- Email: [email protected]
Expression of β1,4-galactosyltransferase in the development of mouse brain
Biochimica et Biophysica Acta 1425 (1998) 204^208
Expression of L1;4 -galactosyltransferase in the development of mouse brain Dapeng Zhou a
1;a
, Chun Chen
1;b
, Songmin Jiang a , Zonghou Shen a , Zhengwu Chi b , Jianxin Gu a; *
Gene Research Center and Department of Biochemistry, Shanghai Medical University, and Key Laboratory of Glycoconjugate, Ministry of Public Health, Shanghai 200032, PR China b Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, PR China Received 9 March 1998; accepted 9 June 1998
Abstract L1;4 -Galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of L1;4 -galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL14GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1) L1;4 -galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development ; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: L1;4 -Galactosyltransferase; Brain development
1. Introduction L1;4 -Galactosyltransferase (GalTase, EC 2.4.1.38, reviewed in [1]) catalyzes the transfer of galactose from UDP-galactose to terminal N-acetylglucosamine residues on elongating oligosaccharide chains, Abbreviations: HPLC, high performance liquid chromatography; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; PA, 2-aminopyridine; PMSF, phenylmethylsulfonyl £uoride * Corresponding author. Fax: +86 (21) 64039987; E-mail: [email protected] 1 These authors contributed equally to this work.
which are suggested to be involved in various cellular functions including cell-cell and cell-matrix interactions during embryogenesis (reviewed in [2]). Several reports suggest that galactose (Gal) containing complex N-glycans are particularly important in early embryogenesis. The poly-N-acetyllactosamine structure expressed in preimplantation embryos is important for cellular communication and oligosaccharides containing this structure will competitively inhibit embryo implantation [3]. Treatment of early embryos with UDP-galactose can also inhibit compaction [4]. More interestingly and distinct from other glycotransferases, GalTase has also been found located not only in the Golgi compartment, but also on the
0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 0 7 0 - 1
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D. Zhou et al. / Biochimica et Biophysica Acta 1425 (1998) 204^208
cell surface, which is thought to function as a recognition molecule through binding to speci¢c ligands on adjacent cell surface and extracellular matrix, directly related to cell adhesion and migration [5,6]. Cell surface L1;4 -galactosyltransferase variation in mouse preimplantation development and morula compa is also reported [7]. More interestingly, a L1;4 -galactosyltransferase `family' has been found and at least two isoenzymes of the L1;4 -galactosyltransferase have been cloned and expressed in Chinese hamster ovary cells (K. Furukawa, personal communication). In this study, we compared the activity of GalTase in the development of mouse brain. The contents of messenger RNA were also measured. 2. Materials and methods 2.1. Materials Swiss white mice (Mus musculus) were from the Department of Experimental Animals, Shanghai Medical University. DEAE-cellulose (DE-52) and Sephadex G-25 were from Whatman. Pronase E, L-galactosidase, UDP-galactose and 2-aminopyridine (PA) were from Merck. HPLC (LC-9A) and ODS120T column (6U150 mm, 5 Wm) were products of Shimadzu. Standard PA-labeled GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA, GlcNAcL1-2ManK1-6(GalL1-4GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA, GalL1-4GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA, and GalL1-4GlcNAcL1-2ManK1-6(GalL1-4GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA (con¢rmed with NMR) were from Prof. N. Taniguchi (Department of Biochemistry, Osaka University). Trizol reagent for RNA extraction was from Gibco. Prime-A-Gene random primer labeling kit was from Promega. Nylon membrane and [K-32 P]dATP were from Amersham. pMGT plasmid containing the full length of the cDNA of mouse GalTase was from Dr. B.D. Shur (Department of Anatomy and Cell Biology, Emory University).
