Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis
2006; Elsevier BV; Volume: 281; Issue: 19 Linguagem: Inglês
10.1074/jbc.m601453200
ISSN1083-351X
AutoresYasuhiko Kizuka, Takahiro Matsui, Hiromu Takematsu, Yasunori Kozutsumi, Toshisuke Kawasaki, Shogo Oka,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoHNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate. HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate. Glycosylation is one of the major post-translational protein modifications, especially for cell surface proteins, that play important roles in a variety of cellular functions, including recognition and adhesion. Among them, human natural killer-1 (HNK-1) 2The abbreviations used are: HNK-1, human natural killer-1; ASOR, asialo-orosomucoid; CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; GAG, glycosaminoglycan; GlcAT, glucuronyltransferase; HRP, horseradish peroxidase; mAb, monoclonal antibody; pAb, polyclonal antibodies; PBS, phosphate-buffered saline; ST, sulfotransferase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; ER, endoplasmic reticulum. carbohydrate, which is recognized by HNK-1 monoclonal antibody, is predominantly expressed in the nervous system (1Schwarting G.A. Jungalwala F.B. Chou D.K. Boyer A.M. Yamamoto M. Dev. Biol. 1987; 120: 65-76Crossref PubMed Scopus (142) Google Scholar, 2Yoshihara Y. Oka S. Watanabe Y. Mori K. J. Cell Biol. 1991; 115: 731-744Crossref PubMed Scopus (57) Google Scholar). HNK-1 carbohydrate is expressed on several glycoproteins and glycolipids and is considered to be involved in cell migration, recognition, and adhesion. The unique structural feature of this carbohydrate is a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc) (3Voshol H. van Zuylen C.W. Orberger G. Vliegenthart J.F. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 4Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar). Recently, we cloned two glucuronyltransferases (GlcAT-P and GlcAT-S) (5Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 6Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar, 7Yamamoto S. Oka S. Saito-Ohara F. Inazawa J. Kawasaki T. J. Biochem. (Tokyo). 2002; 131: 337-347Crossref PubMed Scopus (21) Google Scholar, 8Mitsumoto Y. Oka S. Sakuma H. Inazawa J. Kawasaki T. Genomics. 2000; 65: 166-173Crossref PubMed Scopus (45) Google Scholar, 9Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and one sulfotransferase (HNK-1ST) (10Bakker H. Friedmann I. Oka S. Kawasaki T. Nifant'ev N. Schachner M. Mantei N. J. Biol. Chem. 1997; 272: 29942-29946Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) that are involved in the biosynthesis of HNK-1 carbohydrate. To investigate the biological function of HNK-1 carbohydrate in vivo, we generated GlcAT-P gene-deficient mice and revealed that HNK-1 carbohydrate is involved in synaptic plasticity (11Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). More recently, we found a nonsulfated form of HNK-1 carbohydrate in mouse kidney (12Tagawa H. Kizuka Y. Ikeda T. Itoh S. Kawasaki N. Kurihara H. Onozato M.L. Tojo A. Sakai T. Kawasaki T. Oka S. J. Biol. Chem. 2005; 280: 23876-23883Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), where only GlcAT-S was expressed and not HNK-1ST. In the nervous system, however, all the HNK-1 carbohydrate structures identified so far are sulfated forms because of the presence of glucuronyltransferases (GlcAT-P and GlcAT-S) and HNK-1ST (13Gallego R.G. Blanco J.L. Thijssen-van Zuylen C.W. Gotfredsen C.H. Voshol H. Duus J.O. Schachner M. Vliegenthart J.F. J. Biol. Chem. 2001; 276: 30834-30844Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Liedtke S. Geyer H. Wuhrer M. Geyer R. Frank G. Gerardy-Schahn R. Zahringer U. Schachner M. Glycobiology. 