Control of Bisecting GlcNAc Addition to N-Linked Sugar Chains

Abstract

In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between β-1,4-GalT (UDP-galactose:N-acetylglucosamine β-1,4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:β-d-mannoside β-1,4-N-acetylglucosaminyltransferase) was examined. We isolated a β-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed β-1,4-GalT-I or GnT-III. In the β-1,4-GalT-I-single knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where β-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the β-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that β-1,4-GalT competes with GnT-III for substrate in the cells. In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between β-1,4-GalT (UDP-galactose:N-acetylglucosamine β-1,4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:β-d-mannoside β-1,4-N-acetylglucosaminyltransferase) was examined. We isolated a β-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed β-1,4-GalT-I or GnT-III. In the β-1,4-GalT-I-single knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where β-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the β-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that β-1,4-GalT competes with GnT-III for substrate in the cells. UDP-N-acetylglucosamine:β-d-mannoside β-1,4-N-acetylglucosaminyltransferase 4-GalT, UDP-galactose:N-acetylglucosamine β-1,4-galactosyltransferase pyridylamino- fetal bovine serum polymerase chain reaction high performance liquid chromatography ganciclovir A β-1,4-linked N-acetylglucosamine residue attached to the mannose-β-1,4- in the trimannosyl core of N-linked sugar chains has been described in complex-type and hybrid-type sugar chains of various glycoproteins such as IgA, IgG, IgM, etc. (1Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7260-7269Abstract Full Text PDF PubMed Google Scholar, 2Mizuochi T. Taniguchi T. Shimizu A. Kobata A. J. Immunol. 1982; 129: 2016-2020PubMed Google Scholar, 3Cahour A. Debeire P. Hartmann L. Montreuil J. van Halbeek H. Vliegenthart J.F.G. FEBS Lett. 1984; 170: 343-349Crossref PubMed Scopus (23) Google Scholar). This GlcNAc residue has been termed a “bisecting” GlcNAc and is formed by β-d-mannoside β-1,4-N-acetylglucosaminyltransferase (GnT-III).1 GnT-III is presumed to be involved in pathological conditions, because an increase in its expression is accompanied by malignant transformation or oncofetal changes (4Miyoshi E. Nishikawa A. Ihara Y. Gu J. Sugiyama T. Hayashi N. Fusamoto H. Kamada T. Taniguchi N. Cancer Res. 1993; 53: 3899-3902PubMed Google Scholar, 5Yoshimura M. Nishikawa A. Ihara Y. Nishiura T. Nakao H. Kanayama Y. Matuzawa Y. Taniguchi N. Int. J. Cancer. 1995; 60: 443-449Crossref PubMed Scopus (26) Google Scholar). Although the function of the bisecting GlcNAc is not well understood, this modification can inhibit the action of some enzymes (α-mannosidase-II, GnT-II, GnT-V, core α-1,6-fucosyltransferase) in the subsequent biosynthesis of N-linked sugar chains, suggesting a regulatory role in the formation of complex-type and hybrid-type sugar chains (6Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (489) Google Scholar). It is also known that the occurrence of bisecting GlcNAc-containing sugar chains on IgG increases with age (7Yamada E. Tsukamoto Y. Sasaki R. Yagyu K. Takahashi N. Glycoconj. J. 1997; 14: 401-405Crossref PubMed Scopus (128) Google Scholar).Here, we studied a method to control the addition of bisecting GlcNAc to N-linked sugar chains. We used human monoclonal IgM as a model glycoprotein for control of the attachment of the bisecting GlcNAc residue. Human IgM is a glycoprotein containing 7–12% carbohydrate distributed at five N-glycosylation sites in the constant region of the heavy chain at positions Asn-171, Asn-332, Asn-395, Asn-402, and Asn-563 (8Shimizu A. Putnam F.W. Paul C. Clamp J.R. Johnson I. Nat. New Biol. 1971; 231: 73-76Crossref PubMed Scopus (55) Google Scholar, 9Putnam F.W. Florent G. Paul C. Shinoda T. Shimizu A. Science. 1973; 182: 287-291Crossref PubMed Scopus (159) Google Scholar, 10Ohbayashi H. Endo T. Mihaesco E. Gonzales M.G. Kochibe N. Kobata A. Arch. Biochem. Biophys. 