Structural Basis for the Inactivity of Human Blood Group O2 Glycosyltransferase

The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fucα1-2Galβ-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O2 enzyme was discovered that was a full-length form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O2 blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected. The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fucα1-2Galβ-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O2 enzyme was discovered that was a full-length form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O2 blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected. The human A and B blood groups are produced by two closely related glycosyltransferase enzymes (1Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (869) Google Scholar, 2Watkins W.M. Adv. Hum. Genet. 1980; 10: 1-136PubMed Google Scholar, 3Palcic M.M. Seto N.O. Hindsgaul O. Transfus. Med. 2001; 11: 315-323Crossref PubMed Scopus (17) Google Scholar). Blood type A structures are synthesized by an α1-3 N-acetylgalactosaminyltransferase (GTA, 1The abbreviations used are: GTA, glycosyltransferase A; GTB, glycosyltransferase B; MOPS, 4-morpholinepropanesulfonic acid; aa, amino acids. 1The abbreviations used are: GTA, glycosyltransferase A; GTB, glycosyltransferase B; MOPS, 4-morpholinepropanesulfonic acid; aa, amino acids. EC 2.4.1.40) that transfers GalNAc from UDP-GalNAc to Fucα1-2Galβ-R (H)-terminating acceptors producing the A antigen GalNAcα1-3[Fucα1-2]Galβ-R. The B-synthesizing α1-3 galactosyltransferase (GTB, EC 2.4.1.37) transfers Gal from UDP-Gal to the same acceptors, producing the B antigen Galα1-3[Fucα1-2]Galβ-R. Individuals with blood type O do not express enzymes capable of modifying the H antigen (1Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (869) Google Scholar). GTA and GTB exhibit characteristic mammalian glycosyltransferase topologies; they are type II integral membrane proteins with a short N-terminal cytoplasmic tail, a transmembrane domain, a proteolytically sensitive stem region, and a catalytic domain (4Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar). GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268 (1Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (869) Google Scholar, 5Yamamoto F. Hakomori S. J. Biol. Chem. 1990; 265: 19257-19262Abstract Full Text PDF PubMed Google Scholar). Substitution of these four critical amino acids converts the donor specificity from that of GTA to that of GTB. Recently, single crystal x-ray diffraction studies of soluble forms of GTA and GTB in complex with H acceptor and UDP have provided a structural basis for substrate recognition (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar). The critical residue Leu/Met-266 dominates donor selection with Gly/Ala-268 having a significant but lesser effect. The origin of blood group O was initially shown to be a deletion or mutation in the GTA or GTB gene that gave inactive truncated enzyme (1Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (869) Google Scholar). More recently, an O2 enzyme (O03) was discovered that was a full-length form of GTA with three substitutions, P74S, R176G, and G268R (7Yamamoto F. McNeill P.D. Yamamoto M. Hakomori S. Bromilow I.M. Duguid J.K. Vox Sang. 1993; 64: 175-178Crossref PubMed Scopus (107) Google Scholar, 8Grunnet N. Steffensen R. Bennett E.P. Clausen H. Vox Sang. 1994; 67: 210-215Crossref PubMed Scopus (83) Google Scholar). The O2 glycosyltransferase showed no measurable transferase activity when expressed in Sf9 cells (9Amado M. Bennett E.P. Carneiro F. Clausen H. Vox Sang. 2000; 79: 219-226Crossref PubMed Scopus (19) Google Scholar). Since the P74S mutation is distant from the active site and the R176G mutation in GTA increases enzyme turnover (10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), it is presumably the replacement of glycine with arginine at position 268 that adversely affects enzyme activity (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar, 11Yamamoto F. McNeill P.D. J. Biol. Chem. 1996; 271: 10515-10520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). GTA transferase-like activity ranging from 0.006 to 0.25% of GTA levels have been detected in some concentrated blood group O serum samples (12Greenwell P. Glycoconj. J. 1997; 14: 159-173Crossref PubMed Scopus (129) Google Scholar). Here we prepare three mutant enzymes derived from truncated soluble R176G GTA: P74S, G268R, and P74S/G268R. For comparison, we also report the structure of the GTA R176G mutant enzyme, which was earlier shown to have an enzyme turnover rate greater than that of wild-type GTA (10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Each of these constructs is based on the same synthetic genes with codons optimized for facile mutagenesis and a high level of expression in Escherichia coli (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar, 10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Kinetics and single crystal x-ray diffraction were used to determine the effect of each mutation on enzyme activity. Materials and General Techniques—All molecular biology procedures were as described previously (10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York1997: 8.5.7-8.5.10Google Scholar, 14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, NY1989Google Scholar, 15Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S. Eur. J. Biochem. 