Advance Publication The Journal of Veterinary Medical Science Accepted Date: 29 May 2012 J-STAGE Advance Published Date: 12 Jun 2012 1 Virology Note 2 3 Generation of human bronchial epithelial cell lines expressing inactive mutants of 4 GALNT3 5 6 7 Shoko Nakamura1,2, Masayuki Horie1,¶, Kan Fujino1, Yusuke Matsumoto1, Tomoyuki Honda1, and 8 Keizo Tomonaga1,* 9 10 11 1Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. 12 2Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate 13 School of Medicine, Suita, Osaka 565-0871, Japan. 14 15 Running Title: Inactive mutants of GALNT3 16 17 ¶Present address; Department of Virology, Institute for Medical Microbiology and Hygiene, University of 18 Freiburg, D-79104 Freiburg, Germany 19 20 *Corresponding author; Keizo Tomonaga; Department of Viral Oncology, Institute for Viral Research, 21 Kyoto University, Sakyo-ku Kyoto 606-8507, Japan. 22 Tel: +81-75-751-3997, Fax: +81-75-751-4000. E-mail: [email protected] 23 1 1 Abstract 2 3 As a tool to understand the role of mucins in the infection of respiratory viruses, we 4 established cell lines stably expressing inactive mutants of UDP-GalNAc:polypeptide 5 N-acetylgalactosaminlytransferase 3 (GALNT3), which initiates O-glycosylation of mucins. 6 We introduced single-amino-acid mutations into the regions essential for the enzyme activity 7 of GALNT3 using the expression plasmid of human GALNT3 and transfected the mutant 8 constructs into the human bronchial epithelial cell line, BEAS-2B. We showed that although 9 the mutants of GALNT3 exhibit an authentic localization at the Golgi apparatus, the 10 glycosylation pattern of the expressing cell lines is appeared to be different from that of the 11 cells expressing wild-type GALNT3. These results suggested that the established cell lines 12 express inactive forms of GALNT3 and might be useful to investigate the significance of 13 O-glycosylation of mucins in respiratory virus infections. 14 15 16 Key Words: GALNT3, Mucin, Respiratory viruses 2 1 2 Viral infections affecting the upper or lower respiratory tract, such as influenza virus, 3 parainfluenza virus, adenovirus and respiratory syncytial virus, commonly induce 4 characteristic alterations in the epithelial surfaces of the respiratory tract [2, 6, 13]. One of 5 such changes is the increased production of mucus [14]. The increased mucus in the 6 respiratory tract is believed to be involved in many characteristic clinical symptoms, as well 7 as the viral eliminations, at the early stage of the virus infections [8]. The mucus is largely 8 formed by mucins, which are complex O-linked glycosylated proteins. Recent studies 9 revealed that the expression of mucins increases in the epithelial cells both in vivo and in vitro 10 soon after some respiratory virus infections [9] and also that the upregulation of mucins may 11 play important roles for the host defense mechanism to virus infections [12]. However, the 12 detailed about how mucins are involved in the host defense mechanism have not yet been 13 elucidated. 14 The O-glycosylation of mucins is initiated by the transfer of GalNAc to serine and 15 threonine (Ser/Thr) residues on target polypeptides by a family of UDP-GalNAc:polypeptide 16 N-acetylgalactosaminlytransferase (GalNAc-T or GALNT). In human, the GALNT family 17 consists of twenty genes, which localize to different chromosomal loci. The genes have 18 different, but overlapping substrates and expression specificities [16]. Among them, GALNT3 19 is considered to mediate O-glycosylation of some mucins, including MUC1 and MUC2, as 20 well as the fibroblast growth factor 23 (FGF23), which is a bone-derived hormone that plays 21 an important role in the regulation of the metabolism of the phosphate and 1,25-dihydroxy 22 vitamin D [1, 7, 15]. Previous studies demonstrated that the expression of GALNT3 is seen in 23 several normal tissues, including the respiratory epithelium cells and bronchial glands cells 3 1 [3], showing suggestive roles of GALNT3 in respiratory virus infections. In this study, to 2 understand the roles of mucin-type O-glycosylation in the infection of respiratory viruses, we 3 tried to generate human bronchial epithelial cell lines expressing inactive mutants of 4 GALNT3. 