Human α1,3/4-Fucosyltransferases
1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês
10.1074/jbc.273.39.25256
ISSN1083-351X
AutoresAnne L. Sherwood, Anton Nguyen, Jeffery M. Whitaker, Bruce A. Macher, Mark R. Stroud, Eric H. Holmes,
Tópico(s)Lysosomal Storage Disorders Research
ResumoAmino acid sequence alignment of human α1,3/4-fucosyltransferases (FucTs) demonstrates that three highly conserved Lys residues are present in the catalytic domain of FucTs III, IV, V, and VI. Two of these sites are conserved in FucT VII, with the third located within the α1,3-FucT motif as a conservative change to Arg at position 223. Site-directed mutagenesis experiments were conducted to change Lys255 of FucT V (equivalent to Arg223 of FucT VII) to either Arg255 or Ala255. Enzyme assays demonstrate that the FucT V K255R mutant has a 34-fold lower specific activity than native FucT V and that the K255A mutant is inactive. Site-directed mutagenesis of FucT VII was also conducted to change Arg223 to Lys223 for analysis of the effect on enzyme kinetic parameters. No differences in acceptor specificities orK m values for either substrate were observed between native FucT VII and the R223K mutant; however, the purified R223K mutant enzyme had a 2-fold increased specific activity compared with purified native FucT VII. No change in GDP-fucose-protectable pyridoxal-P/NaBH4 inactivation was observed for native or mutant FucT V or VII, further supporting the absence of involvement of this residue in sugar nucleotide binding. The results indicate that a basic residue in this position is required for enzyme activity, with a Lys residue providing higher intrinsic activity. The lack of influence of this site on substrate binding parameters and its location within the α1,3-FucT motif suggest that at least some of the residues within this motif are involved in catalysis rather than substrate binding. Amino acid sequence alignment of human α1,3/4-fucosyltransferases (FucTs) demonstrates that three highly conserved Lys residues are present in the catalytic domain of FucTs III, IV, V, and VI. Two of these sites are conserved in FucT VII, with the third located within the α1,3-FucT motif as a conservative change to Arg at position 223. Site-directed mutagenesis experiments were conducted to change Lys255 of FucT V (equivalent to Arg223 of FucT VII) to either Arg255 or Ala255. Enzyme assays demonstrate that the FucT V K255R mutant has a 34-fold lower specific activity than native FucT V and that the K255A mutant is inactive. Site-directed mutagenesis of FucT VII was also conducted to change Arg223 to Lys223 for analysis of the effect on enzyme kinetic parameters. No differences in acceptor specificities orK m values for either substrate were observed between native FucT VII and the R223K mutant; however, the purified R223K mutant enzyme had a 2-fold increased specific activity compared with purified native FucT VII. No change in GDP-fucose-protectable pyridoxal-P/NaBH4 inactivation was observed for native or mutant FucT V or VII, further supporting the absence of involvement of this residue in sugar nucleotide binding. The results indicate that a basic residue in this position is required for enzyme activity, with a Lys residue providing higher intrinsic activity. The lack of influence of this site on substrate binding parameters and its location within the α1,3-FucT motif suggest that at least some of the residues within this motif are involved in catalysis rather than substrate binding. fucosyltransferase polymerase chain reaction pyridoxal-5′-phosphate. Five distinct human α1,3/4-fucosyltransferases (FucTs)1 have been cloned (1Kukowska-Latallo J. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (472) Google Scholar, 2Goelz S.E. Hession C. Goff D. Griffiths B. Tizard R. Newman B. Chi-Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 3Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar, 4Koszdin K.L. Bowen B.R. Biochem. Biophys. Res. Commun. 