1989; 269: 463-475Crossref PubMed Scopus (22) Google Scholar, 11Hickman S. Kornfeld R. Osterland C.K. Kornfeld S. J. Biol. Chem. 1972; 247: 2156-2163Abstract Full Text PDF PubMed Google Scholar, 12Chapman A. Kornfeld R. J. Biol. Chem. 1979; 254: 816-823Abstract Full Text PDF PubMed Google Scholar). The sugar chains at Asn-402 and Asn-563 are high mannose-type chains, and those at Asn-171, Asn-332, and Asn-395 are complex-type chains. Sugar chains of the human IgM produced by hybridoma cells were analyzed at each of the five glycosylation sites on the μ-chain (13Monica T.J. Williams S.B. Goochee C.F. Maiorella B.L. Glycobiology. 1995; 5: 175-185Crossref PubMed Scopus (29) Google Scholar, 14Leibiger H. Kersten B. Albersheim P. Darvill A. Glycobiology. 1998; 8: 497-507Crossref PubMed Scopus (10) Google Scholar). Although the function of the sugar chains of IgM is not well understood, it has been demonstrated that the sugar chain structure at Asn-402 on mouse IgM influences the ability of the IgM-antigen complex to bind complement (15Wright J.F. Shulman M.J. Isenman D.E. Painter R.H. J. Biol. Chem. 1990; 265: 10506-10513Abstract Full Text PDF PubMed Google Scholar). Bazin et al. (16Bazin R. Darveau A. Martel F. Pelletier A. Piche L. St-Laurent M. Thibault L. Demers A. Boyer L. Lemieux G. Lemieux R. J. Immunol. 1992; 149: 3889-3893PubMed Google Scholar) reported that murine hybridoma-produced IgM lacking the sugar chain at Asn-563 had increased avidity for antigen. The function of the sugar chains of antibodies has been well studied in the case of IgG. For example, hypogalactosylation of IgG has been shown to affect some of the effector functions of the IgG molecule including binding to complement C1q and mannose-binding protein (17Tsuchiya N. Endo T. Matsuta K. Yoshinoya S. Aikawa T. Kosuge E. Takeuchi F. Miyamoto T. Kobata A. J. Rheumatol. 1989; 16: 285-290PubMed Google Scholar). Patients with rheumatoid arthritis have a higher frequency of IgG lacking galactose (18Parekh R.B. Roitt I.M. Isenberg D.A. Dwek R.A Ansell B.M. Rademacher T.W. Lancet. 1988; 1: 966-969Abstract PubMed Scopus (196) Google Scholar).Our original interest was to investigate the effect of hypogalactosylation on the function of IgM. For this purpose, we attempted to isolate β-1,4-GalT-I-null B cells producing IgM. Although we failed to isolate β-1,4-GalT-I-null cells, we isolated a single knockout cell clone in which the β-1,4-GalT level was reduced to half due to disruption of one of the two β-1,4-GalT-I alleles. In the case of the IgM produced by this β-1,4-GalT-I single knockout clone, hypogalactosylation was not observed; however, we discovered that the extent of bisecting GlcNAc addition to the sugar chains of the IgM was increased. To explain this unexpected increase in the extent of bisecting GlcNAc addition, we speculated that β-1,4-GalT might compete with GnT-III for substrate in the cells. It has been previously reported that, in an in vitro system using sugar chains in a free form or glycopeptides, GnT-III and β-1,4-GalT react with an agalactosyl nonbisected biantennary sugar chain as a suggesting that GnT-III and β-1,4-GalT compete for the substrate in vitro S. Nishiura T. Nishikawa A. R. Taniguchi N. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the occurrence of such competition in cells was as the of the two as reported previously J. J. Cell Biol. 1982; Scopus Google Scholar, D.E. Kornfeld S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Ihara Y. Y. Taniguchi N. Glycobiology. 1997; PubMed Scopus Google Scholar, Y. T. Nishikawa A. E. M. Taniguchi N. Glycoconj. J. 1995; Scopus Google Scholar). In the present study, we attempted to the competition between β-1,4-GalT and GnT-III in cells. We that the extent of bisecting GlcNAc addition to the sugar chains of IgM is significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. that sugar chain can be and by the levels of expression of attempted to B cells producing IgM to the effect of hypogalactosylation on the function of IgM. For this purpose, we isolated a single knockout clone in which the intracellular level of β-1,4-GalT activity was reduced to half due to disruption of one of the two β-1,4-GalT-I alleles. In the β-1,4-GalT-I single knockout cells, sugar chains were not in the IgM we discovered an the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased in the cells, suggesting that β-1,4-GalT is involved in the formation of the bisecting has been shown in vitro that GnT-III and β-1,4-GalT can react with an agalactosyl nonbisected biantennary sugar chain as a substrate S. Nishiura T. Nishikawa A. R. Taniguchi N. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). β-1,4-GalT the of this sugar chain to a the can be a substrate for indicating that GnT-III and β-1,4-GalT compete for an agalactosyl nonbisected biantennary sugar chain as a the increased extent of bisecting GlcNAc addition in the case of β-1,4-GalT-I single knockout cells be by the competition between GnT-III and β-1,4-GalT for Nishiura et al. T. S. Kanayama Y. Nishikawa A. Y. M. T. Nakao H. T. Taniguchi N. S. Cancer Res. 1990; Google Scholar) also that the extent of bisecting GlcNAc formation and that of galactose addition in IgG are by the between GnT-III activity and β-1,4-GalT activity. For such competition to however, GnT-III and β-1,4-GalT be at the It is well known that are on the of the in the of the β-1,4-GalT is known to be on the J. J. Cell Biol. 1982; Scopus Google Scholar, D.E. Kornfeld S. J. Biol. Chem. Full Text PDF PubMed Google GnT-III is to be present on the K. Ihara Y. Y. Taniguchi N. Glycobiology. 1997; PubMed Scopus Google Scholar, Y. T. Nishikawa A. E. M. Taniguchi N. Glycoconj. J. 1995; Scopus Google Scholar). is to the two enzymes were to be at it has been that β-1,4-GalT not be to compete with GnT-III in cells, the two enzymes compete in vitro are to react with an agalactosyl nonbisected biantennary sugar the present study, we β-1,4-GalT and GnT-III compete for a substrate the cells. described the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased in the case of β-1,4-GalT-I single knockout clone the extent was in the case of the β-1,4-GalT-I In the GnT-III transfectants, the extent increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. the competition between the two enzymes in cells is it was that the addition of the bisecting GlcNAc residue was not by GnT-III was also by the level of expression of β-1,4-GalT relative to that of GnT-III. we have demonstrated that the extent of bisecting GlcNAc addition to sugar chains can be by the levels of expression of the β-1,4-GalT and GnT-III. The demonstrated competition between β-1,4-GalT and GnT-III that two enzymes at the in the cells. we not where the overexpressed β-1,4-GalT-I and GnT-III are on the in the transfectants, is to the competition between two enzymes in the is that a of addition of to be by the of bisecting GlcNAc residue was This might that the of the bisecting GlcNAc residue reaction as reported in vitro M. FEBS Lett. 1995; PubMed Scopus Google between IgM with and the bisecting GlcNAc has not been demonstrated in on the and binding to C1q not The role of the bisecting GlcNAc to the sugar chains of IgM be is well known that sugar chains in the of glycoproteins such as and The functions of glycoproteins are to be by of the sugar chain for the sugar chain are for this we have in sugar chain for the by the levels of expression of the control was in of the attachment of bisecting The present as a for to control sugar chain A β-1,4-linked N-acetylglucosamine residue attached to the mannose-β-1,4- in the trimannosyl core of N-linked sugar chains has been described in complex-type and hybrid-type sugar chains of various glycoproteins such as IgA, IgG, IgM, etc. (1Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7260-7269Abstract Full Text PDF PubMed Google Scholar, 2Mizuochi T. Taniguchi T. Shimizu A. Kobata A. J. Immunol. 1982; 129: 2016-2020PubMed Google Scholar, 3Cahour A. Debeire P. Hartmann L. Montreuil J. van Halbeek H. Vliegenthart J.F.G. FEBS Lett. 1984; 170: 343-349Crossref PubMed Scopus (23) Google Scholar). This GlcNAc residue has been termed a “bisecting” GlcNAc and is formed by β-d-mannoside β-1,4-N-acetylglucosaminyltransferase (GnT-III).1 GnT-III is presumed to be involved in pathological conditions, because an increase in its expression is accompanied by malignant transformation or oncofetal changes (4Miyoshi E. Nishikawa A. Ihara Y. Gu J. Sugiyama T. Hayashi N. Fusamoto H. Kamada T. Taniguchi N. Cancer Res. 1993; 53: 3899-3902PubMed Google Scholar, 5Yoshimura M. Nishikawa A. Ihara Y. Nishiura T. Nakao H. Kanayama Y. Matuzawa Y. Taniguchi N. Int. J. Cancer. 