2001; 11: 373-384Crossref PubMed Scopus (86) Google Scholar). The fact that only the existence of HNK-1ST is sufficient for the sulfation of HNK-1 carbohydrate and that a nonsulfated form has not been detected in brain leads us to hypothesize that GlcAT-P or GlcAT-S interacts with HNK-1ST, resulting in sequential and efficient biosynthesis of the HNK-1 carbohydrate. Oligosaccharides expressed on glycoproteins and glycolipids are biosynthesized by glycosyltransferases in a stepwise manner. Recently, endoplasmic reticulum (ER) or Golgi resident glycosyltransferases and related proteins, such as epimerase, sulfotransferase, and sugar-nucleotide transporters, were found to associate physically and functionally both for retention in the ER or Golgi and for regulation of their enzymatic activities (15de Graffenried C.L. Bertozzi C.R. Curr. Opin. Cell Biol. 2004; 16: 356-363Crossref PubMed Scopus (94) Google Scholar, 16Seko A. Yamashita K. Glycobiology. 2005; 15: 943-951Crossref PubMed Scopus (49) Google Scholar). In many cases, the N-terminal cytoplasmic tail, membrane-spanning domain, or stem region, but not the C-terminal catalytic domain, is needed and sufficient for formation of a complex. In this study, we investigated whether or not GlcAT-P or GlcAT-S interacts with HNK-1ST by using a transient expression system in CHO cells and by performing immunoprecipitation and immunofluorescence staining. Furthermore, we examined the effects of the interaction on their catalytic activities in vitro and in vivo. Monoclonal antibody (mAb) M6749 was a generous gift from Dr. H. Tanaka (Kumamoto University). Anti-HNK-1 mAb was purchased from the American Type Culture Collection. Mouse anti-FLAG M2 mAb and rabbit anti-FLAG polyclonal antibodies (pAb) were purchased from Sigma. Mouse anti-EGFP mAb and rabbit anti-EGFP pAb were purchased from Clontech. HRP-conjugated anti-mouse IgG and HRP-conjugated anti-mouse IgM were purchased from Zymed Laboratories Inc.. Alexa Fluor 568 anti-mouse IgG was purchased from Molecular Probes. Protein G-Sepharose TM4 Fast Flow, IgG-Sepharose TM6 Fast Flow, and Blue-Sepharose TM6 Fast Flow were obtained from Amersham Biosciences. [35S]Adenosine 3′-phosphate,5′-phosphosulfate was purchased from PerkinElmer Life Sciences. Expression vectors p3XFLAG-CMV-10 and p3XFLAG-CMV-14 were from Sigma, and pEGFP-N1 and pIRES were from Clontech. pGIR201protA was kindly provided by Dr. H. Kitagawa (Kobe Pharmaceutical University). Rat HNK-1ST cDNA cloned into pBluescript (pBluescript/HNK-1ST) was kindly provided by Dr. Hans Bakker (Swiss Federal Institute of Technology). Mouse C4ST1 and human GalNAc4ST1 cDNA were kindly provided by Dr. O. Habuchi (Aichi University of Education). Construction of Full-length cDNAs in pIRES—Rat GlcAT-P cDNA was released from pCRII/GlcAT-P (5Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar) with EcoRI and inserted into the EcoRI site of multicloning site A in pIRES (pIRES/P). Rat HNK-1ST cDNA was amplified by PCR with the primer pair listed below using pBluescript/HNK-1ST as a template to create SalI sites at both the 5′ and 3′ ends of the coding sequence of HNK-1ST, and then cloned into the SalI site of multicloning site B in pIRES/P (pIRES/P+ST). Rat GlcAT-S cDNA was released from pEF-BOS/GlcAT-S (6Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar) with XbaI and then inserted into the NheI site of multicloning site A in pIRES (pIRES/S). HNK-1ST cDNA was then released with SalI and NotI from pBluescript/HNK-1ST and cloned into multicloning site B in pIRES/S digested with the same restriction enzymes (pIRES/S+ST). Construction of Epitope-tagged cDNAs in Expression Plasmid—Full-length cDNAs of the rat