1992; 187: 152-157Crossref PubMed Scopus (149) Google Scholar, 5Weston B.W. Smith P.L. Kelly R.J. Lowe J.B. J. Biol. Chem. 1992; 267: 24575-24584Abstract Full Text PDF PubMed Google Scholar, 6Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar, 7Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar), as well as related enzymes from other species (8Hadwiger J.A. Wilkie T.M. Strathmann M. Firtel R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8213-8217Crossref PubMed Scopus (38) Google Scholar, 9Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hirarwa N. Jenkens N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10Smith P.L. Gersten K.M. Petryniak B. Kelly R.J. Rogers C. Natsuka Y. Alford III, J.A. Scheidegger E.P. Natsuka S. Lowe J.B. J. Biol. Chem. 1996; 271: 8250-8259Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 11Ozawa M. Muramatsu T J. Biochem. (Tokyo). 1996; 119: 302-308Crossref PubMed Scopus (21) Google Scholar, 12Oulmouden A. Wierinckx A. Petit J.M. Costache M. Palcic M.M. Mollicone R. Oriol R. Julien R. J. Biol. Chem. 1997; 272: 8764-8773Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Lee K.P. Carlson L.M. Woodcock J.B. Ramachandra N. Schultz T.L. Davis T.A. Lowe J.B. Thompson C.B. Larsen J.B. J. Biol. Chem. 1996; 271: 32960-32967Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 14Sajdel-Sulkowska E.M. Smith F.I. Wiederschain G. McCluer R.H. Glycoconj. J. 1997; 14: 249-258Crossref PubMed Scopus (32) Google Scholar, 15Martin S.L. Edbrooke M.R. Hodgman T.C. van den Eijnden D.H. Bird M.I. J. Biol. Chem. 1997; 272: 21349-21356Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 16Ge Z. Chan N.W.C. Palcic M.M. Taylor D.E. J. Biol. Chem. 1997; 272: 21357-21363Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Analysis of their deduced amino acid sequences demonstrates that a variable number of Lys residues are present. FucTs III, V, and VI are highly homologous and contain 13, 12, and 12 Lys residues, respectively. In contrast, FucTs VII and IV contain only two and five Lys residues, respectively. Sequence alignment analysis of these forms demonstrate that nine of the Lys residues present in FucT III, V, and VI are in equivalent positions. Of these, three Lys residues can be aligned with Lys residues found in the FucT III, IV, V, and VI sequences. The FucT VII sequence contains two of these Lys residues, and a conservative substitution (Arg223) occurs at the third site. The sites corresponding to Arg223 and Lys240 of FucT VII are located in the so-called α1,3-FucT motif of amino acids that are highly conserved among FucT enzymes (15Martin S.L. Edbrooke M.R. Hodgman T.C. van den Eijnden D.H. Bird M.I. J. Biol. Chem. 1997; 272: 21349-21356Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,16Ge Z. Chan N.W.C. Palcic M.M. Taylor D.E. J. Biol. Chem. 1997; 272: 21357-21363Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This distribution of Lys/Arg residues in human FucTs is shown in Fig. 1. The highly conserved nature of these three amino acid sites strongly suggests that one or more are critical for enzymatic activity. We have previously shown that at least one catalytically essential Lys residue is present in human FucTs (17Holmes E.H. Arch. Biochem. Biophys. 1992; 296: 562-568Crossref PubMed Scopus (12) Google Scholar) and is protected from PLP/NaBH4 inactivation by GDP or GDP-fucose. We have initiated a series of site-directed mutagenesis studies to investigate the importance of the Lys (or Arg) residues that are highly conserved among the FucTs. Initial mutagenesis studies were done to evaluate Lys255 in FucT V, which corresponds to Arg223of FucT VII, changing it to an Arg or Ala residue. Further site-directed mutagenesis experiments were conducted to convert Arg223 to Lys223 in FucT VII. The results of these investigations demonstrate that this Lys/Arg residue is critical for FucT activity and that even conservative amino acid substitutions significantly affect enzyme activity. COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD). Sodium taurodeoxycholate, GDP-fucose, and DEAE-dextran were obtained from Sigma. Plasmid pZErO-1 was from Invitrogen (San Diego, CA), and plasmid pCDM8-FucT VII was kindly supplied by