1995; 60: 443-449Crossref PubMed Scopus (26) Google Scholar). Although the function of the bisecting GlcNAc is not well understood, this modification can inhibit the action of some enzymes (α-mannosidase-II, GnT-II, GnT-V, core α-1,6-fucosyltransferase) in the subsequent biosynthesis of N-linked sugar chains, suggesting a regulatory role in the formation of complex-type and hybrid-type sugar chains (6Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (489) Google Scholar). It is also known that the occurrence of bisecting GlcNAc-containing sugar chains on IgG increases with age (7Yamada E. Tsukamoto Y. Sasaki R. Yagyu K. Takahashi N. Glycoconj. J. 1997; 14: 401-405Crossref PubMed Scopus (128) Google Scholar). we studied a method to control the addition of bisecting GlcNAc to N-linked sugar chains. We used human monoclonal IgM as a model glycoprotein for control of the attachment of the bisecting GlcNAc residue. Human IgM is a glycoprotein containing 7–12% carbohydrate distributed at five N-glycosylation sites in the constant region of the heavy chain at positions Asn-171, Asn-332, Asn-395, Asn-402, and Asn-563 (8Shimizu A. Putnam F.W. Paul C. Clamp J.R. Johnson I. Nat. New Biol. 1971; 231: 73-76Crossref PubMed Scopus (55) Google Scholar, 9Putnam F.W. Florent G. Paul C. Shinoda T. Shimizu A. Science. 1973; 182: 287-291Crossref PubMed Scopus (159) Google Scholar, 10Ohbayashi H. Endo T. Mihaesco E. Gonzales M.G. Kochibe N. Kobata A. Arch. Biochem. Biophys. 1989; 269: 463-475Crossref PubMed Scopus (22) Google Scholar, 11Hickman S. Kornfeld R. Osterland C.K. Kornfeld S. J. Biol. Chem. 1972; 247: 2156-2163Abstract Full Text PDF PubMed Google Scholar, 12Chapman A. Kornfeld R. J. Biol. Chem. 1979; 254: 816-823Abstract Full Text PDF PubMed Google Scholar). The sugar chains at Asn-402 and Asn-563 are high mannose-type chains, and those at Asn-171, Asn-332, and Asn-395 are complex-type chains. Sugar chains of the human IgM produced by hybridoma cells were analyzed at each of the five glycosylation sites on the μ-chain (13Monica T.J. Williams S.B. Goochee C.F. Maiorella B.L. Glycobiology. 1995; 5: 175-185Crossref PubMed Scopus (29) Google Scholar, 14Leibiger H. Kersten B. Albersheim P. Darvill A. Glycobiology. 1998; 8: 497-507Crossref PubMed Scopus (10) Google Scholar). Although the function of the sugar chains of IgM is not well understood, it has been demonstrated that the sugar chain structure at Asn-402 on mouse IgM influences the ability of the IgM-antigen complex to bind complement (15Wright J.F. Shulman M.J. Isenman D.E. Painter R.H. J. Biol. Chem. 1990; 265: 10506-10513Abstract Full Text PDF PubMed Google Scholar). Bazin et al. (16Bazin R. Darveau A. Martel F. Pelletier A. Piche L. St-Laurent M. Thibault L. Demers A. Boyer L. Lemieux G. Lemieux R. J. Immunol. 1992; 149: 3889-3893PubMed Google Scholar) reported that murine hybridoma-produced IgM lacking the sugar chain at Asn-563 had increased avidity for antigen. The function of the sugar chains of antibodies has been well studied in the case of IgG. For example, hypogalactosylation of IgG has been shown to affect some of the effector functions of the IgG molecule including binding to complement C1q and mannose-binding protein (17Tsuchiya N. Endo T. Matsuta K. Yoshinoya S. Aikawa T. Kosuge E. Takeuchi F. Miyamoto T. Kobata A. J. Rheumatol. 1989; 16: 285-290PubMed Google Scholar). Patients with rheumatoid arthritis have a higher frequency of IgG lacking galactose (18Parekh R.B. Roitt I.M. Isenberg D.A. Dwek R.A Ansell B.M. Rademacher T.W. Lancet. 1988; 1: 966-969Abstract PubMed Scopus (196) Google Scholar). original interest was to investigate the effect of hypogalactosylation on the function of IgM. For this purpose, we attempted to isolate β-1,4-GalT-I-null B cells producing IgM. Although we failed to isolate β-1,4-GalT-I-null cells, we isolated a single knockout cell clone in which the β-1,4-GalT level was reduced to half due to disruption of one of the two β-1,4-GalT-I alleles. In the case of the IgM produced by this β-1,4-GalT-I single knockout clone, hypogalactosylation was not observed; however, we discovered that the extent of bisecting GlcNAc addition to the sugar chains of the IgM was increased. To explain this unexpected increase in the extent of bisecting GlcNAc addition, we speculated that β-1,4-GalT might compete with GnT-III for substrate in the cells. It has been previously reported that, in an in vitro system using sugar chains in a free form or glycopeptides, GnT-III and β-1,4-GalT react with an agalactosyl nonbisected biantennary sugar chain as a suggesting that GnT-III and β-1,4-GalT compete for the substrate in vitro S. Nishiura T. Nishikawa A. R. Taniguchi N. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the occurrence of such competition in cells was as the of the two as reported previously J. J. Cell Biol. 1982; Scopus Google Scholar, D.E. Kornfeld S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Ihara Y. Y. Taniguchi N. Glycobiology. 1997; PubMed Scopus Google Scholar, Y. T. Nishikawa A. E. M. Taniguchi N. Glycoconj. J. 1995; Scopus Google Scholar). In the present study, we attempted to the competition between β-1,4-GalT and GnT-III in cells. We that the extent of bisecting GlcNAc addition to the sugar chains of IgM is significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. that sugar chain can be and by the levels of expression of attempted to B cells producing IgM to the effect of hypogalactosylation on the function of IgM. For this purpose, we isolated a single knockout clone in which the intracellular level of β-1,4-GalT activity was reduced to half due to disruption of one of the two β-1,4-GalT-I alleles. In the β-1,4-GalT-I single knockout cells, sugar chains were not in the IgM we discovered an the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased in the cells, suggesting that β-1,4-GalT is involved in the formation of the bisecting has been shown in vitro that GnT-III and β-1,4-GalT can react with an agalactosyl nonbisected biantennary sugar chain as a substrate S. Nishiura T. Nishikawa A. R. Taniguchi N. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). β-1,4-GalT the of this sugar chain to a the can be a substrate for indicating that GnT-III and β-1,4-GalT compete for an agalactosyl nonbisected biantennary sugar chain as a the increased extent of bisecting GlcNAc addition in the case of β-1,4-GalT-I single knockout cells be by the competition between GnT-III and β-1,4-GalT for Nishiura et al. T. S. Kanayama Y. Nishikawa A. Y. M. T. Nakao H. T. Taniguchi N. S. Cancer Res. 1990; Google Scholar) also that the extent of bisecting GlcNAc formation and that of galactose addition in IgG are by the between GnT-III activity and β-1,4-GalT activity. For such competition to however, GnT-III and β-1,4-GalT be at the It is well known that are on the of the in the of the β-1,4-GalT is known to be on the J. J. Cell Biol. 1982; Scopus Google Scholar, D.E. Kornfeld S. J. Biol. Chem. Full Text PDF PubMed Google GnT-III is to be present on the K. Ihara Y. Y. Taniguchi N. Glycobiology. 1997; PubMed Scopus Google Scholar, Y. T. Nishikawa A. E. M. Taniguchi N. Glycoconj. J. 1995; Scopus Google Scholar). is to the two enzymes were to be at it has been that β-1,4-GalT not be to compete with GnT-III in cells, the two enzymes compete in vitro are to react with an agalactosyl nonbisected biantennary sugar the present study, we β-1,4-GalT and GnT-III compete for a substrate the cells. described the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased in the case of β-1,4-GalT-I single knockout clone the extent was in the case of the β-1,4-GalT-I In the GnT-III transfectants, the extent increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. the competition between the two enzymes in cells is it was that the addition of the bisecting GlcNAc residue was not by GnT-III was also by the level of expression of β-1,4-GalT relative to that of GnT-III. we have demonstrated that the extent of bisecting GlcNAc addition to sugar chains can be by the levels of expression of the β-1,4-GalT and GnT-III. The demonstrated competition between β-1,4-GalT and GnT-III that two enzymes at the in the cells. we not where the overexpressed β-1,4-GalT-I and GnT-III are on the in the transfectants, is to the competition between two enzymes in the is that a of addition of to be by the of bisecting GlcNAc residue was This might that the of the bisecting GlcNAc residue reaction as reported in vitro M. FEBS Lett. 1995; PubMed Scopus Google