GlcAT-P, rat GlcAT-S, human GlcAT-I, and rat ST3GalIV coding sequences were amplified by PCR using the primers listed below to create a 5′-EcoRI site (skipping initiation ATG codon) and a 3′-EcoRV site, and then cloned into p3XFLAG-CMV-10 (SIGMA) using the EcoRI and EcoRV sites. The rat HNK-1ST, mouse C4ST1, and human GalNAc4ST1 coding sequences were cloned into pEGFP-N1 (Clontech) using HindIII and AgeI sites (skipping stop codon) in the same way as described above. The catalytic domain of human HNK-1ST (HNK-1STcat, from Leu63 to the C terminus) was cloned into pGIR201-prot.A using cDNAs amplified by PCR using the primers listed below. pGIR201-prot.A-HNK-1STcat was then digested with NheI. The NheI DNA fragment consisting of the insulin signal sequence, the IgG-binding domain of protein A, and HNK-1STcat was subcloned into a mammalian expression vector, pEF-BOS, that had been digested with XbaI (pEF-BOS-prot.A-STcat). Construction of plasmids for expression of FLAG-Pcat (from T58 to the C terminus) and FLAG-Scat (from A50 to the C terminus) was described previously (17Kakuda S. Oka S. Kawasaki T. Protein Expression Purif. 2004; 35: 111-119Crossref PubMed Scopus (14) Google Scholar). The primers used were as follows: FLAG-GlcAT-P, TGCGAATTCGGGTAATGAGGAGCTGTGGG and TGTGATATCTCAGATCTCCACCGAGGGGT; FLAG-GlcAT-S, GCGGAATTCGAAGTCCGCGCTGTGCAACC and TCCGATATCCTACACCTCGATGTTCACTG; FLAG-GlcAT-I, CGCGAATTCGAAGCTGAAGCTGAAGAACG and TGGGATATCGCCGCCATCACACCTCAATT; FLAG-ST3GalIV, GAGGAATTCGACCAGCAAATCTCACTGGA and GGAGATATCCTCTCATCCAAGTCAGAAGT; HNK-1ST-EGFP, CGGAAGCTTCCTGTTTGACAACATGCACC and ATGACCGGTTTTAGCAAAAAATCTGGTTT; prot.A-STcat, TCCGAATTCACTGAAGCCAACTGGGAAGG and TAGGAATTCTGCATTAGTTTAGCAAAAAG; HNK-1ST (for pIRES/P+ST), CGGGTCGACCCTGTTTGACAACATGCACC and GCAGTCGACAGGAGTCTGATGCCTTAATT; C4ST1-EGFP, AAGCTTGCCGCCATGAAGCCGGCGCTG and TCTAGAGACCGGTGGATCCAACTTCAGGTAG; and GalNAc4ST1-EGFP, AAGCTTGCCGCCATGACCCTGCGACCTG and TCTAGAGACCGGTGGGTACAGATCTGCAAAGG. CHO cells were cultured in α-minimum Eagle's medium supplemented with 10% fetal bovine serum at 37 °C until 50-80% confluency. For transfection, cells were plated on 100-mm tissue culture dishes, grown overnight, and then transfected with various expression constructs using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. Briefly, a 2.5-fold volume of FuGENE 6 and 4 μgof each DNA were incubated with 300 μlof α-minimum Eagle's for 15 min at room temperature in a polystyrene tube, and then the mixture was added to the tissue culture dishes. Cells were collected 24 h after transfection and lysed with lysis buffer consisting of 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.5% deoxycholate, and protease inhibitor mixture (Nacalai Tesque). After centrifugation, the clarified lysate was incubated with anti-FLAG rabbit pAb (Sigma) or anti-EGFP rabbit pAb (Clontech) for 1 h. The mixture was then incubated with protein G-Sepharose TM4 Fast Flow (Amersham Biosciences) for 2 h with gentle shaking. The beads were precipitated by centrifugation (500 × g for 3 min) and then washed three times with an excess volume of wash buffer consisting of 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.1% Triton X-100. Proteins bound to the Sepharose beads were eluted by boiling in Laemmli sample buffer. 