹ RNA was amplified by means of RT-PCR using primers designed from the conserved amino acids between bovine membrane and rat cytosolic sialidases. The deduced amino acid sequence of a PCR product RBI showed 89% and 82% identity to the corresponding regions of mouse (21) and human membrane (25) ganglioside sialidases, respectively. By screening of the gt10 rat brain cDNA library (1.2 ⫻ 10 6) with the RBI probe, we isolated four individual clones of about 0.9 to 2.4 kb. Sequence analysis of the clones revealed that the longest clone consisted of 2307 nucleotides having an open reading frame (ORF) encoding 418 amino acids with a molecular mass of 47.0 kDa (Fig. 1). There was a putative hydrophobic segment at nucleotides 716 to 784, and the sequence motif YRIP (amino acids 24 –27) and three copies of Asp-box consensus sequences (SXDXGXS/TW) (26) were also present. The highest sequence identity was observed with mouse membrane sialidase (81.3%, Accession No. AB026842), with a slightly less identity evident with human (67.5%, AB008185) and bovine (66.0%, AB008184) membrane sialidases. It should be noted that the sequence identities of cytosolic and lysosomal sialidases between the rat (Accession No. D164300 and AB035722, respectively) and mouse (AB028023 and Y11412, respectively) are higher (90 –95%) than for the membrane sialidase case. Northern blot analysis with poly (A)-
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riched RNAs from rat brain, cardiac muscle and liver exhibited a major 3.5 kb transcript with minor 3.1 and 1.6 kb forms expressed most highly in cardiac muscle but at very low levels in liver (data not shown). Transient expression in COS-1 cells showed an over 400fold increase in sialidase activity toward gangliosides as compared with mock-transfected cells (Table 1). Most of the activity (91%) in the crude homogenates was recovered in the particulate fractions, indicating it to be membrane-bound. Like other membrane sialidases, the expressed sialidase hydrolyzed gangliosides GD3 and GM3 preferentially, rather than GM2, and at a significant degree 4MU-NeuAc and oligosaccharides such as sialyllactose in the presence of Triton X-100 with an optimum pH at 4.8 to 5.0. The similarities in primary structure and enzymatic properties of mouse, bovine, and human membrane sialidases imply that this rat sialidase is a homologue of the other two sialidases. Developmental changes in sialidase activity. To determine how the expression of the membrane sialidase is altered during rat development, we evaluated the level by activity assays and competitive RT-PCR. Developmental changes of sialidase activity in rat brain and liver examined were then compared with those of cytosolic and lysosomal sialidases as shown in Fig. 2. Based on the previous studies of sialidase substrate specificity (23, 27, 28), we attempted differential activity assays for three sialidases. Activity toward gangliosides at pH4.6 in the presence of TritonX-100 can be considered as due to membrane-associated sialidase, whereas the activities toward 4MU-NeuAc at pH 6.0 in the cytosolic fraction and at pH 4.5 in the particulate fraction are for cytosolic and lysosomal sialidases, respectively, as described in the “Materials and Methods.” We first measured the membrane sialidase activity toward endogenous and endogenous plus exogenous (total) ganglioside substrates when only the particulate fraction of rat brain was used as the enzyme source, because the fraction is highly abundant in gangliosides, possibly acting as endogenous substrates for the sialidase. As shown in Fig. 2, the total activity of the membrane sialidase was somewhat higher than the endogenous activity in the developing brain. In brain, a gradual increase of the sialidase activity was observed by 3.6-fold (total) and 4.3-fold (endogenous) between gestational day 17 and the 18th day after birth. This was followed by slight temporary decline by the 30th day then a slight rise by the 50th day. This
FIG. 1. Nucleotide and deduced amino acid sequences of rat membrane-associated sialidase. The predicted amino acid sequence is shown by the single letter amino acid code under the nucleotide
sequence. The hydrophobic domain in membrane sialidase is underlined. Asp-boxes are boxed and the F/YRIP motif is indicated by a dotted line. The nucleotide sequence has been submitted to the DDBJ, EMBL and GenBank Databases with accession number AB026841.