between IgM with and the bisecting GlcNAc has not been demonstrated in on the and binding to C1q not The role of the bisecting GlcNAc to the sugar chains of IgM be is well known that sugar chains in the of glycoproteins such as and The functions of glycoproteins are to be by of the sugar chain for the sugar chain are for this we have in sugar chain for the by the levels of expression of the control was in of the attachment of bisecting The present as a for to control sugar chain We attempted to B cells producing IgM to the effect of hypogalactosylation on the function of IgM. For this purpose, we isolated a single knockout clone in which the intracellular level of β-1,4-GalT activity was reduced to half due to disruption of one of the two β-1,4-GalT-I alleles. In the β-1,4-GalT-I single knockout cells, sugar chains were not in the IgM we discovered an the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased in the cells, suggesting that β-1,4-GalT is involved in the formation of the bisecting It has been shown in vitro that GnT-III and β-1,4-GalT can react with an agalactosyl nonbisected biantennary sugar chain as a substrate S. Nishiura T. Nishikawa A. R. Taniguchi N. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). β-1,4-GalT the of this sugar chain to a the can be a substrate for indicating that GnT-III and β-1,4-GalT compete for an agalactosyl nonbisected biantennary sugar chain as a the increased extent of bisecting GlcNAc addition in the case of β-1,4-GalT-I single knockout cells be by the competition between GnT-III and β-1,4-GalT for Nishiura et al. T. S. Kanayama Y. Nishikawa A. Y. M. T. Nakao H. T. Taniguchi N. S. Cancer Res. 1990; Google Scholar) also that the extent of bisecting GlcNAc formation and that of galactose addition in IgG are by the between GnT-III activity and β-1,4-GalT activity. For such competition to however, GnT-III and β-1,4-GalT be at the It is well known that are on the of the in the of the β-1,4-GalT is known to be on the J. J. Cell Biol. 1982; Scopus Google Scholar, D.E. Kornfeld S. J. Biol. Chem. Full Text PDF PubMed Google GnT-III is to be present on the K. Ihara Y. Y. Taniguchi N. Glycobiology. 1997; PubMed Scopus Google Scholar, Y. T. Nishikawa A. E. M. Taniguchi N. Glycoconj. J. 1995; Scopus Google Scholar). is to the two enzymes were to be at it has been that β-1,4-GalT not be to compete with GnT-III in cells, the two enzymes compete in vitro are to react with an agalactosyl nonbisected biantennary sugar In the present study, we β-1,4-GalT and GnT-III compete for a substrate the cells. described the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased in the case of β-1,4-GalT-I single knockout clone the extent was in the case of the β-1,4-GalT-I In the GnT-III transfectants, the extent increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular β-1,4-GalT activity relative to that of GnT-III. the competition between the two enzymes in cells is it was that the addition of the bisecting GlcNAc residue was not by GnT-III was also by the level of expression of β-1,4-GalT relative to that of GnT-III. we have demonstrated that the extent of bisecting GlcNAc addition to sugar chains can be by the levels of expression of the β-1,4-GalT and GnT-III. The demonstrated competition between β-1,4-GalT and GnT-III that two enzymes at the in the cells. we not where the overexpressed β-1,4-GalT-I and GnT-III are on the in the transfectants, is to the competition between two enzymes in the cells. It is that a of addition of to be by the of bisecting GlcNAc residue was This might that the of the bisecting GlcNAc residue reaction as reported in vitro M. FEBS Lett. 1995; PubMed Scopus Google Scholar). A between IgM with and the bisecting GlcNAc has not been demonstrated in on the and binding to C1q not The role of the bisecting GlcNAc to the sugar chains of IgM be It is well known that sugar chains in the of glycoproteins such as and The functions of glycoproteins are to be by of the sugar chain for the sugar chain are for this we have in sugar chain for the by the levels of expression of the control was in of the attachment of bisecting The present as a for to control sugar chain We Kobata and Endo of for We are also to Taniguchi for GnT-III and to N. for human β-1,4-GalT-I We also for