Dr. John Lowe (Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI). PCR primers were made on a Beckman Oligo 1000 Synthesizer or obtained from Operon (Alameda, CA). DNA sequencing was done using the Sequenase version 2.0 DNA sequencing kit or the Sequenase PCR product sequencing kit obtained from U.S. Biochemical Corp. GDP-[14C]fucose (270 mCi/mmol) was obtained from Amersham Pharmacia Biotech. [35S]dATP was obtained from NEN Life Science Products. Sialosyllactoneotetraosylceramide 2Glycolipids are designated according to the recommendations of the IUPAC Nomenclature Committee (35IUPAC-IUB Commission on Biochemical Nomenclature Lipids. 1977; 12: 455-463Crossref PubMed Scopus (259) Google Scholar), but the suffix OseCer is omitted. The major glycolipids considered in this study are as follows: sialosyllactoneotetraosylceramide (IV3NeuAcnLc4), NeuAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer; sialosylneolactonorhexaosylceramide (VI3NeuAcnLc6), and NeuAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer.and sialosylneolactonorhexaosylceramide were prepared from bovine erythrocytes (18Chien J.L. Li S.-C. Laine R.A. Li Y.-T. J. Biol. Chem. 1978; 253: 4031-4035Abstract Full Text PDF PubMed Google Scholar). All other reagents were of the highest purity commercially available. Parental, native FucT coding sequences for site-directed mutagenesis were either full-length forms expressed in the pCDM8 vector or truncated catalytic domain forms in the pPROTA vector. Properties of pPROTA-expressed enzymes have been shown to be very similar to those of full-length enzymes (26De Vries T. Srnka C.A. Palcic M.M. Swiedler S.L. van den Eijnden D.H. Macher B.A. J. Biol. Chem. 1995; 270: 8712-8722Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Site-directed mutagenesis of FucT V in pPROTA forming the two mutants K255R and K255A was conducted as follows via recombinant PCR (19Higuchi R. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., New York1990: 177-183Google Scholar). The two flanking primers used for the two FucT V mutants were B14 (which contains an EcoRI site and nucleotides flanking the truncated form of FucT V, i.e. from Leu76) and C6 (containing nucleotides flanking the COOH-terminal end of FucT V,i.e. Thr374, a STOP codon, and anEcoRI site). The flanking primers were B14 (5′-CCCGAATTCGCTACTGATCCTGCTGTGGACG-3′) and C6 (5′-GCTGAATTCTCAGGTGAACCAAGCCGCTA-3′). K255R mutagenic primers were V5K255R (5′-ACGCTGTCCCGGTACAGGTTCTATCTGGCCTTC-3′) and V3K255R (5′-GAAGGCCAGATAGAACCTGTACCGGGACAGCGT-3′). K255A mutagenic primers were V5K255A (5′-ACGCTGTCCCGGTACGCGTTCTATCTGGCCTTC-3′) and V3K255A (5′-GAAGGCCAGATAGAACGCGTACCGGGACAGCGT-3′). In the first step, a specific mutation was introduced by using a pair of mutagenic primers and a pair of opposite end flanking primers. For the K255R mutant, for example, one PCR mixture included the B14 and V3K255R primers to generate the N-terminal half of the “mutated” FucT V; another PCR mixture included V5K255R and C6 primers to generate the C-terminal half of the “mutated” FucT V. The PCR products were gel-purified and used together with both flanking primers for the second-step PCR mixture. The resulting PCR product was a 916-base pair DNA fragment encoding the truncated FucT V (Leu76–Thr374). The 916-base pair DNA fragment was then purified, cut with EcoRI, and subcloned into pPROTA. Bacterial clones containing plasmids in the correct orientation were screened with HindIII and StuI double digestions. The mutation was confirmed by DNA sequencing using an Applied Biosystems model 373A DNA sequencing system. Site-directed mutagenesis of FucT VII forming the R223K mutant was performed using the same approach with the following primers. The flanking primers produced a 939-base pair product encompassing amino acids Thr40–Ala342 of FucT VII in the pPROTA vector. Flanking primers were 5′-CTGAATTCGACCCCGGCACCCCAGCCC-3′ (upper) and 5′-TCCGAATTCCAGCGGATCTCAGGCCTG-3′ (lower). R223K mutagenic primers were VII5R223K (5′-GCCAGCTGCCTGGTGCCCACCGTGGCCCAGTACAAATTC-3′) and VII3R223K (5′-GTAGAATTTGTACTGGGCCACGGTGGGCAC-3′). The PCR