24 h post-transfection, the cells were rinsed with PBS, fixed with ice-cold methanol, and then incubated in PBS with 3% bovine serum albumin. The cells were incubated with anti-FLAG M2 mouse mAb (Sigma) followed by incubation with Alexa Fluor 568 anti-mouse IgG antibodies (Molecular Probes). The cells were then visualized and digitized with a Fluoview laser confocal microscope system (Olympus, Japan). Proteins were separated by 5-20% gradient SDS-PAGE with the buffer system of Laemmli and then transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk in PBS containing 0.05% Tween 20, the membranes were incubated with primary antibodies, followed by HRP-conjugated secondary antibodies, and then protein bands were detected with ECL (Pierce) using a Luminoimage Analyzer LAS-3000 (Fuji). COS-1 cells plated on 100-mm tissue culture dishes were transfected with pEF-BOS-prot.A or pEF-BOS-prot.A-HNK-1STcat (10 μg each) using FuGENE 6 transfection reagent. After 6 h of incubation, the culture medium was replaced with serum-free Opti-MEM I (Invitrogen), followed by incubation for another 3 days. Culture medium containing secreted proteins was applied to a normal human IgG-Sepharose column (Amersham Biosciences). Unbound proteins were washed out with more than 10 column volumes of wash buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.05% Tween 20) and sequentially with 2 column volumes of 10 mm glycine-HCl, pH 5.0. Bound proteins were eluted with 100 mm glycine-HCl, pH 3.4, and then immediately neutralized with 1 m Tris-HCl, pH 8.0. The catalytic domains of human GlcAT-P and human GlcAT-S were expressed in and purified from Escherichia coli as described previously (17Kakuda S. Oka S. Kawasaki T. Protein Expression Purif. 2004; 35: 111-119Crossref PubMed Scopus (14) Google Scholar). Prot.A-STcat and protein A were purified from COS-1 cell culture media as described above. An aliquot (125 ng) of each protein was added to 250 μl of buffer (100 mm MES, pH 6.5, 0.2% Nonidet P-40, 5 mm MnCl2), and then the mixture was incubated for 1 h at 37°C. A fraction of the mixture was recovered for Western blot analysis, and normal IgG-Sepharose was added to the rest of the mixture followed by incubation for 2 h. The beads were precipitated by centrifugation (500 × g for 3 min) and then washed three times with an excess volume of buffer. Proteins bound to beads were eluted by boiling in Laemmli sample buffer, followed by SDS-PAGE and Western blot analysis with anti-FLAG M2 mAb. Glucuronyltransferase activity toward asialo-orosomucoid (ASOR) was measured essentially as described previously (18Kakuda S. Sato Y. Tonoyama Y. Oka S. Kawasaki T. Glycobiology. 2005; 15: 203-210Crossref PubMed Scopus (26) Google Scholar) with slight modification. Before adding to the reaction mixture, FLAG-Pcat or FLAG-Scat was incubated with or without an equivalent amount of prot.A-STcat at 37 °C for 30 min for enzyme complex formation. Then the preincubated enzyme solution was added to the assay reaction mixture as described previously (18Kakuda S. Sato Y. Tonoyama Y. Oka S. Kawasaki T. Glycobiology. 2005; 15: 203-210Crossref PubMed Scopus (26) Google Scholar). For this assay, we prepared an acceptor substrate, GlcA-ASOR, by transferring glucuronic acid to ASOR with GlcAT-P and GlcAT-S. FLAG-Pcat and FLAG-Scat (750 ng each) were added to 3 ml of the reaction mixture (100 mm MES, pH 6.5, 0.2% Nonidet P-40, 2.5 mm ATP, 20 mm MnCl2, 3 mg of ASOR, 1.2 μmol of UDP-GlcA), followed by incubation at 37 °C for 6 h. 375 ng of FLAG-Pcat and FLAG-Scat were then added to the mixture, followed by incubation at 37 °C overnight. The prepared mixture was dialyzed against an excess volume of sterile water. FLAG-Pcat and FLAG-Scat were then removed with a Blue-Sepharose column (Amersham Biosciences). Under these conditions, about 9.1 mol of GlcA was transferred to 1 mol of ASOR. The purified prot.A-STcat was incubated with or without an equivalent amount of FLAG-Pcat or FLAG-Scat at 37 °C for 30 min for enzyme complex formation. The preincubated enzyme solution was then incubated at 37 °C for 1 h in a reaction mixture with a final volume of 50 μl consisting of 20 mm