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Sialidase Activities Toward Various Substrates in the Homogenates of COS-1 Cells Transfected with the Mouse Ganglioside Sialidase cDNA pME18S
pMErmSD a
a b
a
Substrate
Specific activity (units/mg protein)
Specific activity (units/mg protein)
Hydrolysis relative b to GD3 (%)
GD3 GD1a GM3 GM2 Fetuin Sialyllactose 4MU-NeuAc
3.5 2.1 4.4 2.9 1.9 3.7 24.2
1727 1295 1476 348 66 294 397
100 75 85 20 4 17 27
The values are means of three experiments. The values are expressed as the percentage of (specific activity with pMErmSD minus the activity with pME18S) relative to that for GD3.
decline during the period of most intensive protein synthesis is likely due to the dilution of specific enzyme activity following insertion of newly synthesized proteins other than sialidase (29). The previous observations on membrane-bound sialidase in developing mouse brain reported by Segler-Stahl et al. (29)
FIG. 2. Developmental changes of membrane sialidase activity in comparison with those of cytosolic and lysosomal sialidases. The cytosolic and particulate fractions were prepared from rat brain and liver at embryonic and postnatal stages, and assayed for respective sialidase activities under the appropriate conditions described in Materials and Methods. F, membrane-associated sialidase (total activity); E, membrane-associated sialidase (endogeneous activity); ‚, cytosolic sialidase; 䊐, lysosomal sialidase. The data given are mean values from three experiments ⫾ standard deviations.
were essentially in line with our data. The developmental pattern in liver was similar to that in brain, although the activity level was much lower. To compare the activity level with those of cytosolic and lysosomal sialidases, we then performed assays using 4MU-NeuAc as substrate under appropriate conditions. With regard to cytosolic sialidase, only a trace level was detected before birth in both brain and liver. During postnatal development, the brain cytosolic sialidase increased gradually and showed a peak value (1.2-fold vs. adult value) at the 30th day and levellingoff thereafter to the adult value. In contrast, the liver enzyme showed a rapid rise in the perinatal stage and after reaching the maximum (1.2-fold vs. adult value) at postnatal day 18 the activity remained fairly constant. The activity pattern obtained for cytosolic sialidase closely resembled that in a previous report by Carubelli and Tulsiani (30), who used sialyllactose as the substrate. Lysosomal sialidase in brain appeared to be fully active in late embryonic stages. The activity exhibited a sharp decrease prior to birth and relatively high levels were maintained throughout, with a maximum at the 11th postnatal day. The study on two (light and heavy) lysosomal sialidases in developing mouse brain described by Fiorilli et al. (31) revealed the light sialidase to show marked increase of specific activity in the first 10 days, similar to our rat brain enzyme case, although they only measured enzyme activity at 4 stages of postnatal development. The lysosomal sialidase in liver, on the other hand, showed a rapid increase of activity from birth and reached a maximum (2.3-fold vs. adult value) at the 4th postnatal day and remained almost constant during adulthood. Our previous study (27) of rat liver lysosomal sialidase demonstrated two forms, located mainly in lysosomal matrix and membrane, respectively, the former hydro-
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FIG. 3. Developmental changes in membrane sialidase mRNA levels in comparison with those of cytosolic and lysosomal sialidases. The expression profiles of three sialidase genes in developing rat brain and liver were quantitatively estimated by competitive RT-PCR. (A) Agarose gel electrophoretic patterns of the products of competitive PCR analyses. mSD, membrane-associated sialidase; cSD, cytosolic sialidase; lSD, lysosomal sialidase. Black and white arrowheads indicate target and competitor DNA fragments, respectively. (B) Densitometric analyses of the results for electrophoretic patterns (A). F, membrane-associated sialidase; ‚, cytosolic sialidase; 䊐, lysosomal sialidase. The data given are mean values from three experiments ⫾ standard deviations.