205
2.2. Preparation of PA-labeled substrate for L1;4 -galactosyltransferase The substrate was prepared as described [8]. Brie£y, human IgG was puri¢ed with a DEAE-cellulose column. After hydrolysis with pronase E, the IgG was eluted through Sephadex G-25. The sugar contents were quantitated with the phenol-sulfuric method and peaks rich in saccharides collected. Then the glycans were hydrolyzed in 0.1 N HCl to remove sialic acid and fucose, followed by hydrazination, N-acetylation, pyridylamination, and digestion with L-galactosidase. After this, the pyridylaminated sugar chains were puri¢ed with HPLC. This was performed at 55³C using a 0.01 M ammonium acetate bu¡er (pH 4.0) with a linear n-butanol concentration gradient established between 0.1 and 0.5% for 60 min. The £ow rate was 1 ml/min. The substrate was collected and its concentration measured by comparison with the standard sugar chain. 2.3. Preparation of microsomes All procedures were at 4³C. The mice were anesthetized and killed, and the brain was washed with 0.9% sodium chloride three times, then homogenized in 4 vols. homogenizing solution (50 mM sodium maleate, 0.4 M sucrose, 1 mM PMSF, and 5 mM magnesium chloride) with a glass homogenizer tube. Cell debris was removed by spinning and the supernatant centrifuged at 50 000Ug for 1 h. The pellet was collected and resuspended in homogenizing solution. The protein was quantitated with Lowry's method [9]. 2.4. Assay of GalTase The activity of GalTase was assayed as described [8]. The reaction mixture contained the following components in a ¢nal volume of 50 Wl: 25 mM dimethyl sodium arsine, 25 mM manganese chloride, 0.2 M galactose, 0.25 mM UDP-Gal, 0.5% Triton X100, 100 Wg enzyme protein, and 40 nmol acceptor substrate. After incubation at 37³C for 3 h, the reaction mixture was boiled for 3 min and centrifuged at 10 000Ug for 10 min. An aliquot of 20 Wl was
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applied to HPLC. The condition for HPLC was the same as that for the preparation of substrate. The product was quantitated as described [8] and the activity of the enzyme expressed as pmol of product per mg protein per minute. 2.5. Northern blot of GalTase The full length of mouse GalTase cDNA between HindIII (3239) and EcoRI (2615) was cut from pMGT plasmid as the template for labeling with a random primer method using a kit from Gibco. The 25 Wl labeling reaction mixture contained 5 Wl 5Ubu¡er (random primers included), 1 Wl dCTP, dTTP, dGTP (1.5 mM each), 100 ng GalTase template, 1 Wl BSA (1 mg/ml), 2.5 Wl [K-32 P]dATP (25 WCi), 5 U Klenow enzyme. The mixture was incubated at 25³C for 1 h. The probes were heated for 2 min and incubated on ice before use. The total RNA was isolated with Trizol reagent. Twenty micrograms RNA were electrophoresed in 1% agarose gel containing formaldehyde and transferred to nylon membrane. After RNA was ¢xed with a UV crosslinker, the membrane was prehybridized in buffer A (0.2 M PBS, pH 7.2, containing 1 mM EDTA, 1% BSA, 7% SDS, 15% formamide) at 65³C for 3 h, then the prehybridizing bu¡er was discarded and the membrane hybridized in hybridizing bu¡er (bu¡er A added with radiolabeled probes, 10U106 cpm) at 65³C for 3 h. The membrane was washed in bu¡er B (40 mM PBS, pH 7.2, containing 1 mM EDTA, 1% SDS) at 65³C for three times, 30 min each. The membrane was exposed to Kodak X-ray ¢lm for 4 days at 370³C.
3. Results and discussion 3.1. Product pattern of GalTase during development As reported before [8], three products appeared for the GalTase in all the reactions from 18-day-old fetal mice to adult mice (Scheme 1) and no signi¢cant changes for the ratio of product A and product B were found (Fig. 1). These ¢ndings suggest similar branch speci¢city. No signi¢cant kinetic changes were found using this substrate. 3.2. Activity of GalTase in fetal brain As shown in Fig. 2, the activity of GalTase in 18-day-old fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually during the course of development. 3.3. Levels of messenger RNA of GalTase The expression of L1;4 -galactosyltransferase in brain was very low. We could not detect mRNA in adult murine brain with Northern blot (Fig. 3). However, Northern blot in 18-day-old fetal and young mouse brain gave positive results, although the bands were weak. We could not quantitate the relative contents of all mRNA since there were no bands for 14-day-old, 21-day-old, and adult mice. The ratio of mRNA for 18-day-old fetal, newborn, and 7-day-old mice is about 40:20:1. It showed that the mRNA level was highest in fetus mice and decreased abruptly after birth. On the contrary, enzyme activity decreased gradually. This suggests that after
Scheme 1. Products of GalTase.