BisTris-HCl, pH 6.6, 0.1% Triton X-100, 10 mm MnCl2, 2.5 mm ATP, 20 μg of GlcA-ASOR, and 100 μm [35S]adenosine 3′-phosphate, 5′-phosphosulfate (300,000 dpm). After incubation, the assay mixture was spotted onto a 2.5-cm Whatman No.1 disk. The disk was washed with a 10% (w/v) trichloroacetic acid solution three times, followed by ethanol/ether (2:1, v/v), and then with ether. The disk was air-dried, and then the radioactivity of [35S]HSO3-GlcA-ASOR was counted with a liquid scintillation counter (Beckman LS-6000). Co-immunoprecipitation of GlcAT-P or GlcAT-S and HNK-1ST— HNK-1 carbohydrate is highly expressed in the nervous system. The HNK-1 carbohydrate structures on P0 and NCAM, which are major carrier glycoproteins in the peripheral and central nervous systems, respectively, have been determined (13Gallego R.G. Blanco J.L. Thijssen-van Zuylen C.W. Gotfredsen C.H. Voshol H. Duus J.O. Schachner M. Vliegenthart J.F. J. Biol. Chem. 2001; 276: 30834-30844Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Liedtke S. Geyer H. Wuhrer M. Geyer R. Frank G. Gerardy-Schahn R. Zahringer U. Schachner M. Glycobiology. 2001; 11: 373-384Crossref PubMed Scopus (86) Google Scholar). All the HNK-1 carbohydrate structures so far determined are sulfated, i.e. sulfoglucuronic acid attached to N-acetyllactosamine, a nonsulfated form of HNK-1 carbohydrate not having been found on such molecules. These results indicate that most of the HNK-1 carbohydrate in the nervous system is expressed as a sulfated form. These results suggest the possibility that the HNK-1 carbohydrate biosynthetic key enzymes, glucuronyltransferases (GlcAT-P and GlcAT-S) and sulfotransferase (HNK-1ST), associate with each other, which results in efficient sequential biosynthesis. To investigate the interaction between the glucuronyltransferases (GlcAT-P and GlcAT-S) and HNK-1ST, FLAG-tagged glucuronyltransferases (FLAG tag fused at N terminus of each glucuronyltransferase, FLAG-P or FLAG-S) and EGFP-tagged HNK-1ST (EGFP fused at C terminus of HNK-1ST, ST-EGFP) were transiently expressed in CHO cells. Western blot analysis using the cell lysates revealed that each enzyme was detected at around the position of the expected molecular weight with anti-FLAG mAb (40-50 kDa) and anti-EGFP mAb (70 kDa), respectively (Fig. 1A). However, one major band and a few minor bands were detected for each enzyme. The major band corresponded to a fully glycosylated enzyme and the minor bands to partially glycosylated enzymes because they converged as one band with N-glycosidase F digestion (data not shown). As shown in Fig. 1A, bottom panel, HNK-1 carbohydrate was expressed on several glycoproteins in CHO cells co-expressing both FLAG-P or FLAG-S and ST-EGFP (lanes 4 and 5), indicating that these fusion enzymes have enzymatic activity. However, the immunoreactive bands for the cells expressing FLAG-S (Fig. 1A, bottom panel, lane 5) were weaker than for those expressing FLAG-P (lane 4), suggesting that the activity of FLAG-P is greater than that of FLAG-S and/or that the N-terminal FLAG tag may have a little effect on the GlcAT-S activity. Next, these enzymes in CHO cell lysates were immunoprecipitated with anti-FLAG rabbit pAb or anti-EGFP rabbit pAb, and then Western blot analyses were performed with anti-FLAG mouse mAb and anti-EGFP mouse mAb, respectively. As shown in Fig. 1B, both FLAG-P and FLAG-S were co-precipitated with ST-EGFP (upper panel, lanes 7 and 9) and vice versa (lower panel, lanes 8 and 10), indicating that each glucuronyltransferase bound with HNK-1ST and formed an enzyme complex in CHO cells. Co-localization of Glucuronyltransferases and HNK-1ST in CHO Cells—To investigate co-localization of glucuronyltransferases and HNK-1ST