lyzing 4MU-NeuAc effectively but the latter attacking gangliosides preferentially. Although we were not able to distinguish between them completely under the
present assay conditions, the activity obtained here would be expected to reflect lysosomal-matrix sialidase activity because of the use of 4MU-NeuAc as substrate
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in the absence of any detergent. Carubelli and Tulsiani (30) also described developmental changes of rat particulate-bound sialidase using sialyllactose as a substrate, the sialidase seemingly being of lysosomal nature. The present data are very similar to their results for brain, but inconsistent with liver data showing a fairly constant level during all stages, the reason for this difference being unclear. Sialidase gene expression in developing brain and liver. To estimate the amounts of membrane sialidase transcripts in developing rat brain and liver, competitive PCR was carried out as summarized in Fig. 3A (gel photo) and B (densitogram). Throughout the late embryonic and postnatal stages, lysosomal sialidase maintained the highest expression among the three sialidases, and the cytosolic sialidase showed the lowest expression. The relative mRNA levels of membrane, cytosolic and lysosomal sialidase genes at day 50 were 1.0, 0.4 and 1.6 in brain and 1.0, 0.25 and 2.0 in liver, respectively, after normalization for GAPDH as the internal control. General trends in the developmental changes of cytosolic and lysosomal sialidase expression were almost paralleled by results obtained from enzyme activity assays. In both brain and liver, transcripts of cytosolic sialidase were barely detectable in the late embryonic stage and gradually increased reaching a plateau at the 6 –11th postnatal days. On the other hand, the expression of lysosomal sialidase in brain and liver showed a slight decline before birth, and after immediate restoration relatively high levels were maintained with some fluctuation throughout the postnatal period. In addition, it is interesting that, at the second postnatal day, there was a spike increase in the amount of transcript of brain lysosomal sialidase, corresponding to a small peak in the enzyme activity at the same time point. Membrane-associated sialidase exhibited distinct differences in profiles. First, the mRNA level in brain showed a sharp increase in the perinatal stage, and soon reached a plateau level much faster (about 2 weeks) than the enzyme activity. Secondly, the enzyme activity of liver membrane sialidase was low in the late embryonal stage and even in postnatal and adult periods, whereas the mRNA level estimated was not commensurately low. This discrepancy between the level of mRNA and the enzyme activity may be partly due to posttranslational regulation of the enzyme, such as phosphorylation (32). In conclusion, this is the first report of expression patterns of three sialidase genes during development evaluated by both RT-PCR methods and activity assays. The results indicate that the genes show developmental stage-specific expression, possibly contributing to provide glycoconjugate components required during development in collaboration with specific sialyltransferases. The present results should con-
tribute to a better understanding of how sialoglycoconjugate metabolism is developmentally regulated and how these sialidases are involved in metabolic regulation. ACKNOWLEDGMENTS We are grateful to Dr. Kazuo Maruyama, Tokyo Medical and Dental University, for kindly providing the pME18S vector. We also thank Ms. Setsuko Moriya, Division of Biochemistry, Miyagi Prefectural Cancer Center, for her expert technical assistance.
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H., and Miyagi, T. (1999) Biochem. Biophys. Res. Commun. 261, 21–27. 26. Roggentin, P., Rothe, B., Kaper, J. B., Galen, J., Lawrisuk, L., Vimr, E. R., and Schauer, R. (1989) Glycoconjugate J. 6, 349 – 353. 27. Miyagi, T., and Tsuiki, S. (1984) Eur. J. Biochem. 141, 75– 81. 28. Miyagi, T., Sagawa, J., Konno, K., Handa, S., and Tsuiki, S. (1990) J. Biochem. 107, 787–793.
29. Segler-Stahl, K., Ro¨sner, H., Rahmann, H. (1981) Life Sci. 29, 1363–1367. 30. Carubelli, R., and Tulsiani, D. R. P. (1971) Biochim. Biophys. Acta 237, 78 – 87. 31. Fiorilli, A., Siniscalco, C., Chiarini, A., Francesco, L. D., Venerando, B., and Tettamanti, G. (1991) FEBS Lett. 282, 235–238. 32. Palestini, P., Pitto, M., Ferraretto, A., Tettamanti, G., and Masserini, M. (1998) Biochemistry 37, 3143–3148.
