Cloning and Characterization of a Ninth Member of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family, ppGaNTase-T9
2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês
10.1074/jbc.m009638200
ISSN1083-351X
AutoresKelly G. Ten Hagen, Gurrinder S. Bedi, Daniel Tétaert, Paul D. Kingsley, Fred K. Hagen, Marlene Balys, Thomas M. Beres, Pierre Degand, Lawrence A. Tabak,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoWe have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situhybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development.AF241241 We have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situhybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development. AF241241 UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase polymerase chain reaction sublingual gland base pair(s) kilobase(s) trans-epoxysuccinyl-l-leucylamido-3-methyl butane matrix-assisted laser desorption ionization-time of flight high performance liquid chromatography polyacrylamide gel electrophoresis N-tris(hydroxymethyl)methylglycine phenylthiohydantoin Mucin type O-linked glycosylation is initiated by the action of a family of UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferases (ppGaNTase,1 EC 2.4.1.41), which catalyze the transfer of GalNAc from the nucleotide sugar UDP-GalNAc to the hydroxyl group of either serine or threonine. A number of functional roles for O-glycans have been suggested (reviewed in Ref. 1Van den Steen P. Rudd P.M. Dwek R.A. Opdenakker G. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 151-208Crossref PubMed Scopus (602) Google Scholar), including protection from proteolytic degradation (2Marinaro J.A. Neumann G.M. Russo V.C. Leeding K.S. Bach L.A. Eur. J. Biochem. 2000; 267: 5378-5386Crossref PubMed Scopus (44) Google Scholar), alteration of substrate structural conformation (3Woodward H.D. Ringler N.J. Selvakumar R. Simet I.M. Bhavanandan V.P. Davidson E.A. Biochemistry. 1987; 26: 5315-5322Crossref PubMed Scopus (41) Google Scholar), aiding in sperm-egg binding during fertilization in mice (4Kinloch R.A. Sakai Y. Wasserman P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 263-267Crossref PubMed Scopus (95) Google Scholar), and coordination of leukocyte rolling along endothelial cells upon inflammation and injury (5Dowbenko D. Andalibi A. Young P.E. Lusis A.J. Lasky L.A. J. Biol. Chem. 1993; 268: 4525-4529Abstract Full Text PDF PubMed Google Scholar). However, the exact biological functions of O-linked glycosylation remain largely unknown, as studies involving chemical/enzymatic cleavage of sugars and/or mutagenesis of acceptor residues on proteins can result in secondary effects unrelated to sugar removal or absence. Since carbohydrates can only be "mutated" indirectly by modifying the enzymatic activities of the glycosyltansferases responsible for their synthesis, our efforts have focused on the characterization of the enzyme family responsible for the initiation of O-glycan addition. Thus far, seven distinct mammalian isoforms from this gene family have been identified and functionally characterized: ppGaNTase-T1 (6Hagen F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 7Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar), -T2 (8White T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clauser H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), -T3 (9Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 10Zara J. Hagen F.K. Ten Hagen K.G. VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar), -T4 (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), -T5 (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), -T6 (13Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and -T7 (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 15Bennett E.P. Hassan H. Hollingsworth M.A. Clausen H. FEBS Lett. 1999; 460: 226-230Crossref PubMed Scopus (113) Google Scholar). An eighth putative isoform was ablated in mice without any obvious phenotypic effects (16Hennet T. Hagen F.K. Tabak L.A. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12070-12074Crossref PubMed Scopus (224) Google Scholar, 17Marth J.D. Glycobiology. 