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The Cytoplasmic, Transmembrane, and Stem Regions of Glycosyltransferases Specify Their in Vivo Functional Sublocalization and Stability in the Golgi

1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês

10.1074/jbc.274.51.36107

ISSN

1083-351X

Autores

Eckart Grabenhorst, Harald S. Conradt,

Tópico(s)

Lysosomal Storage Disorders Research

Resumo

We provide evidence for the presence of targeting signals in the cytoplasmic, transmembrane, and stem (CTS) regions of Golgi glycosyltransferases that mediate sorting of their intracellular catalytic activity into different functional subcompartmental areas of the Golgi. We have constructed chimeras of human α1,3-fucosyltransferase VI (FT6) by replacement of its CTS region with those of late and early acting Golgi glycosyltransferases and have stably coexpressed these constructs in BHK-21 cells together with the secretory reporter glycoprotein human β-trace protein. The sialyl Lewis X:Lewis X ratios detected in β-trace protein indicate that the CTS regions of the early acting GlcNAc-transferases I (GnT-I) and III (GnT-III) specify backward targeting of the FT6 catalytic domain, whereas the CTS region of the late acting human α1,3-fucosyltransferase VII (FT7) causes forward targeting of the FT6in vivo activity in the biosynthetic glycosylation pathway. The analysis of the in vivo functional activity of nine different CTS chimeras toward β-trace protein allowed for a mapping of the CTS donor glycosyltransferases within the Golgi/trans-Golgi network: GnT-I < (ST6Gal I, ST3Gal III) < GnT-III < ST8Sia IV < GalT-I < (FT3, FT6) < ST3Gal IV < FT7. The sensitivity or resistance of the donor glycosyltransferases toward intracellular proteolysis is transferred to the chimeric enzymes together with their CTS regions. Apparently, there are at least three different signals contained in the CTS regions of glycosyltransferases mediating: first, their Golgi retention; second, their targeting to specific in vivofunctional areas; and third, their susceptibility toward intracellular proteolysis as a tool for the regulation of the intracellular turnover. We provide evidence for the presence of targeting signals in the cytoplasmic, transmembrane, and stem (CTS) regions of Golgi glycosyltransferases that mediate sorting of their intracellular catalytic activity into different functional subcompartmental areas of the Golgi. We have constructed chimeras of human α1,3-fucosyltransferase VI (FT6) by replacement of its CTS region with those of late and early acting Golgi glycosyltransferases and have stably coexpressed these constructs in BHK-21 cells together with the secretory reporter glycoprotein human β-trace protein. The sialyl Lewis X:Lewis X ratios detected in β-trace protein indicate that the CTS regions of the early acting GlcNAc-transferases I (GnT-I) and III (GnT-III) specify backward targeting of the FT6 catalytic domain, whereas the CTS region of the late acting human α1,3-fucosyltransferase VII (FT7) causes forward targeting of the FT6in vivo activity in the biosynthetic glycosylation pathway. The analysis of the in vivo functional activity of nine different CTS chimeras toward β-trace protein allowed for a mapping of the CTS donor glycosyltransferases within the Golgi/trans-Golgi network: GnT-I < (ST6Gal I, ST3Gal III) < GnT-III < ST8Sia IV < GalT-I < (FT3, FT6) < ST3Gal IV < FT7. The sensitivity or resistance of the donor glycosyltransferases toward intracellular proteolysis is transferred to the chimeric enzymes together with their CTS regions. Apparently, there are at least three different signals contained in the CTS regions of glycosyltransferases mediating: first, their Golgi retention; second, their targeting to specific in vivofunctional areas; and third, their susceptibility toward intracellular proteolysis as a tool for the regulation of the intracellular turnover. N-acetylglucosaminyltransferase amino acid residues baby hamster kidney β-trace protein cytoplasmic, transmembrane, and stem fucosyltransferase β1,4-galactosyltransferase high pH anion exchange chromatography with pulsed amperometric detection Lewis X (Gal(β1→4)[Fuc(α1→3)]GlcNAc-R) polymerase chain reaction polysialyltransferase sialyl Lewis X (NeuAc (α2→3)Gal(β1→4)[Fuc(α1→3)] GlcNAc-R) trans-Golgi network The assembly of protein- or lipid-linked oligosaccharides is mediated by the reactions of a series of glycosidases and glycosyltransferases that localize in the subcompartments of the secretory pathway of mammalian cells (1Kobata A. Eur. J. Biochem. 