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Differential Expression of Three Sialidase Genes in Rat Development Takafumi Hasegawa,* ,† Carmen Feijoo Carnero,* Tadashi Wada,* Yasuto Itoyama,† and Taeko Miyagi* ,1 *Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, Natori, Miyagi 981-1293, Japan; and †Department of Neurology, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan
Received November 24, 2000
Mammalian sialidases have been reported to give a great influence on a number of cellular functions including cell differentiation and cell growth by removal of sialic acids from glycoproteins and gangliosides. To understand the roles of the sialidases during development, we investigated expression pattern of three types of sialidase in developing rat brain and liver. For this purpose we cloned a new membrane-associated sialidase cDNA from rat brain. The cDNA encodes 418 amino acids containing three ASP-boxes characteristic of sialidases and the major transcript of 3.5 kb is highly expressed in brain and cardiac muscle but low in liver. Competitive polymerase chain reaction methods were developed to evaluate the mRNA level together with activity assays in comparison with cytosolic and lysosomal sialidases previously obtained. The results indicate that the expression of individual sialidase genes is spatiotemporally controlled with distinct roles in determining the concentration and components of sialo-glycoconjugates during development. © 2001 Academic Press Key Words: sialidase; gangliosides; sialic acid; cDNA cloning; development; competitive PCR.
Sialic acids and their derivatives are ubiquitous at the terminal positions of oligosaccharides of glycoproteins and glycolipids in mammalian cells, and the roles have been implicated in various biological phenomena, such as cell proliferation, differentiation, signal transduction, cell surface interactions (1–5). Studying the developmental patterns of sialo-glycoconjugates in mammalian tissues is of interest, especially in the nervous system, because growth spurts such as neurogenesis, neuritogenesis, synaptogenesis and myelination 1 To whom correspondence should be addressed at Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, 47-1 Nodayama, Medeshima-shiode, Natori, Miyagi 981-1293, Japan. Fax: 81-22-381-1195. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
are characterized by striking alterations in concentration and composition of cell surface gangliosides (6 –9). In fact, during the second and third weeks of life, there is both an accumulation and a change in composition of gangliosides and sialoglycoproteins in mammalian brain (7, 8), and the turnover rate of rat brain gangliosides was found to be highest in this period (10). The contents of gangliosides and sialo-glycoproteins are mainly determined by the levels of sialyltransferase and sialidase and also by their proximity to and levels of substrate-oligosaccharides (11–15). Although not a few such enzymes have been identified and characterized so far, observations on the developmental pattern in neural and extraneural tissues remains scanty. To our knowledge, no information is available on the molecular basis of developmental changes of sialidase. In the light of recently obtained information on sialidase molecular cloning, we have now examined the expression patterns of three types of rat sialidase, including a newly cloned membrane sialidase. Mammalian sialidases have been classified based on their subcellular distribution and three types have recently been cloned: cytosolic, lysosomal and plasma membrane sialidases (16). In the present work, we have isolated cDNAs encoding rat membraneassociated sialidase and expression of this and two other rat sialidase genes was evaluated by the competitive reverse transcription-polymerase chain reaction (RT-PCR) method (17) as well as by activity assays in developing rat brain and liver. MATERIALS AND METHODS Animals and tissue preparation. Sprague-Dawley rats were purchased from Japan SLC (Shizuoka, Japan). Brain and liver tissues were obtained from the embryos at gestational days 17 and 19, postnatal (days 0, 2, 4, 6, 9, 11, 18, 30), and adult (day 50) rats. They were pooled from 8-9 embryonic and 3-4 postnatal rats, yielding about 0.4-0.5 g to avoid irregularity among the animals, and kept frozen at ⫺80°C until use.