product from the second step PCR mixture was briefly treated with mung bean nuclease and cloned into the EcoRV site of pZErO-1 plasmid, and the mutation was confirmed by DNA sequencing using the Sequenase version 2.0 sequencing system. The insert containing the desired mutation was then excised withEcoRI and cloned into pPROTA as described above for FucT V mutants. The correct orientation was established byHindIII/MscI double digestion. Plasmids were transiently transfected into COS-7 cells grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum using the DEAE-dextran method (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 York1993Google Scholar), and the expressed protein was isolated on IgG-agarose beads as described previously (21Holmes E.H. Xu Z. Sherwood A.L. Macher B.A. J. Biol. Chem. 1995; 270: 8145-8151Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The pCDM8 FucT VII plasmid contains the full coding sequence of FucT VII plus approximately 0.78 kilobase pair of 3′-untranslated region. A NotI cassette of approximately 1.5 kilobase pairs, encompassing NotI (nucleotide 329) within the FucT VII gene through the end of the 3′-untranslated region was excised from parental pCDM8-FucT VII and replaced with a 0.718-kilobase pair NotI cassette from pZErO-1-FucT VII-R223K mutant. This cassette represents the same coding portion of the FucT VII gene (including the R223K mutation), minus the 3′-untranslated sequence. Orientation was established viaEcoRI/MscI digestion. The resultant plasmid, pCDM8-FucT VII-R223K, along with native pCDM8-FucT VII, was subsequently used in transfections using the DEAE-dextran method (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 York1993Google Scholar) and harvested after 5 days. FucT activity using glycolipid acceptors was determined in reaction mixtures containing 2.5 μmol of HEPES buffer, pH 7.2, 1 μmol of MnCl2, 100 μg of taurodeoxycholic acid, 20 μg IV3NeuAcnLc4 or VI3NeuAcnLc6, 15 nmol of GDP-[14C]fucose (15,000 cpm/nmol), and 300–400 μg of protein in a total volume of 0.1 ml. Cells expressing full-length enzymes were homogenized by two strokes of a Potter-Elvehjem homogenizer in a buffer composed of 50 mm HEPES, pH 7.2, containing 25% glycerol. The reaction mixture was incubated at room temperature for 2 h, terminated by the addition of 0.1 ml of CHCl3/CH3OH, 2:1, and quantitated as described previously (22Stroud M.R. Holmes E.H. Biochem. Biophys. Res. Commun. 1997; 238: 165-168Crossref PubMed Scopus (13) Google Scholar). FucT enzyme activities utilizing oligosaccharide acceptors were determined in reaction mixtures composed of 1 μmol of HEPES buffer, pH 7.2, 6 nmol of GDP-[14C]fucose (15,000 cpm/nmol), 0.4 μmol of LacNAc or 0.072 μmol of 8-methoxycarbonyloctyl glycoside derivatives, 2 μmol of NaCl, 0.125 μmol of MnCl2, 10 μg of bovine serum albumin, 0.01 μmol of ATP, and chimeric enzyme bound to IgG-agarose beads in a total volume of 0.02 ml. The reaction mixture was incubated for 1 h at room temperature and stopped and quantitated as described previously utilizing Dowex-1 for oligosaccharide acceptors (26De Vries T. Srnka C.A. Palcic M.M. Swiedler S.L. van den Eijnden D.H. Macher B.A. J. Biol. Chem. 1995; 270: 8712-8722Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) or C18 columns for 8-methoxycarbonyl glycoside derivatives as acceptors (26De Vries T. Srnka C.A. Palcic M.M. Swiedler S.L. van den Eijnden D.H. Macher B.A. J. Biol. Chem. 1995; 270: 8712-8722Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The pPROTA-expressed recombinant FucT enzymes were separated on 12% Tris/glycine polyacrylamide gels, transferred to nitrocellulose membranes, and probed as described in an accompanying paper (34Nguyen A.T. Holmes E.H. Whitaker J.M. Ho S. Shetterly S. Macher B.A. J. Biol. Chem. 1998; 273: 25244-25249Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 0.5 g of PLP, was initially purified by Dowex-1 chromatography with elution by an acetic acid gradient. Fractions containing PLP were pooled, lyophilized, dissolved in 0.1 m NaHCO3, and adjusted to pH 7.0 for use in the following