1992; 209: 483-501Crossref PubMed Scopus (381) Google Scholar). According to the current consensus, the enzymes should be arranged in a sequential manner within the Golgi stacks. The control mechanisms that underlie the distribution of glycosyltransferases into different Golgi subcompartments are not understood. Some key enzymes like α-mannosidase II and GnT-I1 have been localized in the medial Golgi and trans-Golgi, whereas several terminal glycosyltransferases (GalT-I, ST6Gal I, ST3Gal III, FT5, FT6) have been localized in the trans-Golgi/TGN (2Nilsson T. Pypaert M. Hoe M.H. Slusarewicz P. Berger E.G. Warren G. J. Cell Biol. 1993; 120: 5-13Crossref PubMed Scopus (183) Google Scholar, 3Nilsson T. Hoe M.H. Slusarewicz P. Rabouille C. Watson R. Hunte F. Watzele G. Berger E.G. Warren G. EMBO J. 1994; 13: 562-574Crossref PubMed Scopus (226) Google Scholar, 4Rabouille C. Hui N. Hunte F. Kieckbusch R. Berger E.G. Warren G. Nilsson T. J. Cell Sci. 1995; 108: 1617-1627Crossref PubMed Google Scholar, 5Borsig L. Kleene R. Dinter A. Berger E.G. Eur. J. Cell Biol. 1996; 70: 42-53PubMed Google Scholar, 6Borsig L. Katopodis A.G. Bowen B.R. Berger E.G. Glycobiology. 1998; 8: 259-268Crossref PubMed Scopus (33) Google Scholar, 7Burger P.C. Lötscher M. Streiff M. Kleene R. Kaissling B. Berger E.G. Glycobiology. 1998; 8: 245-257Crossref PubMed Scopus (22) Google Scholar). Evidence is provided in the literature that most glycosyltransferases show an overlapping distribution into more than only one morphological defined subcompartment (e.g. GnT-I has been localized to the medial and trans-Golgi, and GalT-I and ST6Gal I have been localized to the trans-Golgi as well as the TGN (2Nilsson T. Pypaert M. Hoe M.H. Slusarewicz P. Berger E.G. Warren G. J. Cell Biol. 1993; 120: 5-13Crossref PubMed Scopus (183) Google Scholar, 4Rabouille C. Hui N. Hunte F. Kieckbusch R. Berger E.G. Warren G. Nilsson T. J. Cell Sci. 1995; 108: 1617-1627Crossref PubMed Google Scholar)). The transmembrane region as well as the flanking domains of type II Golgi resident glycosyltransferases have been identified to maintain Golgi retention (8Colley K. Glycobiology. 1997; 7: 1-13Crossref PubMed Scopus (285) Google Scholar, 9Munro S. Trends Cell Biol. 1998; 8: 11-15Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 10Gleeson P.A. Histochem. Cell Biol. 1998; 109: 517-532Crossref PubMed Scopus (68) Google Scholar). The bilayer thickness model for Golgi retention of glycosyltransferases (11Bretscher M.S. Munro S. Science. 1993; 261: 1280-1281Crossref PubMed Scopus (735) Google Scholar, 12Munro S. EMBO J. 1995; 14: 4695-4704Crossref PubMed Scopus (342) Google Scholar) postulates that the length of the transmembrane region of transferases mediates Golgi retention. A second hypothesis (3Nilsson T. Hoe M.H. Slusarewicz P. Rabouille C. Watson R. Hunte F. Watzele G. Berger E.G. Warren G. EMBO J. 1994; 13: 562-574Crossref PubMed Scopus (226) Google Scholar, 13Nilsson T. Slusarewicz P. Warren G. FEBS Lett. 1993; 330: 1-4Crossref PubMed Scopus (192) Google Scholar, 14Weisz O.A. Swift A.M. Machamer C.E. J. Cell Biol. 1993; 122: 1185-1196Crossref PubMed Scopus (125) Google Scholar) proposes a disulfide-linked homo-/hetero-oligomerization of the enzymes to function as a Golgi retention signal by preventing the large complexes from being delivered to secretory vesicles and ongoing transport to the plasma membrane. Neither model provides sufficient information about the mechanisms that control the in