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Cloning of a membrane-associated sialidase from rat brain. A rat membrane-associated sialidase cDNA was obtained by homologybased RT-PCR using rat brain poly (A) ⫹ RNA prepared by acid guanidium-phenol-chloroform extraction methods (18) followed by oligo (dT) column chromatography. The primers 7S (5⬘-GGACACCGGACCATGAACCCCTGTCCT-3⬘) and 9A (5⬘-CCTGGCCC CACAGCAAAAGTGGCCCA-3⬘) were designed from sequences in regions in which the amino acids of bovine membrane sialidase (19) are identical to those of rat cytosolic sialidase (20). They were used for PCR with the template cDNA of rat brain under the following conditions; initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min and extension at 72°C for 2 min with final elongation at 72°C for 10 min. The PCR products were subcloned into pBluescript and sequenced with an ALF DNA autosequencer (Amersham Pharmacia) using a Thermo Sequenase Cycle Sequencing Kit (Amersham Pharmacia). The 245 bp fragment (RBI) thus obtained was used as a probe for library screening. RBI was radiolabeled with [␣- 32P]-dCTP using a Random Primer DNA Labeling Kit (Takara, Japan) for screening of a 5⬘-stretch gt10 cDNA rat brain library (Clontech). Hybridization with the radiolabeled RBI probe was carried out on Hybond-N ⫹ membranes (Amersham Pharmacia) at 65°C overnight. The filters were washed and used to expose to an X-ray films at ⫺80°C overnight. Positive clones were isolated, subcloned, and sequenced as described previously (21). Expression of rat membrane-associated sialidase cDNA in COS-1 cells. The entire open reading frame (ORF ) of rat membrane sialidase cDNA was PCR-amplified using primers, 1S (5⬘-CCCGAATTCGTCATGGAAGAAGTTT CATCC-3⬘) and 2A (5⬘-CCCGAATTCTTAGTTGCTACTAGGGCTGGTA-3⬘) and subcloned into a pME18S expression vector. COS-1 cells (Riken Cell Bank, Japan) were transfected by electroporation (at 240 V, 975 F) with the empty vector and the vector containing entire ORF (pMErmSD) of the membrane sialidase cDNA using Bio-Rad Genepulser. After 48 h, cells were pelleted, lysed in 9 volumes of ice-cold PBS by sonication and centrifuged at 1,000 ⫻ g, 4°C for 10 min. The supernatant was assayed for sialidase activity with various substrates, including gangliosides. Sialidase assay. Tissue homogenates (1:8, w/v) were prepared in ice-cold sucrose solution containing 0.25 mM sucrose, 10 mM phosphate buffer (pH 7.0), 1 mM EDTA and 0.5 mM phenylmethanesulfonyl fluoride using a glass homogenizer at 1000 rev./min with 7-strokes. After centrifugation at 1000 ⫻ g, 4°C for 10 min, the crude homogenates were further centrifuged at 100000 ⫻ g, 4°C for 1 h. The resultant supernatant was used as the enzyme fraction for cytosolic sialidase, and the sediment homogenized with 0.25 M sucrose solution was applied as the enzyme source for membrane and lysosomal sialidases. The reaction mixture (200 l) for sialidase assay contained 30 nmol of substrate as bound sialic acid, 0.2 mg of BSA, 10 mol of sodium acetate buffer (pH 4.6 or 5.5). The membrane sialidase activity was assayed by incubating the particulate fraction in pH 4.6 buffer with (for total activity) or without (for endogeneous activity) bovine brain mixed gangliosides (Sigma) in the presence of 0.1% Triton X-100, whereas for the assays of cytosolic and lysosomal sialidases the cytosolic and particulate fractions were incubated at pH 5.5 and 4.6, respectively, with synthetic substrate 4-methylumbellyferone-neuraminic acid (4MU-NeuAc). After incubation at 37°C for 2 h, released sialic acids were determined by the thiobarbituric acid method after passing through an AG1X-2 minicolumn and 4MU by fluorometric measurement, respectively, as described elsewhere (22). Quantitative PCR methods. A rat lysosomal sialidase cDNA (Accession No. AB035722) was obtained with homology-based hybridization by screening a rat liver cDNA library using human lysosomal sialidase cDNA as a probe, which was generously provided by Dr. Milner (23), and was confirmed to encode an acidic sialidase for
4MU-NeuAc in the same manner described above. The cDNA competitors were prepared by single- or double-digestion of the Bluescript vectors containing entire ORFs of each sialidase cDNAs with BstPI and BglII for membrane sialidase, XcmI for cytosolic (20), and BbsI for lysosomal sialidase (Accession No. AB035722), respectively. From 5 g of total RNA, cDNA was synthesized using 200 units of Moloney murine leukemia virus-reverse transcriptase and oligo (dT) primers in a final volume of 20-l. The 25-fold diluted cDNA (10 l )was subjected to quantitative PCR analysis with the competitors adjusted to the following concentrations: 40 fg (brain) and 6 fg (liver) for membrane, 8 fg (brain) and 0.4 fg (liver) for cytosolic, 50 fg (brain) and 25 fg (liver) for lysosomal sialidase. PCR was performed with primers rm4S (5⬘-CCTACTTGATATCATGCTGG-3⬘) and rm4A (5⬘TTAGTTGCTACTAGGGCTGG-3⬘) for membrane (target: 718 bp, competitor: 484 bp), rc3S (5⬘-CCGTCCAGGACCTCACAGAG-3⬘) and rc3A (5⬘-TCACTGAGCACCATGTACTG-3⬘) for cytosolic (target: 727 bp, competitor: 387 bp), rlSD12S (5⬘-TTGGAGTAAG GATGACGGCC-3⬘) and rlSD10A (5⬘-TCAAAGCGTGCCGTAGACGC-3⬘) for lysosomal sialidases (target: 707 bp, competitor: 401 bp), respectively. PCRs were conducted under the conditions described previously (21). The annealing temperatures and PCR cycles were 52, 55, and 57°C and 40, 36 and 38 cycles for membrane sialidase, cytosolic sialidase, and lysosomal sialidase, respectively. To normalize for sample variation, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (24) was also measured as an internal control using a GAPDH competitor, prepared by digestion of the 0.5 kb cDNA (nucleotides 566 to 1017) with StyI and BspMI. After amplification, 10-l aliquots were electrophoresed in 1.5% agarose gels. Gel photos were scanned and densitometric analyses were performed using the NIH-image program, version 1.61.
RESULTS AND DISCUSSION Isolation of a cDNA encoding rat membraneassociated sialidase. cDNA from rat brain poly(A) ⫹ RNA was amplified by means of RT-PCR using primers designed from the conserved amino acids between bovine membrane and rat cytosolic sialidases. The deduced amino acid sequence of a PCR product RBI showed 89% and 82% identity to the corresponding regions of mouse (21) and human membrane (25) ganglioside sialidases, respectively. By screening of the gt10 rat brain cDNA library (1.2 ⫻ 10 6) with the RBI probe, we isolated four individual clones of about 0.9 to 2.4 kb. Sequence analysis of the clones revealed that the longest clone consisted of 2307 nucleotides having an open reading frame (ORF) encoding 418 amino acids with a molecular mass of 47.0 kDa (Fig. 1). There was a putative hydrophobic segment at nucleotides 716 to 784, and the sequence motif YRIP (amino acids 24 –27) and three copies of Asp-box consensus sequences (SXDXGXS/TW) (26) were also present. The highest sequence identity was observed with mouse membrane sialidase (81.3%, Accession No. AB026842), with a slightly less identity evident with human (67.5%, AB008185) and bovine (66.0%, AB008184) membrane sialidases. It should be noted that the sequence identities of cytosolic and lysosomal sialidases between the rat (Accession No. D164300 and AB035722, respectively) and mouse (AB028023 and Y11412, respectively) are higher (90 –95%) than for the membrane sialidase case. Northern blot analysis with poly (A)-
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riched RNAs from rat brain, cardiac muscle and liver exhibited a major 3.5 kb transcript with minor 3.1 and 1.6 kb forms expressed most highly in cardiac muscle but at very low levels in liver (data not shown). Transient expression in COS-1 cells showed an over 400fold increase in sialidase activity toward gangliosides as compared with mock-transfected cells (Table 1). Most of the activity (91%) in the crude homogenates was recovered in the particulate fractions, indicating it to be membrane-bound. Like other membrane sialidases, the expressed sialidase hydrolyzed gangliosides GD3 and GM3 preferentially, rather than GM2, and at a significant degree 4MU-NeuAc and oligosaccharides such as sialyllactose in the presence of Triton X-100 with an optimum pH at 4.8 to 5.0. The similarities in primary structure and enzymatic properties of mouse, bovine, and human membrane sialidases imply that this rat sialidase is a homologue of the other two sialidases. Developmental changes in sialidase activity. To determine how the expression of the membrane sialidase is altered during rat development, we evaluated the level by activity assays and competitive RT-PCR. Developmental changes of sialidase activity in rat brain and liver examined were then compared with those of cytosolic and lysosomal sialidases as shown in Fig. 2. Based on the previous studies of sialidase substrate specificity (23, 27, 28), we attempted differential activity assays for three sialidases. Activity toward gangliosides at pH4.6 in the presence of TritonX-100 can be considered as due to membrane-associated sialidase, whereas the activities toward 4MU-NeuAc at pH 6.0 in the cytosolic fraction and at pH 4.5 in the particulate fraction are for cytosolic and lysosomal sialidases, respectively, as described in the “Materials and Methods.” We first measured the membrane sialidase activity toward endogenous and endogenous plus exogenous (total) ganglioside substrates when only the particulate fraction of rat brain was used as the enzyme source, because the fraction is highly abundant in gangliosides, possibly acting as endogenous substrates for the sialidase. As shown in Fig. 2, the total activity of the membrane sialidase was somewhat higher than the endogenous activity in the developing brain. In brain, a gradual increase of the sialidase activity was observed by 3.6-fold (total) and 4.3-fold (endogenous) between gestational day 17 and the 18th day after birth. This was followed by slight temporary decline by the 30th day then a slight rise by the 50th day. This