experiments, all conducted under subdued light. PLP treatment of enzymes expressed as pPROTA fusion proteins bound to IgG-agarose beads were conducted in reaction mixtures containing 5 μl of packed enzyme beads in a phosphate-buffered saline (8.1 mm Na2HPO4, 1.5 mmKH2PO4, 137 mm NaCl, 2.7 mm KCl, pH 7.4) slurry and PLP varying from 0.5 to 2 mm final concentration in a total volume of 50 μl. In some reaction mixtures, GDP-fucose was added at a final concentration of 300 μm. After incubation for 15 min at room temperature, a 15-fold excess of NaBH4 dissolved in water was added, and the reaction mixtures were incubated for an additional 15 min. The enzyme activity remaining was then determined using assay conditions described above. Site-directed mutagenesis of FucT V was performed by replacing the sequence for codon 255 (AAG) with AGG for Arg or GCG for Ala and with FucT VII to change the sequence for codon 223 (CGC) to AAA for Arg. Sequencing on both strands of all mutants confirmed that there were no other nucleotide modifications compared with the respective native enzyme (results not shown). Table I shows the effect of the K255R and K255A mutations of FucT V on enzyme specific activity. For this comparison, the enzymes were expressed as soluble fusion proteins of the catalytic domain of the enzymes with the protein A Ig binding domain using the pPROTA vector. These enzyme forms have the important advantage of allowing convenient protein isolation and protein determinations needed for accurate specific activity measurements of the purified enzyme. Replacement of the Lys residue by Arg reduced the enzyme's specific activity 34-fold compared with the native enzyme. Replacement by an Ala residue inactivated the enzyme. Western blot analysis demonstrated that each mutant was expressed at levels comparable with that of the wild type enzyme and that each produced a single band with a molecular weight corresponding to that of the wild type enzyme (results not shown). The dramatic decrease in specific activity for the K255R mutant precluded a kinetic analysis of this enzyme to accurately determine the nature of the changes in kinetic parameters caused by this mutation. To test kinetic parameters and to determine if a Lys residue in this site was generally optimal for activity, an Arg to Lys mutant at the corresponding site of FucT VII (Arg223) was prepared for a kinetic evaluation.Table ISpecific activity of pPROTA-expressed native and mutant FucT V enzymesFucT V enzyme formActivitynmol/h/mg proteinNative48.0K255R mutant1.4K255A mutantNDAssays were conducted as described under “Experimental Procedures” using LacNAc as the acceptor.ND, none detected. Open table in a new tab Assays were conducted as described under “Experimental Procedures” using LacNAc as the acceptor. ND, none detected. An analysis of acceptor specificity and enzyme-specific activity of native FucT VII and the R223K mutant was conducted by expression of the catalytic domains for both enzymes in the pPROTA vector. The results shown in Table IIconfirm the requirement of a terminal sialyl substitution on the acceptor for optimal activity for both the native FucT VII and the R223K mutant. Product formation occurred with 3′-sulfo-LacNAc at a rate 20–25% of that found with 3′-sialyl-LacNAc. This demonstrates the importance of a negative charge at the nonreducing end of the acceptor for activity. The FucT VII R223K enzyme form had 2-fold higher specific activity than native FucT VII when assayed with sialyl-LacNAc-R1 acceptor. Overall, the results demonstrate that the change of Arg223 to Lys223 in FucT VII had no effect on acceptor specificity but had a substantial effect on the intrinsic activity of the enzyme in a manner predicted by the results with native FucT V and its K255R mutant.Table IIAcceptor specificity of pPROTA-expressed native and R223K mutant FucT VII enzymesAcceptorActivityNativeR223K mutantnmol/h/mg proteinFucα1→2Galβ1→4GlcNAc-R15.213.4SO4→3Galβ1→4GlcNAc-R118.030.8NeuAcα2→3Galβ1→4GlcNAc-R171.2143.6Galβ1→4GlcNAc-R10.82.8Galβ1→3GlcNAc-R1NDNDacceptors used were the indicated