vivo functional organization of the different members of the glycosyltransferase families and how their sequential arrangement within different subcompartments might be accomplished. Immunochemical localization techniques lack the sensitivity to resolve in detail the distribution of, for example, the many late acting Golgi glycosyltransferases in the functional network of thetrans-Golgi/TGN (10Gleeson P.A. Histochem. Cell Biol. 1998; 109: 517-532Crossref PubMed Scopus (68) Google Scholar). A further complication with immunodetection of enzymes in defined subcompartments results from the migration of the newly synthesized membrane-bound glycosyltransferases from the endoplasmic reticulum through the compartments of the secretory pathway until they arrive at their final destination in individual functional Golgi stacks. It has been shown that glycosyltransferases themselves undergo complex-typeN-glycosylation including terminal sialylation (15Bosshart H. Berger E.G. Eur. J. Biochem. 1992; 208: 341-349Crossref PubMed Scopus (28) Google Scholar, 16Costa J. Grabenhorst E. Nimtz M. Conradt H.S. J. Biol. Chem. 1997; 272: 11613-11621Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In addition, there are several reports describing different levels of intracellular proteolytically cleaved forms of certain glycosyltransferases that might change under different physiological conditions in different cells (18Strous G.J.A.M. Berger E.G. J. Biol. Chem. 1982; 257: 7623-7628Abstract Full Text PDF PubMed Google Scholar, 19Weinstein J. Lee E.U. McEntee K. Lai P.-H. Paulson J.C. J. Biol. Chem. 1987; 262: 17735-17743Abstract Full Text PDF PubMed Google Scholar, 20Masri K.A. Appert H.E. Fukuda M.N. Biochem. Biophys. Res. Commun. 1988; 157: 657-663Crossref PubMed Scopus (130) Google Scholar, 21Paulson J.C. Colley K. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar, 22Johnson P.H. Watkins W.M. Glycoconj. J. 1992; 9: 241-249Crossref PubMed Scopus (62) Google Scholar, 23Johnson P.H. Donald A.S.R. Watkins W.M. Glycoconj. J. 1993; 10: 152-164Crossref PubMed Scopus (22) Google Scholar). A major problem of immunohistochemical methods is that they do not provide any information concerning the in vivo functional activity of the detected enzyme species. Assuming a defined in vivo acceptor substrate specificity for glycosyltransferases, their sequential action and possibly also their sequential distribution along the secretory pathway should be reflected in the final oligosaccharide structure of the biosynthetic products of a cell (e.g. in a secretory glycoprotein). Therefore, the structural characterization of a reporter glycoprotein expressed at a constant level from cells transfected with new glycosyltransferase genes should allow to identify the position of the newly introduced enzyme within the biosynthetic reaction sequence of the host cell. We have recently analyzed in detail the in vivo biosynthetic activity of the human α1,3/4-fucosyltransferases III–VII (FT3–FT7) in BHK-21 cells (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) by stable coexpression of each individual enzyme together with human β-TP, which is decorated exclusively with diantennary complex-type N-glycans (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Hoffmann A. Nimtz M. Wurster U. Conradt H.S. J. Neurochem. 1994; 63: 2185-2196Crossref PubMed Scopus (75) Google Scholar, 25Grabenhorst E. Hoffmann A. Nimtz M. Zettlmeissl G. Conradt H.S. Eur. J. Biochem. 1995; 232: 718-725Crossref PubMed Scopus (61) Google Scholar, 26Hoffmann A. Nimtz M. Conradt H.S. Glycobiology. 