in CHO cells, we examined the immunofluorescence staining of the CHO cells. FLAG-P and FLAG-S were stained with anti-FLAG mAb (Fig. 2, A and D), and ST-EGFP was visualized according to its own EGFP fluorescence (Fig. 2, B and E). As shown in Fig. 2, C and F, both FLAG-P and FLAG-S were co-localized with HNK-1ST in CHO cells. These enzymes were mainly localized in the Golgi apparatus because they were co-localized with a Golgi marker, wheat germ agglutinin (data not shown). Binding Specificities as to Formation of These Enzyme Complexes—To investigate the binding specificities of these interactions, we performed immunoprecipitation experiments using other glycosyltransferases instead of GlcAT-P or GlcAT-S. We used FLAG-tagged GlcAT-I (Fig. 3, A and B) involved in the biosynthesis of the linkage region of glycosaminoglycans, and GlcAT-I exhibits amino acid sequence similarity with GlcAT-P and GlcAT-S (19Kitagawa H. Tone Y. Tamura J. Neumann K.W. Ogawa T. Oka S. Kawasaki T. Sugahara K. J. Biol. Chem. 1998; 273: 6615-6618Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). We expected that GlcAT-I would not bind to HNK-1ST, but FLAG-GlcAT-I (FLAG-I) was detected in the anti-EGFP pAb immunoprecipitate (Fig. 3B, upper panel, lane 5), and ST-EGFP was detected in anti-FLAG pAb immunoprecipitate (Fig. 3B, lower panel, lane 6), indicating that GlcAT-I also bound with HNK-1ST, as in the case of GlcAT-P and GlcAT-S. Next we used FLAG-tagged ST3GalIV (Fig. 3, C and D), which is a sialyltransferase and transfers sialic acid to the nonreducing terminal of N-acetyllactosamine residues on glycoproteins (20Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 1394-1401Abstract Full Text PDF PubMed Google Scholar). This enzyme is thought to compete with GlcAT-P or GlcAT-S for the acceptor substrates, i.e. the nonreducing terminal of N-acetyllactosamine residues. As shown in Fig. 3D (upper panel, lane 5, and lower panel, lane 6), the interaction of ST3GalIV with HNK-1ST was almost negligible compared with that of GlcAT-P or GlcAT-S with HNK-1ST, indicating that ST3GalIV did not form a complex with HNK-1ST. These results suggest that GlcAT-P and GlcAT-S bind specifically with HNK-1ST and that the interaction of GlcAT-I with HNK-1ST may have some biological significance in vivo. To examine the binding specificity of sulfotransferases, we used two sulfotransferases, C4ST1 and GalNAc4ST1, both of which belong to the HNK-1ST family (21Yamauchi S. Mita S. Matsubara T. Fukuta M. Habuchi H. Kimata K. Habuchi O. J. Biol. Chem. 2000; 275: 8975-8981Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Okuda T. Mita S. Yamauchi S. Fukuma M. Nakano H. Sawada T. Habuchi O. J. Biol. Chem. 2000; 275: 40605-40613Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). An EGFP tag was fused with these sulfotransferases at their C termini, and then they were co-expressed with FLAG-tagged GlcAT-P in CHO cells. By using these cell lysates, immunoprecipitation and Western blot analyses were carried out (Fig. 4, A and B). As expected, FLAG-P was not detected in the anti-EGFP pAb immunoprecipitate (Fig. 4B, upper panel, lanes 3 and 5), and both C4ST1-EGFP and GalNAc4ST1-EGFP were barely detected in the anti-FLAG immunoprecipitate (Fig. 4B, lower panel, lanes 4 and 6), suggesting that the interactions of both C4ST1 and GalNAc4ST1 with GlcAT-P are considerably weaker than that of HNK-1ST with GlcAT-P. We also confirmed that GlcAT-S was not co-immunoprecipitated with either C4ST1 or GalNAc4ST1 (data not shown). As described above, ST3GalIV, C4ST1, and GalNAc4ST1 did not bind or barely bound with HNK-1 biosynthetic enzymes (GlcAT-P and GlcAT-S and HNK-1ST). However, it is possible that these overexpressed enzymes