5 To generate expression plasmid of human GALNT3, cDNA corresponding GLANT3 6 coding region was amplified from total RNA isolated from human alveolar basal epithelial 7 cells, A549, by PCR and cloned into the restriction enzyme sites (Kpn I and Not I) of the 8 pEF4 plasmid to yield pEF-hGALNT3. Detailed information about the primers and PCR 9 procedures used to generate the plasmid is available from the authors. Nucleotide sequence of 10 the recombinant plasmid was confirmed by DNA sequencing. To confirm the expression of 11 recombinant GALNT3, the plasmid was transfected into a human bronchial epithelial cell line, 12 BEAS-2B, and detected by anti-GALNT3 polyclonal antibody (SIGMA-Aldrich) (Fig. 2B, 13 see below). The inactive mutants of GALNT3 were constructed by introducing 14 single-amino-acid substitutions using pEF-hGALNT3. To predict the reactive residues within 15 GALNT3, we used information of earlier studies [4, 16, 17], in which the amino acid residues 16 responsible for the enzyme activity of murine and bovine GalNAc-T1 (GALNT1) were 17 determined. As the GALNT family is known to be highly homologues and the amino acid 18 residues essential for the enzyme activity seems to be significantly conserved among the 19 family [16], we introduced mutations in human GALNT3 in the sites corresponding to those 20 identified in murine and bovine GALNT1 (Fig. 1). In previous studies, some residues (mostly 21 histidines) were identified as essential for the enzyme activity of GALNT1 [4]. Among them, 22 mutants of aspartate and histidine at positions 209 and 211 of murine GALNT1, respectively, 4 1 consist of DxH motif, which was also described in many other glycosyltransferase, showed no 2 detectable enzyme activity [17]. In addition, a point mutation of histidine at position 344 was 3 shown to significantly decrease the activity of bovineGALNT1 [17]. Therefore, we introduced 4 the point mutations in the corresponding residues, D277, H279, and H415, of human 5 GALNT3 (Fig. 1). According to the previous studies, the aspartate and histidine residues were 6 changed to asparagine (D277N), aspartate (H279D), and alanine (H415A), respectively. The 7 mutant forms of the expression plasmid were generated using PCR amplification and 8 recloning or a PCR-based site-directed mutagenesis technique. Oligonucleotide primers and 9 PCR conditions to create the plasmids are available on request. Nucleotide sequences of the 10 recombinant constructs were also confirmed by DNA sequencing. 11 To generate cell clones stably expressing the GALNT3 mutants, we first tested the 12 susceptibility of BEAS-2B cells to the respiratory virus infection. The A/Puerto Rico/8/34 13 (PR8) strain of influenza A virus was inoculated into the parental BEAS-2B cell line at a 14 multiplicity of infection (MOI) of 3.0, and the infectivity was confirmed by indirect 15 immunofluorescence assay (IFA) using anti-nucleoprotein (NP) monoclonal antibody. As 16 shown in Fig. 2A, at 12 h postinfection, the parental BEAS-2B cells were shown to be 17 susceptible to influenza A virus. In addition, endogenous expression of GALNT3 was 18 undetectable in the parental BEAS-2B cells by both western blot analysis and IFA (Fig. 2B 19 and 2C), indicating that BEAS-2B cells are relevant for studying the role of GALNT3 in the 20 respiratory virus infection. 21 The mutant plasmids were transfected into BEAS-2B cells and, cell clones stably 22 expressing the wild-type and mutant GALNT3 were selected by 200 µg/ml Zeocin 5 1 (Invitrogen) in D-MEM (Dulbecco's Modified Eagle's Medium) (Nacalai tesque). We 2 obtained 4, 3, and 7 cell clones expressing D277N, H279D, and H415A mutants, respectively, 3 in which the expression level, as well as intracellular localization, of the mutant protein was 4 comparable to those of the wild-type GALNT3 (data not shown). Among them, we used a 5 representative clone in each mutant, and the expression of the mutant enzymes was verified 6 by western blotting and IFA using anti-GALNT3 polyclonal antibody (SIGMA-Aldrich). 7 Immunoblotting using 12% SDS-PAGE revealed that the mutants produce recombinant 8 proteins at the expected sizes (Fig. 2B). Previous studies revealed that the 9 glycosyltransferases commonly localize at the Golgi apparatus [11]. As shown in Fig. 2C, the 10 all mutants, D277N, H279D, and H415A, as well as the wild-type protein, exhibited a clear 11 distribution in the Golgi apparatus. These results indicated that the single-amino-acid 12 substitutions exert no effects on the expression and localization of GALNT3 in the transfected 13 cells. 14 We next investigated whether the GALNT3 mutants reduce its enzyme activity in the 15 cells. To this end, we performed lectin blot analysis using the lysates of BEAS-2B cell lines 16 stably expressing the wild-type and mutant GALNT3. Briefly, the cells were washed with 17 PBS and then lysed. Insoluble materials were removed by centrifugation, and equal amounts 18 of protein were separated using 12% SDS-PAGE and then transferred to nitrocellulose 19 membranes (Millipore). As a control of protein loading, we performed Coomassie Brilliant 20 Blue (CBB) staining using CBB Stain One (Nacalai tesque) (Fig. 3). For lectin blotting, 21 membranes were blocked with a Carbo-Free Blocking Solution (Vector Laboratories, Inc.) 22 and incubated with a different biotinylated lectin; 1.0 mg/ml PNA (Arachis hypogaea 6 1 agglutinin) (SIGMA-Aldrich) or 1.0 mg/ml ABA (Agaricus bisporus agglutinin) (Vector 2 Laboratries, Inc.) in Carbo-Free Blocking Solution for 1 h at room temperature.