1997; 7: 499-506Crossref PubMed Scopus (117) Google Scholar). We found that each human α1,3/4-fucosyltransferase is characterized in vivo by the synthesis of an individual ratio of sLex:Lex, with FT7 forming exclusively sLex and FT4 preponderantly (90%) Lex, whereas FT6 expression results in a 1.1:1 mixture of sLex and Lex motifs in the oligosaccharides of the coexpressed reporter glycoprotein β-TP. The in vitro specificity data of the enzymes clearly support the exclusive sLex-forming specificity of the FT7 catalytic domain and the Lex-forming specificity of the FT4 catalytic domain. Consequently, in order to get access to its Gal(β1→4)GlcNAc-R substrate, FT4 should be localized in a cellular subcompartment before α2,3-sialylation occurs, since, according to all data available, the human α2,3-sialyltransferase ST3Gal III does not transfer NeuAc to Lex (compare Fig. 1). Likewise, FT7 action strictly depends on the proper supply with α2,3-sialylated acceptors. FT7 should either colocalize with ST3Gal III in the same functional area, or, more preferably, should reside in a later subcompartment in order to get access to high acceptor substrate concentrations. The human FT6 catalytic domain recognizes both, in vivo and in vitro, the sialylated or unsialylated acceptor motifs with a high efficiency (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 27Nimtz M. Grabenhorst E. Gambert U. Costa J. Wray V. Morr M. Thiem J. Conradt H.S. Glycoconj. J. 1998; 15: 873-883Crossref PubMed Scopus (15) Google Scholar); therefore, this enzyme should either colocalize with ST3Gal III, resulting in competition for the common Gal(β1→4)GlcNAc-R substrate, or should have a broader subcompartmental distribution. We have also shown that a variant of FT6 constructed by replacement of its CTS region with the signal peptide sequence of human interleukin-2 is efficiently secreted from cells but does not show in vivo functional activity when expressed at a total activity level comparable with wt-FT6 cells (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This result as well as reports by other groups suggesting the transmembrane and flanking regions of several glycosyltransferases (ST6Gal I, GalT-I, GnT-I, and α1,2-fucosyltransferase) as playing an important role in their Golgi retention (12Munro S. EMBO J. 1995; 14: 4695-4704Crossref PubMed Scopus (342) Google Scholar, 28Nilsson T. Lucocq J.M. Mackay D. Warren G. EMBO J. 1991; 10: 3567-3575Crossref PubMed Scopus (161) Google Scholar, 29Munro S. EMBO J. 1991; 10: 3577-3588Crossref PubMed Scopus (218) Google Scholar, 30Aoki D. Lee N. Yamaguchi N. Dubois C. Fukuda M.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4319-4323Crossref PubMed Scopus (124) Google Scholar, 31Burke J. Pettitt J.M. Schachter H. Sarkar M. Gleeson P.A. J. Biol. Chem. 1992; 267: 24433-24440Abstract Full Text PDF PubMed Google Scholar, 32Colley K. Lee E.U. Paulson J.C. J. Biol. Chem. 1992; 267: 7784-7793Abstract Full Text PDF PubMed Google Scholar, 33Russo R.N Shaper N.L. Taatjes D.J. Shaper J.H. J. Biol. Chem. 1992; 267: 9241-9247Abstract Full Text PDF PubMed Google Scholar, 34Tang B.L. Wong S.H. Low S.H. Hong W. J. Biol. Chem. 1992; 267: 10122-10126Abstract Full Text PDF PubMed Google Scholar, 35Teasdale R.D. D'Agostaro G. Gleeson P.A. J. Biol. Chem. 1992; 267: 4084-4096Abstract Full Text PDF PubMed Google Scholar, 36Wong S.H. Low S.H. Hong W. J. Cell Biol. 1992; 117: 245-258Crossref PubMed Scopus (88) Google Scholar, 37Masibay A.S. Balaji P.V. Boeggeman E.E. Qasba P.K. J. Biol. Chem. 1993; 268: 9908-9916Abstract Full Text PDF PubMed Google Scholar, 38Nilsson T. Rabouille C. Hui N. Watson R. Warren G. J. Cell Sci. 1996; 109: 1975-1989Crossref PubMed Google Scholar, 39Osman N. McKenzie I.F.C. Mouhtouris E. Sandrin M.S. J. Biol. Chem. 1996; 271: 33105-33109Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 40Skrincosky D. Kain R. El-Battari A. Exner M. Kerjaschki D. Fukuda M. J. Biol. Chem. 1997; 272: 22695-22702Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) prompted us to investigate the properties of glycosyltransferase CTS regions in thein vivo functional targeting of the human FT6 catalytic domain to different biosynthetically active Golgi subcompartments. If localizing to early Golgi compartments, the FT6 catalytic domain would be expected to encounter low levels of sialylated N-glycans and preferentially would transfer Fuc to Gal(β1→4)GlcNAc-R, resulting in increased Lex synthesis detectable in the secreted