1996; 6: 701-705Crossref PubMed Scopus (62) Google Scholar); however, the enzymatic activity and the gene encoding this isoform remain uncharacterized. Whereas some isoforms display a broad range of expression in adult tissues and act on a robust set of substrates (ppGaNTase-T1, -T2, and -T3), others are more restricted in both expression and substrate preference (ppGaNTase-T4, -T5, and -T7). ppGaNTase-T7 (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) has the distinction of being the only transferase identified thus far that requires a GalNAc-containing glycopeptide as a substrate; glycosylation of the peptide substrate by ppGaNTase-T1 is required before ppGaNTase-T7 will further glycosylate additional residues. This result indicates that not all O-linked glycosylation occurs simultaneously and suggests that a hierarchy of action within this family may be responsible for the complex patterns of multisite substrate glycosylation seen in vivo. Here, we report the cloning of another member of this transferase family, termed ppGaNTase-T9. In common with ppGaNTase-T7, ppGaNTase-T9 demonstrated no transferase activity against a panel of unmodified peptide substrates in vitro. However, when the MUC5AC peptide substrate was first glycosylated by ppGaNTase-T1, the resultant glycopeptides were readily glycosylated further by ppGaNTase-T9 in a manner distinct from that of ppGaNTase-T7. ppGaNTase-T9 and ppGaNTase-T7 transcript expression patterns differed as well; ppGaNTase-T9 was expressed more widely across adult tissues and exhibited distinct expression patterns within developing mouse embryos. These results suggest that glycosylation of multisite substrates occurs through the specific and hierarchical action of multiple members of this enzyme family, whose expression is uniquely regulated both during development and in adult tissues. Previously, the conserved amino acid regions EIWGGEN and VWMDEYK were used to design sense and antisense PCR primers to amplify products from rat sublingual gland (rat SLG) cDNA. These products were cloned, sequenced and used to screen a rat SLG cDNA library as described (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). A probe previously used to clone the rat ppGaNTase-T5 cDNA (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) resulted in the detection of additional isoforms when screening an oligo(dT)-primed Uni-Zap XR rat SLG cDNA library according to standard procedures (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A novel isoform, designated ppGaNTase-T9, was identified by cross-hybridization with the probe derived from positions 2076–2240 of ppGaNTase-T5 (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). One clone containing a truncated 3′ end was initially isolated (rTA-0). An oligonucleotide (d(AGACGTTGTGGCCCAGAAAAAACTCCGAGGCTCC)) based on the 3′-most sequence of this partial cDNA clone was end-labeled and used to screen the cDNA library a second time. A cDNA clone containing a complete open reading frame was isolated (rTA-3). The coding region within this clone was completely sequenced and given the designation ppGaNTase-T9. The N-terminal transmembrane domain was determined by a Kyte-Doolittle hydrophobicity plot. Amino acid sequences were aligned, one pair at a time, using the pairwise ClustalW (1.4) algorithm in MacVector (Oxford Molecular Group). The following alignment modes and parameters were used: slow alignment, open gap penalty = 10, extended gap penalty = 0.1, similarity matrix = blosum, delay divergence = 40%, and no hydrophilic gap penalty. The percentage of amino acid sequence similarity displayed in Tables I and II represents the sum of the percent identities and similarities. Sequences comprising the conserved domains used in TableI begin with the conserved region FNXXXSD in the putative catalytic domain (amino acid position 84 in ppGaNTase-T1 (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), 100 in ppGaNTase-T2 (8White T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clauser H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), 150 in ppGaNTase-T3 (10Zara J. Hagen F.K. Ten Hagen K.G. VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar), 102 in ppGaNTase-T4 (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), 454 in ppGaNTase-T5 (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), 142 in ppGaNTase-T6 (13Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), 175 in ppGaNTase-T7 (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and 113 in ppGaNTase-T9) and end with a conserved proline (amino acid position 425 in ppGaNTase-T1, 440 in ppGaNTase-T2, 500 in ppGaNTase-T3, 438 in ppGaNTase-T4, 796 in ppGaNTase-T5, 492 in ppGaNTase-T6, 526 in ppGaNTase-T7, and 451 in ppGaNTase-T9). The segment of conserved sequences is ∼340 amino acids in length in the various isoforms and corresponds to the putative catalytic domain based on structural modeling and mutagenesis studies (19Hagen F.K. Hazes B. Raffo R. deSa D. Tabak L.A. J. Biol. Chem. 1999; 274: 6797-6803Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Sequences aligned in Table II consisted of the C-terminal ricin-like lectin motif (19Hagen F.K. Hazes B. Raffo R. deSa D. Tabak L.A. J. Biol. Chem. 1999; 274: 6797-6803Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) (amino acids 430–558 in ppGaNTase-T1, 444–569 in ppGaNTase-T2, 505–632 in ppGaNTase-T3, 444–577 in ppGaNTase-T4, 801–929 in ppGaNTase-T5, 498–622 in ppGaNTase-T6, 533–656 in ppGaNTase-T7, and 459–597 in ppGaNTase-T9).Table IAmino acid similarity between ppGaNTase isoforms within a 340-amino acid (aa) conserved domainrT1hT2mT3mT4rT5hT6rT7rT9rT1NA70717068696168hT270NA666865675964mT37166NA6964855761mT4706869NA65685966rT568656465NA625766hT66967856862NA5858rT7615957595758NA63rT968646166665863NAPercentage of amino acid similarity is shown for an ∼340-aa conserved domain, using pairwise ClustalW alignments, as described under "Experimental Procedures." rT1, rat ppGaNTase-T1; hT2, human ppGaNTase-T2; mT3, mouse ppGaNTase-T3; mT4, mouse ppGaNTase-T4; rT5, rat ppGaNTase-T5; hT6, human ppGaNTase-T6; rT7, rat ppGaNTase-T7 (previously called rat ppGaNTase-T6); rT9, rat ppGaNTase-T9; NA, not applicable. Open table in a new tab Table IIAmino acid similarity between ppGaNTase isoforms within the ricin-like lecin motifrT1hT2mT3mT4rT5hT6rT7rT9rT1NA44444032484645hT244NA403736394033mT34440NA5437683451mT4403754NA35473339rT532363735NA372837hT64839684737NA3439rT7464034332834NA40rT945335139373940NAPercentage of amino acid similarity is shown using pairwise ClustalW alignments, as described under "Experimental Procedures." rT1, rat ppGaNTase-T1; hT2, human ppGaNTase-T2; mT3, mouse ppGaNTase-T3; mT4, mouse ppGaNTase-T4; rT5, rat ppGaNTase-T5; hT6, human ppGaNTase-T6; rT7, rat ppGaNTase-T7 (previously called rat ppGaNTase-T6); rT9, rat ppGaNTase-T9; NA, not applicable. Open table in a new tab Percentage of amino acid similarity is shown for an ∼340-aa conserved domain, using pairwise ClustalW alignments, as described under "Experimental Procedures." rT1, rat ppGaNTase-T1; hT2, human ppGaNTase-T2; mT3, mouse ppGaNTase-T3; mT4, mouse ppGaNTase-T4; rT5, rat ppGaNTase-T5; hT6, human ppGaNTase-T6; rT7, rat ppGaNTase-T7 (previously called rat ppGaNTase-T6); rT9, rat ppGaNTase-T9; NA, not applicable. Percentage of amino acid similarity is shown using pairwise ClustalW alignments, as described under "Experimental Procedures." rT1, rat ppGaNTase-T1; hT2, human ppGaNTase-T2; mT3, mouse ppGaNTase-T3; mT4, mouse ppGaNTase-T4; rT5, rat ppGaNTase-T5; hT6, human ppGaNTase-T6; rT7, rat ppGaNTase-T7 (previously called rat ppGaNTase-T6); rT9, rat ppGaNTase-T9; NA, not applicable. Total RNA from Wistar rat tissues was extracted according to the single-step isolation method described by Ausubel et al. (20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley % Sons, Inc., New York1995: 4.2.4-4.2.8Google Scholar). Following electrophoresis in a 1% formaldehyde-agarose gel, rat total RNA samples were transferred to Hybond-N membranes (Amersham Pharmacia Biotech) according to Sambrooket al. (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A 325-bp segment of the ppGaNTase-T9 cDNA region (from the vector pBSmTA-423, containing a 325-bp ppGaNTase-T9 insert in the HindIII site of pBluescriptKS+) from nucleotides 1334–1756 of the amino acid coding region was labeled using the Random Primers DNA labeling system (Life Technologies, Inc.) according to manufacturer's instructions and used as a probe for ppGaNTase-T9 transcripts. ppGaNTase-T7 and -T1 were detected as described previously (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Antisense 18 S ribosomal subunit oligonucleotide d(TATTGGAGCTGGAATTACCGCGGCTGCTGG) was end-labeled as described (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and used to normalize sample loading by hybridizing with 5 m excess of probe. All hybridizations were performed in 5× SSPE, 50% formamide at 42 °C with two final washes in 2× SSC, 0.1% SDS at 65 °C for 20 min. In situ hybridization studies were performed using a modification of procedures described by Wilkinson and Green (21Wilkinson D.G. Green J. Copp A.J. Cockroft D.L. Postimplantation Mammalian Embryos: A Practical Approach.in: IRL Press, New York1990: 155-171Google Scholar). Mouse embryos were fixed overnight in freshly prepared ice-cold 4% paraformaldehyde in phosphate-buffered saline. The embryos were dehydrated through ethanol into xylene and embedded in paraffin using a Tissue-Tek V.I.P. automatic processor (Miles). Sections (5 μm) were adhered to commercially modified glass slides (Super Frost Plus, VWR), dewaxed in xylene, rehydrated through graded ethanols, and treated with proteinase K (to enhance probe accessibility) and with acetic anhydride (to reduce nonspecific background). Single-stranded RNA probes were prepared by standard techniques with specific activities of 5 × 109dpm/μg. ppGaNTase-T9 was detected using the plasmid pBSrT9-IS as a template for RNA production, ppGaNTase-T7-specific RNA probes were prepared using the plasmid pBSrT7-IS, and ppGaNTase-T1 transcripts were detected using the plasmid pBSmT1-IS. pBSrT9-IS contains nucleotides 199–381 of the rat ppGaNTase-T9 amino acid coding region generated by PCR amplification using the primers mTAIS+ (d(ATAGGTACCAAGCTTGCTGAACAAAGGCTGAAGGA) and mTAIS− (d(ATAGAGCTCGAGAGAGCGATTCAGGGAGATT). pBSrT7-IS contains a segment of the rat ppGaNTase-T7 (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) amino acid coding region from nucleotide position 1759 to 1964 generated by PCR amplification using the primers mT5IS+ (d(ATAGGTACCAAGCTTGACCAAGGGACCCGACGGATCC) and mT5IS− (d(ATAGAGCTCGAGGATGTTATTCATCTCCCACTTCTGAT). pBSmT1-IS contains nucleotides 1376–1676 of the mouse ppGaNTase-T1 amino acid coding region generated by PCR amplification using the primers mT1insitu+ (d(ATAGGTACCAAGCTTGTCATGGTATGGGAGGTAATCAGG)) and mT1in situ− (d(ATAGAGCTCGAGAATATTTCTGGAAGGGTGACAT)). All of the above mentioned PCR products were cloned into the KpnI andSacI sites of pBluescript KS+. All vectors were linearized at the introduced HindIII site and transcribed with T7 RNA polymerase to produce labeled antisense RNA. Sections were hybridized at Tm −15 °C, washed at high stringency (Tm −7 °C) and treated with RNase A to further diminish nonspecific adherence of probe. Autoradiography with NBT-2 emulsion (Eastman Kodak Co.) was performed for 25 days. Slides were developed with D19 (Eastman Kodak), and the tissue counterstained with hematoxylin. Brightfield and darkfield images were captured with a Polaroid Digital Microscope camera and processed using Adobe Photoshop (Adobe Systems) with Image Processing Toolkit (Reindeer Games, Asheville, NC). cDNA clones containing the 1.8-kb coding region of ppGaNTase-T9 were isolated from the rat sublingual gland cDNA library described previously (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). An MluI site was introduced into cDNA clone rTA-0 by PCR amplification using the primers rTA-MluI-S (d(CCTACGCGTCTCCTGGGGGTTCCGG)) and rTA-PCR-AS (d(GGTCAAGCAAAGGGGGGAGCCAGTT)). This amplified product was digested with MluI and EagI and cloned into the vector pBS-IMKF3 to create the vector, pBS-rTAmut#7. Sequencing was performed to verify that no PCR-induced mutations had been sustained in the cloned product. A 650-bp MluI-EagI(blunt) fragment from pBS-rTAmut#7 was then cloned into theMluI-Bsp120(blunt) sites of pIMKF4 to generate the vector pF4-rTA-Mut-7. (pIMKF4 is identical to pIMKF3 (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) except that the multiple cloning site is expanded between the BglII andNotI sites using the annealed oligonucleotides, Bgl-Not-S (d(GATCTAGAGCTCACCGGTAAGC)) and Not-Bgl-AS (d(GGCCGCTTACCGGTGAGCTCTA)). A 1.2-kb BspEI-Bsu36I(blunt) fragment from the cDNA clone rTA-3 was then cloned into theBspEI-Ecl136II sites of pF4-rTA-Mut-7 to generate the mammalian expression vector, pF4-rT9. pF4-rT9 is an SV40-based expression vector, which generates a fusion protein containing the following, in order: an