FIG. 1. Nucleotide and deduced amino acid sequences of rat membrane-associated sialidase. The predicted amino acid sequence is shown by the single letter amino acid code under the nucleotide
sequence. The hydrophobic domain in membrane sialidase is underlined. Asp-boxes are boxed and the F/YRIP motif is indicated by a dotted line. The nucleotide sequence has been submitted to the DDBJ, EMBL and GenBank Databases with accession number AB026841.
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Sialidase Activities Toward Various Substrates in the Homogenates of COS-1 Cells Transfected with the Mouse Ganglioside Sialidase cDNA pME18S
pMErmSD a
a b
a
Substrate
Specific activity (units/mg protein)
Specific activity (units/mg protein)
Hydrolysis relative b to GD3 (%)
GD3 GD1a GM3 GM2 Fetuin Sialyllactose 4MU-NeuAc
3.5 2.1 4.4 2.9 1.9 3.7 24.2
1727 1295 1476 348 66 294 397
100 75 85 20 4 17 27
The values are means of three experiments. The values are expressed as the percentage of (specific activity with pMErmSD minus the activity with pME18S) relative to that for GD3.
decline during the period of most intensive protein synthesis is likely due to the dilution of specific enzyme activity following insertion of newly synthesized proteins other than sialidase (29). The previous observations on membrane-bound sialidase in developing mouse brain reported by Segler-Stahl et al. (29)
FIG. 2. Developmental changes of membrane sialidase activity in comparison with those of cytosolic and lysosomal sialidases. The cytosolic and particulate fractions were prepared from rat brain and liver at embryonic and postnatal stages, and assayed for respective sialidase activities under the appropriate conditions described in Materials and Methods. F, membrane-associated sialidase (total activity); E, membrane-associated sialidase (endogeneous activity); ‚, cytosolic sialidase; 䊐, lysosomal sialidase. The data given are mean values from three experiments ⫾ standard deviations.
were essentially in line with our data. The developmental pattern in liver was similar to that in brain, although the activity level was much lower. To compare the activity level with those of cytosolic and lysosomal sialidases, we then performed assays using 4MU-NeuAc as substrate under appropriate conditions. With regard to cytosolic sialidase, only a trace level was detected before birth in both brain and liver. During postnatal development, the brain cytosolic sialidase increased gradually and showed a peak value (1.2-fold vs. adult value) at the 30th day and levellingoff thereafter to the adult value. In contrast, the liver enzyme showed a rapid rise in the perinatal stage and after reaching the maximum (1.2-fold vs. adult value) at postnatal day 18 the activity remained fairly constant. The activity pattern obtained for cytosolic sialidase closely resembled that in a previous report by Carubelli and Tulsiani (30), who used sialyllactose as the substrate. Lysosomal sialidase in brain appeared to be fully active in late embryonic stages. The activity exhibited a sharp decrease prior to birth and relatively high levels were maintained throughout, with a maximum at the 11th postnatal day. The study on two (light and heavy) lysosomal sialidases in developing mouse brain described by Fiorilli et al. (31) revealed the light sialidase to show marked increase of specific activity in the first 10 days, similar to our rat brain enzyme case, although they only measured enzyme activity at 4 stages of postnatal development. The lysosomal sialidase in liver, on the other hand, showed a rapid increase of activity from birth and reached a maximum (2.3-fold vs. adult value) at the 4th postnatal day and remained almost constant during adulthood. Our previous study (27) of rat liver lysosomal sialidase demonstrated two forms, located mainly in lysosomal matrix and membrane, respectively, the former hydro-
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FIG. 3. Developmental changes in membrane sialidase mRNA levels in comparison with those of cytosolic and lysosomal sialidases. The expression profiles of three sialidase genes in developing rat brain and liver were quantitatively estimated by competitive RT-PCR. (A) Agarose gel electrophoretic patterns of the products of competitive PCR analyses. mSD, membrane-associated sialidase; cSD, cytosolic sialidase; lSD, lysosomal sialidase. Black and white arrowheads indicate target and competitor DNA fragments, respectively. (B) Densitometric analyses of the results for electrophoretic patterns (A). F, membrane-associated sialidase; ‚, cytosolic sialidase; 䊐, lysosomal sialidase. The data given are mean values from three experiments ⫾ standard deviations.