product. Likewise, its targeting to a later compartment would be expected to lead to preferential formation of sLexmotifs by the enzyme from the availability of already α2,3-sialylated precursor substrates. In the present report, we have fused CTS regions of different donor glycosyltransferases to the N terminus of the FT6 catalytic domain and have stably expressed the constructs in BHK-21 cells together with human β-TP, resulting in expression levels comparable with those in wt-FT6 cells. The characterization of the oligosaccharides attached to the secreted reporter glycoprotein should also allow the identification of any possible interference with the integrity of the cellular glycosylation pathway that could have resulted from the genetic engineering procedure. Peptide-N 4-(N-acetyl-β-d-glucosaminyl)asparagine amidase F (from Flavobacterium meningosepticum) from recombinant Escherichia coli was bought from Roche Molecular Biochemicals, fetal bovine serum was purchased from ITM (Munich, Germany), GDP-[14C]Fuc (285 Ci/mol) was from Amersham Pharmacia Biotech, and GDP-Fuc and G418-sulfate were bought from Sigma. Dulbecco's modified Eagle's medium (Life Technologies, Inc.) was supplemented prior to use with 10 mm Hepes, pH 7.2, 45 mm NaHCO3, 2 mm glutamine, 0.061 g/liter ampicillin, 0.1 g/liter streptomycin sulfate, and 0, 2, or 10% fetal bovine serum. The Gal(β1→4)GlcNAc-O-(CH2)8-COOCH3acceptor was a gift from Dr. O. Hindsgaul (University of Alberta, Canada). The cDNAs encoding the human α1,3/4-fucosyltransferases FT3, FT6, and FT7 as well as the human ST6Gal I were those from previous publications (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,25Grabenhorst E. Hoffmann A. Nimtz M. Zettlmeissl G. Conradt H.S. Eur. J. Biochem. 1995; 232: 718-725Crossref PubMed Scopus (61) Google Scholar). Plasmids containing the cDNAs of GnT-I (41Kumar R. Yang J. Larsen R.D. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9948-9952Crossref PubMed Scopus (142) Google Scholar) and GnT-III (42Ihara Y. Nishikawa A. Tohma T. Soejima H. Niikawa N. Taniguchi N. J. Biochem. (Tokyo). 1993; 113: 692-698Crossref PubMed Scopus (103) Google Scholar) were kindly provided by Professor Geyer (University of Giessen, Giessen, Germany) and Professor Taniguchi (Osaka University Medical School, Osaka, Japan), respectively. A human GalT-I cDNA (43Watzele G. Berger E.G. Nucleic Acids Res. 1990; 18: 7174Crossref PubMed Scopus (29) Google Scholar) was cloned from reverse-transcribed HL-60 mRNA as described (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) by using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) and the primers (upper/lower) 5′-AAG ATG AGG CTT CGG GAG CCG CTC/5′-CTA GCT CGG TGT CCC GAT GTC CAC (35 cycles of denaturation, 15 s, 94 °C, annealing, 20 s, 45 °C, and extension, 120 s, 72 °C). The cDNAs of three BHK-21 cell sialyltransferases were cloned by PCR using primers homologous to human ST3Gal III (44Kitagawa H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (123) Google Scholar), human ST3Gal IV (45Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar), and Chinese hamster ovary cell ST8Sia IV (46Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar). BHK-21 cell mRNA isolation and cDNA synthesis was essentially as described above for HL-60 cells. The PCR was performed as above using the following primer pairs (upper/lower): 5′-AG ATG GGA CTC TTG GTA TTT GT/5′-TCA GAT GCC ACT GCT TAG ATC AGT GAT (ST3Gal III), 5′-AAC ATG GTC AGC AAG TCC CGC T/5′-GGT CAG AAG GAC GTG AGG TTC (ST3Gal IV), and 5′-TTA TAC CAA GAG AAG GTG CC/5′-GAT CCT TCA ATA TGT GCT TTA TT (ST8Sia IV) and 35 cycles with 15 s at 94 °C, 20 s at 45 °C/52 °C/50 °C, respectively, and 120 s at 72 °C. The homology of the BHK-21 cDNA sequences to the human sequences was found to be 91% for ST3Gal III, 87% for ST3Gal IV, and 91% for ST8Sia IV. The PCR products were cloned into the vector pCR3.1 (Invitrogen) according to the manufacturer's instructions and were subsequently used as templates for the generation of