showed the abnormal subcellular localization because of their fused tags; therefore, they may not have had the opportunity to encounter their counterparts in CHO cells. To exclude this possibility, we examined their intracellular localization by means of immunofluorescence staining (Fig. 5). FLAG-I and FLAG-ST3GalIV were stained with anti-FLAG mAb (Fig. 5, A and D). C4ST1-EGFP and GalNAc4ST1-EGFP were visualized according to their own EGFP fluorescence (Fig. 5, H and K). As shown in Fig. 5, A, D, H, and K, they were mainly localized in the Golgi apparatus, suggesting that the fused tags had no influence on their intracellular transport. Even if they were considerably co-localized with their counterparts as shown in Fig. 5, C, F, I, and L, they would not interact with each other (Figs. 3D and 4B). These lines of evidence indicate the specific interaction of GlcAT-P or GlcAT-S with HNK-1ST. GlcAT-P (or GlcAT-S) and HNK-1ST Form a Complex through Their Catalytic Domains—As shown in Fig. 3, GlcAT-I as well as GlcAT-P and GlcAT-S interacted with HNK-1ST. These three glucuronyltransferases have a type II membrane topology as in the case of other glycosyltransferases, i.e. an N-terminal short cytoplasmic tail, a transmembrane region, a stem region, and a catalytic region. GlcAT-I exhibits amino acid sequence similarity with GlcAT-P and GlcAT-S, the highest homology being seen in the catalytic domain. Thus, we hypothesized that the C-terminal catalytic domain of these enzymes is involved in the interaction with HNK-1ST. To examine this hypothesis, we prepared truncated enzymes only consisting of their catalytic domains. The FLAG-tagged human GlcAT-P catalytic domain (FLAG-Pcat, T58 to C terminus) and FLAG-tagged human GlcAT-S catalytic domain (FLAG-Scat, A50 to C terminus) were expressed in E. coli cells, and then purified as described previously (17Kakuda S. Oka S. Kawasaki T. Protein Expression Purif. 2004; 35: 111-119Crossref PubMed Scopus (14) Google Scholar). The protein A-tagged human HNK-1ST catalytic domain (prot.A-STcat, Leu63 to C terminus) was expressed in COS-1 cells and purified from the culture medium on a normal human IgG-Sepharose column. We then performed pulldown assays using the purified enzymes (Fig. 6). FLAG-Pcat or FLAG-Scat was mixed in a tube with prot.A-STcat or protein A (as a control), and then the mixture was incubated at 37 °C for 1 h. After the incubation, prot.A-STcat or protein A was precipitated with normal IgG-Sepharose, followed by Western blot analysis with anti-FLAG mAb (Fig. 6, bottom panel). An aliquot of the incubation mixture was taken before adding the IgG-Sepharose and was used for Western blot analysis to show the amounts of FLAG-Pcat, FLAG-Scat, prot.A-STcat, and protein A in the mixture (Fig. 6, top and middle panels). As shown in Fig. 6, bottom panel, no band was detected with anti-FLAG mAb when only FLAG-Pcat (lane 1), FLAG-Scat (lane 2), or prot.A-STcat (lane 3) was present in the mixture, as expected. However, when prot.A-STcat was mixed with FLAG-Pcat (Fig. 6, bottom panel, lane 4) or FLAG-Scat (lane 5), intense signals were clearly detected, suggesting that both FLAG-Pcat and FLAG-Scat bound with prot.A-STcat. Furthermore, no band was detected even if purified protein A instead of prot.A-STcat was used (lanes 6-8), indicating that the fused tags (i.e. FLAG and protein A) were irrelevant as to the binding. These results revealed that the catalytic domain was sufficient for formation of an enzyme complex and that the binding was direct because the pulldown assays were performed using the purified enzymes. Effect of the Interaction on the Glucuronyltransferase and Sulfotransfe
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