oligosaccharides coupled to a hydrophobic tail composed of R1 = O-(CH2)8-COOCH3, each present at a final concentration of 3.6 mm.ND, none detected. Open table in a new tab acceptors used were the indicated oligosaccharides coupled to a hydrophobic tail composed of R1 = O-(CH2)8-COOCH3, each present at a final concentration of 3.6 mm. ND, none detected. Transfer of fucose into multiple glycolipid acceptors was also tested with full-length forms of the native FucT VII and R223K mutant enzymes expressed using the pCDM8 vector in COS-7 cells. Fig. 2 shows results from transfer of labeled fucose into glycolipid acceptors using crude homogenates of COS-7 cells expressing the full-length enzymes. Fucose transfer was only observed into sialylated acceptors IV3NeuAcnLcOse4Cer and VI3NeuAcnLcOse6Cer. No detectable product was observed with nLcOse4Cer as an acceptor or with mock-transfected COS-7 cell extracts (results not shown). Qualitatively identical results were obtained with both the native and R223K enzymes. However, transfer was always much greater with homogenates of COS-7 cells transfected with cDNA encoding the full-length R223K enzyme compared with native FucT VII, consistent with the observation from the pPROTA-expressed enzyme forms that the R223K mutant was an inherently more active enzyme. Only monofucosylated products were obtained with each enzyme, with transfer to VI3NeuAcnLcOse6Cer (lanes 3 and 4) yielding exclusively the product VI3NeuAcV3FucnLcOse6Cer (results not shown; see Ref. 22Stroud M.R. Holmes E.H. Biochem. Biophys. Res. Commun. 1997; 238: 165-168Crossref PubMed Scopus (13) Google Scholar). The increased specific activity of the FucT VII R223K mutant compared with the native form made it possible to conduct a kinetic analysis of these enzymes to determine if the increased specific activity of the R223K enzyme was due to a K m orV max effect on the enzyme. Saturation kinetics with both donor and acceptor substrates was studied for both pPROTA-expressed enzyme forms (Fig. 3). No differences in the K m for GDP-fucose was found for the mutant versus wild type enzyme, with observedK m values of 9.2 ± 0.3 and 10.8 ± 1.4 μm for the native FucT VII and R223K enzyme, respectively. Similarly, the K m for sialyl-LacNAc-R1 was the same for both enzymes at 0.70 ± 0.02 and 0.70 ± 0.05 mm for the native FucT VII and R223K enzymes, respectively. Thus, the increased specific activity of the R223K mutant is a V max effect and indicates that a Lys residue in this position creates an inherently more active enzyme. The presence of a PLP/NaBH4-sensitive Lys residue that lies in or near the GDP-fucose binding site has previously been demonstrated (17Holmes E.H. Arch. Biochem. Biophys. 1992; 296: 562-568Crossref PubMed Scopus (12) Google Scholar). To determine if Lys255 of FucT V is this residue, a series of PLP inactivation studies were conducted with pPROTA-expressed mutant FucT V and VII enzymes. These enzymes were treated with increasing concentrations of PLP followed by NaBH4 reduction to determine if the Lys group in this position was associated with GDP-fucose-protectable enzyme inactivation. The results are shown in Fig. 4. Panels A–Cshow the GDP-fucose-protectable inactivation of FucTs III, IV, and VI as positive controls confirming the general property of PLP/NaBH4 sensitivity among human FucTs. Panels D and E demonstrate that both the native and K255R FucT V and native and R223K FucT VII enzymes, respectively, are inactivated by PLP/NaBH4 to a similar extent. The extent of the inactivation of the FucT VII enzymes was less than that seen for the expressed pPROTA constructs of the other FucT enzymes at the same PLP concentrations (panels A–D), suggesting that the native and R223K FucT VII enzymes have a lower affinity for PLP compared with other FucTs. Analysis of the K i for PLP for both the native and R223K FucT VII enzymes demonstrate that each enzyme had a K i of approximately 2 mm (results not shown). This compares to aK i for PLP of 105 μm previously reported for the enzyme derived from NCI-H69 cells (17Holmes E.H. Arch. Biochem. Biophys. 