CTS mutants of human FT6 as detailed below. Chimeric constructs of FT6 were generated by fusing the CTS regions of nine different mammalian Golgi glycosyltransferases (51–126 aa) to the N terminus of the FT6 catalytic domain (compare Fig. 2and Table I for details). For each variant, a 5′-megaprimer encoding the donor glycosyltransferase CTS region and containing 13–16 bases homologous to the 3′-end of the FT6 coding sequence was prepared from the mammalian glycosyltransferase cDNAs cited above by using the following primer pairs (upper/lower): 5′-AGG ATG CTG AAG AAG CAG TCT GCA G/5′-CAG GGG GAT GGA GTG GGC CGG CGC GGG GGT CAC AGG (GnT-I), 5′-AAG ATG AGA CGC TAC AAG CTC T/5′-CCA CAG CAG GAT CAG GGG CGA GTG GGA GTA (GnT-III), 5′-AAG ATG AGG CTT CGG GAG CCG CTC/5′-CAG GGG GAT GGA GTG GGC GGG CAG CGA CAG TGC (GalT-I), 5′-AG ATG GGA CTC TTG GTA TTT GT/5′-GGG CGG GGG TCC CTG TGC TGT CCA GCT TCA GGA GAA AAC C (ST3Gal III); 5′-AAC ATG GTC AGC AAG TCC CGC T/5′-CAG CAG GAT CAG GGG GAT GGC TAA CAC CCG GAG (ST3Gal IV), 5′-ATT ATG ATT CAC ACC AAC CTG AA/5′-CAG GGG GAT GGA GGC CTC TGG TT (ST6Gal I), 5′-TTA TAC CAA GAG AAG GTG CC/5′-CAG CAG GAT CAG GGG GAT GGT TGA GCC AGC CTT (ST8Sia IV), 5′-ACT CTG ACC CAT GGA TCC CCT/5′-ACA CCT TGC GGT CGG CAG TGA T (FT3), and 5′-TCT CTT GGC TGA CTG ATC CTG GG/5′-AAA AGG CCA CGT CCA GAC AAG GAT GGT GAT (FT7).The megaprimers were used in a second PCR together with the 3′-primer 5′-TCA CTT GCC GCT GTT TGC GAC GTA ATT TTT GTC GAA TCC AGC TCC GGT GAA CCA AGC CGC for the generation of full-length chimeric FT6 cDNA essentially as described (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The resulting cDNAs were cloned into the vector pCR3.1 as described above, and the correct sequences were verified by DNA sequencing.Table IPolypeptide domains used for the construction of chimeric FT6 variantsCTS region donorsSourceAmino acid rangeFT6 chimerasDonorAcceptor (FT6)GnT-IHuman1–10257–359gnt1-FT6GnT-IIIHuman1–7662–359gnt3-FT6GalT-IHuman1–12657–359galt-FT6ST3Gal IIIBHK-211–6552–359st3-FT6ST3Gal IVBHK-211–10359–359st4-FT6ST6Gal IHuman1–9060–359st6-FT6ST8Sia IVBHK-211–6259–359pst-FT6FucT-IIIHuman1–9292–359ft3-FT6FucT-VIIHuman1–5167–359ft7-FT6 Open table in a new tab BHK-21 cells stably expressing human β-TP and CTS variants of FT6 were generated with the calcium phosphate precipitation method and selected with G418-sulfate as described (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In most cases, cell lines showing a similar β-TP expression levels in Western blots and similar FT6 in vitro activity in cellular extracts compared with the well characterized wt-FT6 cell line (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) were used for the further characterization of the reporter glycoprotein. For this, the cells were cultivated for 2–3 weeks in Dulbecco's modified Eagle's medium containing alternatingly 0 or 2% fetal bovine serum, respectively, and about 0.5 mg of recombinant human β-TP was purified from 500–1000 ml of culture supernatants by immunoaffinity chromatography as described (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (47Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207192) Google Scholar) using 12.5 and 3% acrylamide in the resolution and stacking gels, respectively. For Western blot analysis, proteins were transferred to Immobilon NC membranes (Millipore Corp.) by using a TransBlot™ SD transfer cell (Bio-Rad). The membrane was blocked for 1 h with Tris-buffered saline containing 10% horse serum and 3% bovine serum albumin and was incubated overnight with rabbit anti-β-TP antiserum in blocking buffer at a 1:1000 dilution. The second antibody, goat anti-rabbit immunoglobulin coupled to horseradish peroxidase, was used at a 1:500 dilution. The blots were developed with Tris-buffered saline containing 0.5 g/liter 4-chloro-1-naphtol solubilized in methanol and 0.2% H2O2. Immunodetection of secreted FT6 variants was performed essentially as described for β-TP using a rabbit antiserum raised against the human FT6 peptide