1995; 234: 323-328Crossref PubMed Scopus (48) Google Scholar, 16Seto N.O.L. Compston C.A. Evans S.V. Bundle D.R. Narang S.A. Palcic M.M. Eur. J. Biochem. 1999; 259: 770-775Crossref PubMed Scopus (88) Google Scholar). The original GTA and the GTA R176G gene sequences (aa 54-354) have been described (10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S. Eur. J. Biochem. 1995; 234: 323-328Crossref PubMed Scopus (48) Google Scholar, 16Seto N.O.L. Compston C.A. Evans S.V. Bundle D.R. Narang S.A. Palcic M.M. Eur. J. Biochem. 1999; 259: 770-775Crossref PubMed Scopus (88) Google Scholar). All chimeric GTA/GTB enzymes are referred to by a series of four letters corresponding to the origin of each of the four residues where AAAA is GTA and BBBB is GTB. We reported earlier the interesting kinetics and donor specificity of the chimeric enzyme BAAA (aa 54-354, Gly-176, Gly-235, Leu-266, Gly-268) (10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Truncation of a further 10 amino acids from the N terminus gave higher expression levels and solubility; therefore, all mutants in this study are derived from wild-type GTA and GTB enzymes (both corresponding to aa 63-354). Cloning of GTA Arg-176 —GTA R176, also denoted as BAAA (aa 63-354), was made by PCR amplification using the BAAA (aa 54-354) gene as template together with the forward primer MIN-2 (5′-A TAT GAATTC ATG GTT TCC CTG CCG CGT ATG GTT TAC CCG CAG CCG AA), which introduced an EcoRI site (underlined) in the 5′ end, and the reverse primer PCR-3B (5′-ATA ATT AAGCTT CTA TCA CGG GTT ACG AAC AGC CTG GTG GTT TTT), which introduced a HindIII site (underlined) in the 3′ end. The following PCR profile was used for the construction of all the clones: 94 °C, 3 min (94 °C, 30 s, 55 °C, 30 s, 72 °C, 1 min) for 30 cycles. After gel purification, the PCR products were digested with EcoRI and HindIII for 2 h at 37 °C and were ligated into pCWΔlac vector (17Gegner J.A. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 750-754Crossref PubMed Scopus (163) Google Scholar) opened with EcoRI/HindIII for 1 h at room temperature. Each ligation was transformed into BL21 competent cells (Novagen). All insert and plasmid purifications were made by Qiagen Plasmid Purification System (Qiagen Inc., Chatsworth, CA). All ligations were made by the use of T4 DNA ligase (Invitrogen) at room temperature overnight. The clones were characterized by triplicate DNA sequence analysis of the entire coding region. Cloning of BAAA P74S—The BAAA P74S mutant was constructed by PCR using BAAA (aa 63-354) described above as a template and the forward primer HJL01 (5′-A TAT GAA TTC ATG GTT TCC CTG CCG CGT ATG GTT TAC CCG CAG TCC AAA GTT CTG ACC CCA TGC CG-3′), which was designed with a single codon substitution (CCG → TCC) at codon 74 and an EcoRI site at the 5′ end and the reverse primer PCR-3B. The amplified genes were digested by restriction enzymes (EcoRI and HindIII) and cloned as described above. Cloning of BAAA G268R—The BAAA G268R mutant was constructed by recombinant PCR using the BAAA (aa 63-354) clone as a template. The first PCR was performed using the outside forward primer MIN2 together with the internal reverse primer HJL02 (5′-ACC GAA GAA ACG ACC CAG GTA GTA GAA GTC ACC-3′) that contains a single codon substitution (ACC → AGC) at codon 268. A second PCR was performed using the internal forward primer HJL03 (5′-C CTG GGT CGT TTC TTC GGT GGT TCC GTT CAG-3′) that contains a single codon substitution (GGT → CGT) at codon 268 together with the outside reverse primer PCR-3B. The outside forward and reverse primers included the EcoRI and HindIII restriction sites. The two overlapping PCR products were annealed together and amplified by PCR with the outside primers MIN2 and PCR-3B, digested with EcoRI and HindIII, and cloned as described above. Cloning of P74S/G268R—The BAAA P74S/G268R mutant was constructed by PCR using the BAAA G268R clone as a template together with the forward primer HJL01 and the reverse primer PCR-3B. The amplified gene was digested with restriction enzymes EcoRI and HindIII and cloned as described above. Protein Purification—Mutant enzymes were purified from E. coli as described previously (18Seto N.O.L. Compston C.A. Szpacenko A. Palcic M.M. Carbohydr. Res. 2000; 324: 161-169Crossref PubMed Scopus (45) Google Scholar, 19Marcus S.L. Polakowski R. Seto N.O.L. Leinala E. Borisova S. Blancher A. Roubinet F. Evans S.V. Palcic M.M. J. Biol. Chem. 2003; 278: 12403-12405Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Expression levels for all of the mutants were good, and the yields of final purified proteins ranged from 26 mg/liter for the G268R mutant to 108 mg/liter for the P74S/G268R double mutant. Protein concentration was determined with a Bio-Rad protein assay kit based on the Bradford method (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using bovine γ-globulin as a standard. Kinetic Characterization—Kinetic characterizations were carried out on all mutant enzymes using a Sep-Pak radiochemical assay with the hydrophobic acceptor Fucα1-2Galβ-O(CH2)7CH3 (21Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconj. J. 1988; 5: 49-63Crossref Scopus (277) Google Scholar). Assays were carried out at 37 °C in a total volume of 15 μl containing substrates and enzyme in 50 mm MOPS buffer, pH 7.0, with 20 mm MnCl2 and 1 mg/ml bovine serum albumin. Seven different concentrations of the donor or acceptor were used, at a high concentration of the alternate substrate. The amount of substrate consumed was less than 15% to ensure linear initial reaction rates. The kinetic parameters Vmax and Km were derived from the best fit of the data to the Michaelis-Menten equation by using nonlinear regression with the GraphPad PRISM 3.0 program. Crystallography—All mutants related to O2 enzyme were crystallized using conditions similar to the native GTA and GTB enzymes (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar). Data were collected on a Rigaku R-AXIS4++ area detector at distances of 72 and 100 mm and exposure times and min for were produced by an to x-ray with levels of 30 The were and conditions at a temperature of °C using a crystal All structures were by using molecular replacement with wild-type GTA or GTB Data and as a and were using the P.D. P. J. M. T. Biol. PubMed Scopus Google Scholar). Molecular were generated using the S.V. J. 1993; 11: PubMed Scopus Google Scholar). this a series of mutant blood group were produced and characterized to determine the effect of each on enzyme structure and These mutants are based on the gene sequence for an O2 enzyme discovered in blood that yields a mutant of GTA with arginine with 74 with and glycine 268 with These enzymes were all cloned as soluble truncated proteins (aa of the catalytic domain and expressed in E. The by and on a was for all no of UDP binding. Kinetic were determined for each purified enzyme at a high concentration of the alternate substrate The for BAAA enzyme was than that of wild-type GTA, with report for an enzyme with the same mutation but with 10 amino acids on the N terminus (10Seto N.O.L. Palcic M.M. Hindsgaul O. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). for the P74S which was with that of the wild-type GTA, the purified enzymes with corresponding to the human glycosyltransferase O2 showed activity. The for the G268R mutant was 4 times less than that of GTA and times less than that of the P74S mutant. The P74S/G268R mutant showed the activity of with a of only for mutant is the Michaelis-Menten Km for the acceptor determined at 1 mm is the Michaelis-Menten Km for the donor UDP-GalNAc determined at high concentrations of acceptor are from BAAA that the first critical residue on GTA been to the corresponding residue in GTB were based on the BAAA clone as a is the Michaelis-Menten Km for the acceptor determined at 1 mm is the Michaelis-Menten Km for the donor UDP-GalNAc determined at high concentrations of acceptor Data are from 19Marcus S.L. Polakowski R. Seto N.O.L. Leinala E. Borisova S. Blancher A. Roubinet F. Evans S.V. Palcic M.M. J. Biol. Chem. 2003; 278: 12403-12405Abstract Full Text Full Text PDF PubMed Scopus (87) Google The BAAA that the first critical residue on GTA been to the corresponding residue in GTB All were based on the BAAA clone as a template. in a The O2 mutant enzymes also showed an in the Km for acceptor and donor in as and the wild-type GTA and the BAAA enzymes. For the P74S the Km for donor was higher than GTA, whereas the effect on acceptor was The P74S/G268R mutant showed a in Km for donor and a minor effect on acceptor Km The of these turnover that the activity from the mutant enzyme or Biochem. 