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207
birth the expression of L1;4 -galactosyltransferase gene decreases dramatically, but the enzyme is very stable. Thus much of the enzyme produced in fetal stage or on day 1 maintains its catalytic activity on day 7. Enzyme activity may also be contributed by a novel L1;4 -galactosyltransferase. Recently a novel human galactosyltransferase (designated L1;4 -galactosyltransferase II) was found (K. Furukawa, personal communication). Its deduced amino acid sequence showed 37% identity with that of the much studied human L1;4 -galactosyltransferase (designated galacto-
Fig. 2. Brain GalTase activity during Microsomes were prepared from mice were used to assay GalTase activity. nated as nmol product/h/mg protein. (n = 3). The statistical signi¢cance of mice to fetus mice was P 6 0.001.
Fig. 1. Product pattern of GalTase in mouse brain. Microsomes were prepared from mice brain and 100 Wg protein were used to assay GalTase activity. Three products appeared and were designated A, B, and C respectively. Substrate (S): GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA; A: GlcNAcL1-2ManK1-6(GalL1-4GlcNAcL12ManK1-3)ManL1-4GlcNAcL1-4GlcNAc-PA; B: GalL1-4GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3)ManL1-4GlcNAcL1-4GlcNAc-PA; C: GalL1-4GlcNAcL1-2ManK1-6 (GalL1-4GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA.
the development of mice. brain and 100 Wg protein Unit of enzyme is desigResults are means þ S.D. the decrease from adult
syltransferase I). Relatively high levels of the L1;4 -galactosyltransferase II message were detected in human brain, in which no detectable or a very low amount of L1;4 -GalTase I message was found. But so far there are no data on novel L1;4 -GalTase in mouse brain. Murine L1;4 -galactosyltransferase is especially interesting because it has two inframe ATG codons at the 5P end of its cDNA. S1 nuclease protection and primer extension analyses [10^12] have shown the presence of two sets of transcripts (4.1 kb and 3.9 kb), which are controlled by di¡erent promoters. Translation from the two inframe AUG codons results in proteins of 399 amino acids and 386 amino acids respectively. S1 nuclease protection assay shows that the long and short forms of L1;4 -galactosyltransferase are expressed in a tissue-speci¢c manner in mouse [11]. Most somatic mouse tissues express intermediate levels of enzyme and the ratio of long form (4.1 kb) to short form (3.9 kb) is 5:1. Lactating mammary gland expresses a very high level of L1;4 -galactosyltransferase and the ratio of 4.1 kb to 3.9 kb is 1:10. However, in mouse brain, which expresses a low level of L1;4 -galactosyltransferase, only the long form, 4.1 kb, is expressed. It is inter-
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ment of mouse brain (1) L1;4 -galactosyltransferase showed similar branch speci¢city and kinetics using biantenary GlcNAcL1-2ManK1-6(GlcNAcL12ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA as the substrate; (2) the activity of L1;4 -galactosyltransferase decreased gradually in the course of development; (3) Northern blot analysis and enzyme activity determination suggested that the changes in enzyme activity might result from the transcriptional decrease and the stability of the enzyme or enzyme activity cocontributed by putative L1;4 -galactosyltransferase. The detailed mechanisms remain to be discovered. Acknowledgements This work was supported by National Natural Scienti¢c Grants (39570176, 39470164), PR China. References
Fig. 3. Expression of brain GalTase messenger RNA during the development of mice. Upper panel: Northern blot analysis of 40 Wg total RNA isolated from brains of fetus, 1-, 7-, 14-, 21-day-old, and adult mice. The probe used was generated from mouse GalTase cDNA between HindIII (3239) and EcoRI (2615) cut from pMGT plasmid labeled with a random primer method described in the text. Exposure time is 2 days. No band was found in adult mice even after longer exposure time. Lower panel: Northern blot of 18S RNA as an internal standard. The method is described in the text.
esting to examine the expression pattern of L1;4 -galactosyltransferase in fetal mouse brain. In this report, we found that during the develop-
[1] B.D. Shur, Glycobiology 1 (1991) 563^575. [2] A. Varki, Glycobiology 3 (1993) 97^130. [3] B.A. Fenderson, U. Zehavi, S. Hakomori, J. Exp. Med. 160 (1994) 1591^1596. [4] B.D. Shur, P. Oettgen, D. Bennett, Dev. Biol. 73 (1979) 178^ 181. [5] R.B. Runyan, J. Versalovic, B.D. Shur, J. Cell Biol. 107 (1988) 1863^1871. [6] P.A. Appeddu, B.D. Shur, Proc. Natl. Acad. Sci. USA 91 (1994) 2095^2099. [7] E.M. Bayna, J.H. Shaper, B.D. Shur, Cell 53 (1988) 145^ 157. [8] D. Zhou, S. Jiang, Z. Shen, J. Gu, Biochem. J. 320 (1996) 623^625. [9] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265^271. [10] N.L. Shaper, G.F. Hollis, J.G. Douglas, I.R. Kirsch, J.H. Shaper, J. Biol. Chem. 263 (1988) 10420^10428. [11] A. Herduin-Lepers, J.H. Shaper, N.L. Shaper, J. Biol. Chem. 268 (1993) 14348^14359. [12] B. Rajput, N.L. Shaper, J.H. Shaper, J. Biol. Chem. 271 (1996) 5131^5142.