lyzing 4MU-NeuAc effectively but the latter attacking gangliosides preferentially. Although we were not able to distinguish between them completely under the
present assay conditions, the activity obtained here would be expected to reflect lysosomal-matrix sialidase activity because of the use of 4MU-NeuAc as substrate
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in the absence of any detergent. Carubelli and Tulsiani (30) also described developmental changes of rat particulate-bound sialidase using sialyllactose as a substrate, the sialidase seemingly being of lysosomal nature. The present data are very similar to their results for brain, but inconsistent with liver data showing a fairly constant level during all stages, the reason for this difference being unclear. Sialidase gene expression in developing brain and liver. To estimate the amounts of membrane sialidase transcripts in developing rat brain and liver, competitive PCR was carried out as summarized in Fig. 3A (gel photo) and B (densitogram). Throughout the late embryonic and postnatal stages, lysosomal sialidase maintained the highest expression among the three sialidases, and the cytosolic sialidase showed the lowest expression. The relative mRNA levels of membrane, cytosolic and lysosomal sialidase genes at day 50 were 1.0, 0.4 and 1.6 in brain and 1.0, 0.25 and 2.0 in liver, respectively, after normalization for GAPDH as the internal control. General trends in the developmental changes of cytosolic and lysosomal sialidase expression were almost paralleled by results obtained from enzyme activity assays. In both brain and liver, transcripts of cytosolic sialidase were barely detectable in the late embryonic stage and gradually increased reaching a plateau at the 6 –11th postnatal days. On the other hand, the expression of lysosomal sialidase in brain and liver showed a slight decline before birth, and after immediate restoration relatively high levels were maintained with some fluctuation throughout the postnatal period. In addition, it is interesting that, at the second postnatal day, there was a spike increase in the amount of transcript of brain lysosomal sialidase, corresponding to a small peak in the enzyme activity at the same time point. Membrane-associated sialidase exhibited distinct differences in profiles. First, the mRNA level in brain showed a sharp increase in the perinatal stage, and soon reached a plateau level much faster (about 2 weeks) than the enzyme activity. Secondly, the enzyme activity of liver membrane sialidase was low in the late embryonal stage and even in postnatal and adult periods, whereas the mRNA level estimated was not commensurately low. This discrepancy between the level of mRNA and the enzyme activity may be partly due to posttranslational regulation of the enzyme, such as phosphorylation (32). In conclusion, this is the first report of expression patterns of three sialidase genes during development evaluated by both RT-PCR methods and activity assays. The results indicate that the genes show developmental stage-specific expression, possibly contributing to provide glycoconjugate components required during development in collaboration with specific sialyltransferases. The present results should con-
tribute to a better understanding of how sialoglycoconjugate metabolism is developmentally regulated and how these sialidases are involved in metabolic regulation. ACKNOWLEDGMENTS We are grateful to Dr. Kazuo Maruyama, Tokyo Medical and Dental University, for kindly providing the pME18S vector. We also thank Ms. Setsuko Moriya, Division of Biochemistry, Miyagi Prefectural Cancer Center, for her expert technical assistance.
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