R125RQGQRWIWFSMESPSHCWQLK following immunoaffinity purification on the peptide antigen coupled to Affi-Gel 15 (Bio-Rad). Cell culture supernatants of stable cell lines expressing CTS variants of FT6 were analyzed for secreted forms of the enzymes by an in vitro α1,3-fucosyltransferase assay as detailed previously for wt-FT6 cells (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The secreted chimeric enzymes were partially purified by affinity chromatography on a GDP-Fractogel column and characterized by Western blotting. The N terminus of the secreted form of wt-FT6 was determined by gas phase sequencing of the protein following transfer onto an Immobilon-P (Millipore) membrane. Purified β-TP was reduced, carboxamidomethylated, and digested with trypsin, and the free reducingN-glycans were obtained from reverse phase high pressure liquid chromatography-purified glycopeptides by peptide-N 4-(N-acetyl-β-d-glucosaminyl)asparagine amidase F digestion as detailed earlier (24Hoffmann A. Nimtz M. Wurster U. Conradt H.S. J. Neurochem. 1994; 63: 2185-2196Crossref PubMed Scopus (75) Google Scholar). The released oligosaccharide material was subsequently analyzed by HPAE-PAD using conditions identical to those that were applied for the characterization of β-TP glycans from wt-FT6 cells (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Identification and quantitation of the β-TP N-glycans was achieved by comparison with the elution profile of β-TP oligosaccharides from wt-FT6 cells, since this material contained all possible α1,3-fucosylated diantennary structures as was revealed in our previous investigation by using methylation analysis and mass spectrometry of individual oligosaccharide fractions (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Similarly to wt-FT6 cells, more than 90% of the oligosaccharides from CTS variant cells were of the diantennaryN-acetyllactosamine type, differing only in their content and the distribution of α2,3-linked NeuAc and α1,3-linked Fuc residues. The design of human FT6 CTS-variants is based on our previous finding that the catalytic domain of FT6 contains the in vivo specificity to transfer Fuc in α1,3-linkage to GlcNAc in both sialylated and unsialylated type II N-acetyllactosamine oligosaccharide chains of coexpressed human β-TP with a similar efficiency (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The transmembrane domain as well as flanking regions of FT6 are required for its in vivo functional activity, since an engineered truncated form of the enzyme lacking the first 51 aa, when fused to the interleukin-2 signal peptide, is efficiently secreted from BHK-21 cells and does not fucosylate any oligosaccharide of coexpressed β-TP when expressed at an intracellular enzyme activity level comparable with wt-FT6 cells (17Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). As shown in Fig. 2 and Table I, CTS-regions from donor glycosyltransferases of different length were fused to the N terminus of the human FT6 catalytic domain. The precise location of the catalytic domains of many glycosyltransferases is unknown (10Gleeson P.A. Histochem. Cell Biol. 1998; 109: 517-532Crossref PubMed Scopus (68) Google Scholar). In analogy to the data reported for truncated forms of the highly homologous human FT3 (48Xu Z. Vo L. Macher B.A. J. Biol. Chem. 1996; 271: 8818-8823Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), the catalytic domain of human FT6 was defined by the shortest C-terminal part that showed no loss of enzyme activity in vitro and contained the minimum sequence starting from Pro-62 (… PLILLWTW …), with the exception of ft7-FT6 and ft3-FT6, where the FT6 sequence starts with Trp-67 or Ile-92, respectively. Thus, the new CTS regions introduced into the chimeric FT6 variants contained the entire cytoplasmic and transmembrane domain of the corresponding donor glycosyltransferase as well as the stem region or parts thereof replacing 51–91 aa of the N terminus of wt-FT6. In

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