BBAGEN 24664 2-9-98
Expression of L1;4 -galactosyltransferase in the development of mouse brain Dapeng Zhou a
1;a
, Chun Chen
1;b
, Songmin Jiang a , Zonghou Shen a , Zhengwu Chi b , Jianxin Gu a; *
Gene Research Center and Department of Biochemistry, Shanghai Medical University, and Key Laboratory of Glycoconjugate, Ministry of Public Health, Shanghai 200032, PR China b Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, PR China Received 9 March 1998; accepted 9 June 1998
Abstract L1;4 -Galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of L1;4 -galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL14GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1) L1;4 -galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development ; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: L1;4 -Galactosyltransferase; Brain development
1. Introduction L1;4 -Galactosyltransferase (GalTase, EC 2.4.1.38, reviewed in [1]) catalyzes the transfer of galactose from UDP-galactose to terminal N-acetylglucosamine residues on elongating oligosaccharide chains, Abbreviations: HPLC, high performance liquid chromatography; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; PA, 2-aminopyridine; PMSF, phenylmethylsulfonyl £uoride * Corresponding author. Fax: +86 (21) 64039987; E-mail: [email protected] 1 These authors contributed equally to this work.
which are suggested to be involved in various cellular functions including cell-cell and cell-matrix interactions during embryogenesis (reviewed in [2]). Several reports suggest that galactose (Gal) containing complex N-glycans are particularly important in early embryogenesis. The poly-N-acetyllactosamine structure expressed in preimplantation embryos is important for cellular communication and oligosaccharides containing this structure will competitively inhibit embryo implantation [3]. Treatment of early embryos with UDP-galactose can also inhibit compaction [4]. More interestingly and distinct from other glycotransferases, GalTase has also been found located not only in the Golgi compartment, but also on the
0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 0 7 0 - 1
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D. Zhou et al. / Biochimica et Biophysica Acta 1425 (1998) 204^208
cell surface, which is thought to function as a recognition molecule through binding to speci¢c ligands on adjacent cell surface and extracellular matrix, directly related to cell adhesion and migration [5,6]. Cell surface L1;4 -galactosyltransferase variation in mouse preimplantation development and morula compa is also reported [7]. More interestingly, a L1;4 -galactosyltransferase `family' has been found and at least two isoenzymes of the L1;4 -galactosyltransferase have been cloned and expressed in Chinese hamster ovary cells (K. Furukawa, personal communication). In this study, we compared the activity of GalTase in the development of mouse brain. The contents of messenger RNA were also measured. 2. Materials and methods 2.1. Materials Swiss white mice (Mus musculus) were from the Department of Experimental Animals, Shanghai Medical University. DEAE-cellulose (DE-52) and Sephadex G-25 were from Whatman. Pronase E, L-galactosidase, UDP-galactose and 2-aminopyridine (PA) were from Merck. HPLC (LC-9A) and ODS120T column (6U150 mm, 5 Wm) were products of Shimadzu. Standard PA-labeled GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA, GlcNAcL1-2ManK1-6(GalL1-4GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA, GalL1-4GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA, and GalL1-4GlcNAcL1-2ManK1-6(GalL1-4GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA (con¢rmed with NMR) were from Prof. N. Taniguchi (Department of Biochemistry, Osaka University). Trizol reagent for RNA extraction was from Gibco. Prime-A-Gene random primer labeling kit was from Promega. Nylon membrane and [K-32 P]dATP were from Amersham. pMGT plasmid containing the full length of the cDNA of mouse GalTase was from Dr. B.D. Shur (Department of Anatomy and Cell Biology, Emory University).