1992; 296: 562-568Crossref PubMed Scopus (12) Google Scholar). Thus, the observed inactivation results demonstrate that PLP inactivation of native and R223K FucT VII enzymes occurs over a wider PLP concentration range. Fig. 4 also demonstrates that PLP/NaBH4 inactivation of all enzymes tested is protectable by GDP-fucose present at 0.3 mm final concentration. Genes encoding α1,3-FucT enzymes have been cloned from a variety of species (8Hadwiger J.A. Wilkie T.M. Strathmann M. Firtel R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8213-8217Crossref PubMed Scopus (38) Google Scholar, 9Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hirarwa N. Jenkens N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10Smith P.L. Gersten K.M. Petryniak B. Kelly R.J. Rogers C. Natsuka Y. Alford III, J.A. Scheidegger E.P. Natsuka S. Lowe J.B. J. Biol. Chem. 1996; 271: 8250-8259Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 11Ozawa M. Muramatsu T J. Biochem. (Tokyo). 1996; 119: 302-308Crossref PubMed Scopus (21) Google Scholar, 12Oulmouden A. Wierinckx A. Petit J.M. Costache M. Palcic M.M. Mollicone R. Oriol R. Julien R. J. Biol. Chem. 1997; 272: 8764-8773Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Lee K.P. Carlson L.M. Woodcock J.B. Ramachandra N. Schultz T.L. Davis T.A. Lowe J.B. Thompson C.B. Larsen J.B. J. Biol. Chem. 1996; 271: 32960-32967Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 14Sajdel-Sulkowska E.M. Smith F.I. Wiederschain G. McCluer R.H. Glycoconj. J. 1997; 14: 249-258Crossref PubMed Scopus (32) Google Scholar, 15Martin S.L. Edbrooke M.R. Hodgman T.C. van den Eijnden D.H. Bird M.I. J. Biol. Chem. 1997; 272: 21349-21356Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 16Ge Z. Chan N.W.C. Palcic M.M. Taylor D.E. J. Biol. 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Chem. 1998; 273: 25244-25249Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 36Vo L. Lee S. Marcinko M.C. Holmes E.H. Macher B.A. J. Biol. Chem. 1998; 273: 25250-25255Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar)). The results presented in this paper address a region of the catalytic domain that is instead rather highly conserved among enzyme forms. Although variable numbers of Lys residues are present among human FucTs, highly conserved sites exist. In particular, three Lys residues are in identical positions in FucTs III, IV, V, and VI. Two of these positions are occupied by the only Lys residues present in the FucT VII enzyme. The third site is a conservative change to an Arg residue (Arg223). The results presented in this paper focus on enzymatic properties inherent to this Lys/Arg site among human FucT enzymes. Site-directed mutagenesis experiments changing the equivalent Lys255 of FucT V to an Arg residue (K255R mutant) resulted in an enzyme with dramatically reduced specific activity compared with the native enzyme. Changing this site to a nonconservative Ala residue (K255A mutant) abolished activity. Thus, a basic group in this position is required for activity. The poor activity of the resulting K255R mutant required an alternate approach to accurately determine the kinetic effect of this type of substitution on the enzyme. Since native FucT VII has an Arg residue in this position, changing it to a Lys residue and analyzing its affect was deemed to be an ideal approach. Presumably, a more active enzyme would result, allowing kinetic comparisons of both the native and mutant enzymes. Analysis of native FucT VII and the R223K mutant confirmed that this substitution had no effect on acceptor specificity properties of the enzyme, including the enzyme's behavior with sialylated acceptors containing multiple GlcNAc residues. Further, this amino acid change had no effect on the K m for either the donor GDP-fucose or the acceptor oligosaccharide. The only effect caused by this amino acid change was to create an enzyme with inherently greater activity. Taken together, the results strongly suggest that this site does not directly function in substrate binding; however, a basic group in this site is required for activity. Enzymes