1999; PubMed Scopus Google the of an donor Km that the activity that is is not to wild-type of the data and for the enzymes in the and forms are shown in II and data were collected to a resolution of with final ranging from to and ranging from to All structures showed the entire of the with the of the (aa and the final 10 amino residues at the of which were also in the native GTA/GTB structures (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar). The the active site of the mutant is shown in and for mutants in the of and in high resolution in high resolution of were in in high resolution of were in in a and for mutant in the of donor substrates and H and G268R in high resolution in high resolution of were in in high resolution of were in in a this the catalytic domain corresponding to the human blood group O2 glycosyltransferase was produced and The O2 enzyme is a mutant of GTA with arginine with 74 with and glycine 268 with for the P74S the purified mutants for human glycosyltransferase O2 from E. coli showed activity. determine the effect of each mutation on the transferase three mutants G268R, and derived from BAAA were and by enzyme kinetics and single crystal x-ray All enzymes were from and with the BAAA enzyme, which higher A activity than the The crystal structure of the BAAA mutant does not reveal as to this mutant increased A as to the R176G mutation site a to the active site of the enzyme that is to be in GTA/GTB native and mutant enzyme The crystal structure of enzymes containing the G268R the P74S/G268R mutant for blood type the for the of the as completely the donor recognition the wild-type and BAAA structures the critical amino acids Leu/Met-266 by the corresponding and groups on of GalNAc and The second critical residue in donor is not to the donor in GTA, whereas it the A donor in GTB (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar). The G268R mutation this recognition by completely to the active site and to the was for the that the G268R mutation does not this substrate from the binding site These are with the Km for all G268R which that are than wild-type GTA enzyme, but with BAAA and that are 2 the the GalNAc of donor and that to the active The G268R mutant enzyme also showed activity with a of than that of the wild-type GTA, that the mutation was for the of the G268R Km for the donor that are higher than the wild-type and BAAA whereas Km for acceptor are not as The was higher than GTA and times higher than the whereas the was times higher than GTA and 20 times higher than that the mutation at residue 268 affects donor it affects acceptor binding as The in the crystal structure that the mutation completely blocks the donor site that the corresponding to the is the binding of the UDP is by the that the enzyme the UDP was not to the enzyme in the crystal structure using conditions similar to that to the of UDP in the structures of the native GTA and GTB enzymes (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar, 19Marcus S.L. Polakowski R. Seto N.O.L. Leinala E. Borisova S. Blancher A. Roubinet F. Evans S.V. Palcic M.M. J. Biol. Chem. 2003; 278: 12403-12405Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Seto N.O.L. Leinala E. Borisova N. Palcic M.M. Evans S.V. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the G268R mutation does not acceptor as this substrate is to form the same of in the mutant as the wild-type the and binding of the whereas they are in the mutant enzyme the P74S mutation also significant on the reaction The P74S mutation a in of that in it to levels with the wild-type The same mutation in BAAA G268R a in in an enzyme that is times less active than the it is that the mutation at is the to of the O2 enzyme, the effect of the P74S mutation is as this residue in the stem region of the enzyme and not in the catalytic domain and is than 30 distant from the active of the crystal structure a for this as the stem of in the crystal are to to form The the first 15 N-terminal residues of the stem region and is that residues to in of the are in with residues to the active site in the of the the P74S mutation does not significant in crystal structures of the enzyme or the of the active site and characteristic acceptor (6Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar), the mutation from to and some in the The for the soluble of O2 mutant enzyme is also in the wild-type GTA and GTB of with related with structure that this the N-terminal residues is not a but is to the crystal of GTA, GTB, and soluble of GTA and GTB are to in where they have the to form that the in the crystal is also in from of a purified a single of molecular the of and from the in the of a second at higher molecular corresponding to a GTA N-terminal of the it was the effect of the G268R mutation in O2 glycosyltransferase is restriction of the of the monosaccharide GalNAc of donor in the enzyme active turnover is with of than wild-type glycosyltransferase A. binding is also as in an in Km for in the active site is from that of wild-type levels of GTA activity have been reported in the serum of blood type O enzyme on a single O2 serum be for of the activity for recombinant with DNA was provided by the Molecular of of We F. Dahlquist for the for the pCWΔlac O. Hindsgaul for the Fucα1-2Galβ-O(CH2)7CH3 and N. for the gel

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