205
2.2. Preparation of PA-labeled substrate for L1;4 -galactosyltransferase The substrate was prepared as described [8]. Brie£y, human IgG was puri¢ed with a DEAE-cellulose column. After hydrolysis with pronase E, the IgG was eluted through Sephadex G-25. The sugar contents were quantitated with the phenol-sulfuric method and peaks rich in saccharides collected. Then the glycans were hydrolyzed in 0.1 N HCl to remove sialic acid and fucose, followed by hydrazination, N-acetylation, pyridylamination, and digestion with L-galactosidase. After this, the pyridylaminated sugar chains were puri¢ed with HPLC. This was performed at 55³C using a 0.01 M ammonium acetate bu¡er (pH 4.0) with a linear n-butanol concentration gradient established between 0.1 and 0.5% for 60 min. The £ow rate was 1 ml/min. The substrate was collected and its concentration measured by comparison with the standard sugar chain. 2.3. Preparation of microsomes All procedures were at 4³C. The mice were anesthetized and killed, and the brain was washed with 0.9% sodium chloride three times, then homogenized in 4 vols. homogenizing solution (50 mM sodium maleate, 0.4 M sucrose, 1 mM PMSF, and 5 mM magnesium chloride) with a glass homogenizer tube. Cell debris was removed by spinning and the supernatant centrifuged at 50 000Ug for 1 h. The pellet was collected and resuspended in homogenizing solution. The protein was quantitated with Lowry's method [9]. 2.4. Assay of GalTase The activity of GalTase was assayed as described [8]. The reaction mixture contained the following components in a ¢nal volume of 50 Wl: 25 mM dimethyl sodium arsine, 25 mM manganese chloride, 0.2 M galactose, 0.25 mM UDP-Gal, 0.5% Triton X100, 100 Wg enzyme protein, and 40 nmol acceptor substrate. After incubation at 37³C for 3 h, the reaction mixture was boiled for 3 min and centrifuged at 10 000Ug for 10 min. An aliquot of 20 Wl was
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applied to HPLC. The condition for HPLC was the same as that for the preparation of substrate. The product was quantitated as described [8] and the activity of the enzyme expressed as pmol of product per mg protein per minute. 2.5. Northern blot of GalTase The full length of mouse GalTase cDNA between HindIII (3239) and EcoRI (2615) was cut from pMGT plasmid as the template for labeling with a random primer method using a kit from Gibco. The 25 Wl labeling reaction mixture contained 5 Wl 5Ubu¡er (random primers included), 1 Wl dCTP, dTTP, dGTP (1.5 mM each), 100 ng GalTase template, 1 Wl BSA (1 mg/ml), 2.5 Wl [K-32 P]dATP (25 WCi), 5 U Klenow enzyme. The mixture was incubated at 25³C for 1 h. The probes were heated for 2 min and incubated on ice before use. The total RNA was isolated with Trizol reagent. Twenty micrograms RNA were electrophoresed in 1% agarose gel containing formaldehyde and transferred to nylon membrane. After RNA was ¢xed with a UV crosslinker, the membrane was prehybridized in buffer A (0.2 M PBS, pH 7.2, containing 1 mM EDTA, 1% BSA, 7% SDS, 15% formamide) at 65³C for 3 h, then the prehybridizing bu¡er was discarded and the membrane hybridized in hybridizing bu¡er (bu¡er A added with radiolabeled probes, 10U106 cpm) at 65³C for 3 h. The membrane was washed in bu¡er B (40 mM PBS, pH 7.2, containing 1 mM EDTA, 1% SDS) at 65³C for three times, 30 min each. The membrane was exposed to Kodak X-ray ¢lm for 4 days at 370³C.
3. Results and discussion 3.1. Product pattern of GalTase during development As reported before [8], three products appeared for the GalTase in all the reactions from 18-day-old fetal mice to adult mice (Scheme 1) and no signi¢cant changes for the ratio of product A and product B were found (Fig. 1). These ¢ndings suggest similar branch speci¢city. No signi¢cant kinetic changes were found using this substrate. 3.2. Activity of GalTase in fetal brain As shown in Fig. 2, the activity of GalTase in 18-day-old fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually during the course of development. 3.3. Levels of messenger RNA of GalTase The expression of L1;4 -galactosyltransferase in brain was very low. We could not detect mRNA in adult murine brain with Northern blot (Fig. 3). However, Northern blot in 18-day-old fetal and young mouse brain gave positive results, although the bands were weak. We could not quantitate the relative contents of all mRNA since there were no bands for 14-day-old, 21-day-old, and adult mice. The ratio of mRNA for 18-day-old fetal, newborn, and 7-day-old mice is about 40:20:1. It showed that the mRNA level was highest in fetus mice and decreased abruptly after birth. On the contrary, enzyme activity decreased gradually. This suggests that after
Scheme 1. Products of GalTase.