containing a Lys residue in this position are inherently more active compared with those with an Arg residue, suggesting the ionization state of the side chain is significant in maintaining optimal enzyme conformation or may be involved in catalysis. This is consistent with a previous report that suggested that a basic group on human FucT enzymes (i.e.FucT III and VI) is involved in a critical hydrogen bond donor interaction with reactive acceptor hydroxyl groups during the glycosyl transfer reaction (33Hindsgaul O. Kaur K.J. Srivastava G. Blaszczyk-Thurin M. Crawley S.C. Heerze L.D. Palcic M.M. J. Biol. Chem. 1991; 266: 17858-17862Abstract Full Text PDF PubMed Google Scholar). Recently, cloning of FucT enzymes from H. pylori and a comparison of aligned sequences from a wide variety of species have led to the identification of an “α1,3-FucT motif” (15Martin S.L. Edbrooke M.R. Hodgman T.C. van den Eijnden D.H. Bird M.I. J. Biol. Chem. 1997; 272: 21349-21356Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 16Ge Z. Chan N.W.C. Palcic M.M. Taylor D.E. J. Biol. Chem. 1997; 272: 21357-21363Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This motif is composed of certain fully conserved amino acid residues within a portion of the catalytic domain between residues Tyr254and Lys272 of human FucT V and between residues Tyr222 and Lys240 of FucT VII. Interestingly, the Lys/Arg site described in this paper occurs within this segment of the catalytic domain. The behavior of the native and mutant enzymes described in this paper suggests that this motif may have a more complex function in FucT enzymes by directly participating in catalysis, in addition to a possible involvement in binding GDP-fucose or Mn2+ (15Martin S.L. Edbrooke M.R. Hodgman T.C. van den Eijnden D.H. Bird M.I. J. Biol. Chem. 1997; 272: 21349-21356Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). A more complete evaluation of the role of this α1,3-FucT motif is required before firm conclusions can be drawn relating to the functions of the differing amino acids that are present. Previous results using enzyme derived from NCI-H69 cells demonstrated that PLP is a competitive inhibitor with respect to GDP-fucose and, further, that a PLP/NaBH4-sensitive, GDP-fucose-protectable Lys residue is present (17Holmes E.H. Arch. Biochem. Biophys. 1992; 296: 562-568Crossref PubMed Scopus (12) Google Scholar). The present results confirm that this is a common property of FucT enzymes. Thus, it is reasonable to conclude that this is a property of at least one of the highly conserved Lys residues present in FucTs. To determine if FucT VII was also PLP/NaBH4-sensitive and if the site corresponding to Lys255 of FucT V is the reactive site, PLP/NaBH4 inactivation studies were conducted on the native and mutant enzymes. The results confirmed that FucT VII was also inactivated by PLP/NaBH4, although with a lower efficiency than other enzyme forms. The K i for PLP inhibition with respect to GDP-fucose was determined to be 2 mm, compared with 105 μm reported previously (17Holmes E.H. Arch. Biochem. Biophys. 1992; 296: 562-568Crossref PubMed Scopus (12) Google Scholar). Comparisons of the results from both FucT V and VII native and mutant enzymes demonstrate that there was no difference in the GDP-fucose-protectable PLP/NaBH4 inactivation regardless of whether a Lys or Arg residue occupied this amino acid position. Thus, this position does not correspond to the PLP/NaBH4-sensitive site. This is consistent with the results from a kinetic analysis of the native and mutant FucT VII enzymes, which demonstrate that the change from an Arg to Lys had no affect on the K m for GDP-fucose. We thank Drs. Ole Hindsgaul and Monica Palcic for supplying 8-methoxycarbonyloctyl glycoside derivatives used for enzyme assays. Automated DNA sequencing was performed at the San Francisco State University DNA Sequencing Facility (established by National Science Foundation-ARI Grant BIR-9512443). A. T. N. acknowledges the support of Dr. Sergio Pichuantes and Chiron Corp.
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