insulin secretion signal, a metal binding site, a heart muscle kinase site, a FLAG™ epitope tag, and the truncated rat ppGaNTase-T9 cDNA. COS7 cells were grown to 90% confluence in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) + 10% FCS at 37 °C and 5%CO2. One μg of pIMKF1 (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), pF1-mT1 (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), pF1-rT7 (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), or pF4-rT9 and 8 μl of LipofectAMINE (Life Technologies, Inc.) were used to transfect a 35-mm well of COS7 cells as described previously (11Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Recombinant enzymes were assayed and quantitated (data not shown) directly from the culture media of transfected cells as described (12Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The activity of ppGaNTase-T9 was initially measured against the panel of peptide substrates described previously (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar): EA2 (PTTDSTTPAPTTK) (22Albone E.F. Hagen F.K. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1994; 269: 16845-16852Abstract Full Text PDF PubMed Google Scholar), human immunodeficiency virus (RGPGRAFVTIGKIGNMR) (9Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), MUC2 (PTTTPISTTTMVTPTPTPTC) (23Allen A. Hutton D.A. Pearson J.P. Int. J. Biochem. Cell Biol. 1998; 30: 797-801Crossref PubMed Scopus (155) Google Scholar), MUC1b (PDTRPAPGSTAPPAC) (24Gendler S.J. Lancaster C.A. Taylor-Papadimitriou J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E.-N. Wilson D. J. Biol. Chem. 1990; 265: 15268-15293Abstract Full Text PDF Google Scholar), EPO-T (PPDAATAAPLR) (6Hagen F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar), rMUC-2 (SPTTSTPISSTPQPTS) (25Ohmori H. Dohrman A.F. Gallup M. Tsuda T. Kai H. Gum Jr., J.R. Kim Y.S. Basbaum C.B. J. Biol. Chem. 1994; 269: 17833-17840Abstract Full Text PDF PubMed Google Scholar), mG-MUC (QTSSPNTGKTSTISTT) (26Shekels L.L. Lyftogt C. Kieliszewski M. Filie J.D. Kozak C.A. Ho S.B. Biochem. J. 1995; 311: 775-785Crossref PubMed Scopus (74) Google Scholar), and MUC5AC (GTTPSPVPTTSTTSAP) (27Guyonnet-Dupérat V. Audié J.P. Debailleul V. Laine A. Buisine M.P. Galiegue-Zouitina S. Pigny P. Degand P. Aubert J.P. Porchet N. Biochem. J. 1995; 305: 211-219Crossref PubMed Scopus (188) Google Scholar). No enzymatic activity for ppGaNTase-T9 was detected in any of these initial assays. To generate glycopeptide substrates for analysis of ppGaNTase-T9 activity, glycosylated MUC5AC and EA2 were prepared by incubation with Pichia pastoris-derived recombinant ppGaNTase-T1 as described previously (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Briefly, Pichia-derived ppGaNTase-T1 (0.028 μg) was incubated for 72 h at 37 °C with 1 mg of peptide in a 500-μl total volume under the following conditions: 125 mmcacodylate buffer (pH 7.0) containing 0.2% (v/v) Triton X-100, 12.5 mm MnCl2, 1 mm aprotinin, 1 mm leupeptin, 1 mm E64, 1 mmphenylmethanesulfonyl fluoride, 1.25 mm AMP, 6 mm cold UDP-GalNAc. Additional enzyme (0.028 μg) and UDP-GalNAc (3 μmol) were added at the first 24-h interval. The di- and tri-glycosylated MUC5AC reaction products and the mono-glycosylated EA2 reaction product were passed through AG1-X8 resin and purified on a Waters 265 HPLC using a Vydac C-18 reverse phase column (0.46 × 25 cm) with a flow rate of 1 ml/min using a linear gradient of 5% acetonitrile, 0.1% trifluoroacetic acid to 20% acetonitrile, 0.1% trifluoroacetic acid in 20 min at 22 °C. Capillary electrophoresis was performed on a model 270-HT Capillary Electrophoreses System (Applied Biosystems, Foster City, CA) under conditions previously described (14Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). MALDI-TOF mass spectra were acquired in reflector mode (accelerating voltage: 20 kV, grid voltage: 70%) on a Voyager-DE STR biospectrometry work station (PerSeptive Biosystems, Inc.) equipped with delayed extraction technology. Mass spectra were externally calibrated. Samples (1–4 pmol) were mixed with equal volume of matrix solution (α-cyano-4-hydroxycinnamic acid, 10 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid) and deposited on sample plates. The purified products of t
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