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207
birth the expression of L1;4 -galactosyltransferase gene decreases dramatically, but the enzyme is very stable. Thus much of the enzyme produced in fetal stage or on day 1 maintains its catalytic activity on day 7. Enzyme activity may also be contributed by a novel L1;4 -galactosyltransferase. Recently a novel human galactosyltransferase (designated L1;4 -galactosyltransferase II) was found (K. Furukawa, personal communication). Its deduced amino acid sequence showed 37% identity with that of the much studied human L1;4 -galactosyltransferase (designated galacto-
Fig. 2. Brain GalTase activity during Microsomes were prepared from mice were used to assay GalTase activity. nated as nmol product/h/mg protein. (n = 3). The statistical signi¢cance of mice to fetus mice was P 6 0.001.
Fig. 1. Product pattern of GalTase in mouse brain. Microsomes were prepared from mice brain and 100 Wg protein were used to assay GalTase activity. Three products appeared and were designated A, B, and C respectively. Substrate (S): GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA; A: GlcNAcL1-2ManK1-6(GalL1-4GlcNAcL12ManK1-3)ManL1-4GlcNAcL1-4GlcNAc-PA; B: GalL1-4GlcNAcL1-2ManK1-6(GlcNAcL1-2ManK1-3)ManL1-4GlcNAcL1-4GlcNAc-PA; C: GalL1-4GlcNAcL1-2ManK1-6 (GalL1-4GlcNAcL1-2ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA.
the development of mice. brain and 100 Wg protein Unit of enzyme is desigResults are means þ S.D. the decrease from adult
syltransferase I). Relatively high levels of the L1;4 -galactosyltransferase II message were detected in human brain, in which no detectable or a very low amount of L1;4 -GalTase I message was found. But so far there are no data on novel L1;4 -GalTase in mouse brain. Murine L1;4 -galactosyltransferase is especially interesting because it has two inframe ATG codons at the 5P end of its cDNA. S1 nuclease protection and primer extension analyses [10^12] have shown the presence of two sets of transcripts (4.1 kb and 3.9 kb), which are controlled by di¡erent promoters. Translation from the two inframe AUG codons results in proteins of 399 amino acids and 386 amino acids respectively. S1 nuclease protection assay shows that the long and short forms of L1;4 -galactosyltransferase are expressed in a tissue-speci¢c manner in mouse [11]. Most somatic mouse tissues express intermediate levels of enzyme and the ratio of long form (4.1 kb) to short form (3.9 kb) is 5:1. Lactating mammary gland expresses a very high level of L1;4 -galactosyltransferase and the ratio of 4.1 kb to 3.9 kb is 1:10. However, in mouse brain, which expresses a low level of L1;4 -galactosyltransferase, only the long form, 4.1 kb, is expressed. It is inter-
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D. Zhou et al. / Biochimica et Biophysica Acta 1425 (1998) 204^208
ment of mouse brain (1) L1;4 -galactosyltransferase showed similar branch speci¢city and kinetics using biantenary GlcNAcL1-2ManK1-6(GlcNAcL12ManK1-3) ManL1-4GlcNAcL1-4GlcNAc-PA as the substrate; (2) the activity of L1;4 -galactosyltransferase decreased gradually in the course of development; (3) Northern blot analysis and enzyme activity determination suggested that the changes in enzyme activity might result from the transcriptional decrease and the stability of the enzyme or enzyme activity cocontributed by putative L1;4 -galactosyltransferase. The detailed mechanisms remain to be discovered. Acknowledgements This work was supported by National Natural Scienti¢c Grants (39570176, 39470164), PR China. References
Fig. 3. Expression of brain GalTase messenger RNA during the development of mice. Upper panel: Northern blot analysis of 40 Wg total RNA isolated from brains of fetus, 1-, 7-, 14-, 21-day-old, and adult mice. The probe used was generated from mouse GalTase cDNA between HindIII (3239) and EcoRI (2615) cut from pMGT plasmid labeled with a random primer method described in the text. Exposure time is 2 days. No band was found in adult mice even after longer exposure time. Lower panel: Northern blot of 18S RNA as an internal standard. The method is described in the text.
esting to examine the expression pattern of L1;4 -galactosyltransferase in fetal mouse brain. In this report, we found that during the develop-
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