From the Department of Chemistry, University of
Alberta, Edmonton, Alberta T6G 2G2, Canada, § Institute for
Biological Sciences, National Research Council of Canada, Ottawa K1A
0R6, Canada, ¶ Department of Biochemistry, Microbiology and
Immunology, University of Ottawa, Ottawa K1H 8M5, Canada,
Laboratoire d'Immunogénétique Moléculaire,
Université Paul Sabatier, Hôpital de Rangueil, 1 Avenue Jean Poulhes, 31059 Toulouse Cedex 9, France, and
** Laboratoire d'immunohématologie, Etablissement
Français du Sang, Avenue de Grande Bretagne, BP 3210, 31027 Toulouse, France
Received for publication, November 25, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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Blood group A and B antigens are carbohydrate
structures that are synthesized by glycosyltransferase enzymes. The
final step in B antigen synthesis is carried out by an Human blood group A and B antigens are produced by
glycosyltransferase enzymes that catalyze the transfer of a
monosaccharide from a nucleotide donor to Fuc cis-AB enzymes are rare dual specificity hybrid enzymes
capable of utilizing either donor. Several natural and recombinant cis-AB enzymes have been characterized with interchanges in
the four amino acids such as AAAB (Arg-176, Gly-235, Leu-266, and Ala-268) (8), ABBA, AABA and BBBA, and BABA. Two other
cis-AB enzymes result from point mutations of GTB: one at
codon 266 (M266L) (9) and one at position 234 (P234S) (10). Another
mutation at position 234 (P234A) has been reported to modify the
specificity of the GTB to transfer not only Gal but also small amounts
of GalNAc leading to a phenotype called B(A) (11). To elucidate the
structural basis of functional modification induced by the P234S
replacement, recombinant GTB P234S was characterized by kinetic and
x-ray diffraction studies.
Cloning and Characterization of P234S--
The original GTB and
GTA gene sequences (amino acids 54-354) were described previously (12,
13). In this study, GTB (amino acids 63-354) and GTA (amino acids
63-354) are denoted as wild type and were constructed by PCR using the
original GTA and GTB (amino acids 54-354) clones as templates. The
forward primer MIN2 (5'-ATA TGA ATT CAT GGT TTC CCT GCC GCG
TAT GGT TTA CCC GCA GCC GAA-3') introduced an EcoRI site at
the 5' end, and the reverse primer PCR3B (5'-ATA ATT AAG
CTTCTA TCA CGG GTT ACG AAC AGC CTG GTG GTT TTT-3') introduced a
HindIII site in the 3' end of GTA and GTB genes. The
amplified genes were cloned into the expression vector pCW
The P234S GTB mutant was constructed by directed mutagenesis using PCR
(15) and GTB (residues 63-354) plasmid DNA as a template. Two
fragments were amplified with Pfx DNA polymerase
(Invitrogen) by using the forward primer MIN2 together with SM01
(5'-GTA GAA GCT gga GTG CAG GGT ACC GAA CAG CGG-3') and the reverse
primer PCR3B with SM02 (5'-CTG CAC tcc AGC TTC TAC GGT TCC TCC CGT GAA G-3'). SM01 and SM02 were designed so that the two fragments overlap with each other and have a single codon substitution (CCC to TCC) at
codon 234 (lowercase letters). The two overlapping fragments were
isolated, annealed by 3' extension using PCR, and amplified using the
outside primers MIN2 and PCR3B. The resulting fragment containing the
desired mutation was digested with EcoRI and
HindIII (underlined in the previous section) and
inserted into the corresponding sites of pCW
Both wild type and mutant enzymes were expressed and purified
from E. coli as described previously (16) except 50 mM MOPS buffer, pH 7.0, containing 5 mM UDP,
0.5 M NaCl, and 1 mM dithiothreitol were used
for elution from the UDP-hexanolamine affinity column, and the enzyme
was concentrated in a Centriplus 30 filtration unit (Amicon). The P234S
mutant had expression levels of 40 mg/liter of culture compared with
wild type GTB and GTA with expression levels of 50-100 mg/liter.
Protein concentrations were estimated with a Bio-Rad protein assay
procedure that is based on the method of Bradford using bovine gamma
globulin as a protein standard (17). SDS-PAGE was used to confirm homogeneity.
Steady-state kinetic studies were carried out on all enzymes using a
Sep-Pak radiochemical assay using the hydrophobic acceptor Fuc Crystallography--
P234S was crystallized using conditions
similar to the native GTB enzyme (4). Data were collected at beamline
X8C at the National Synchrotron Light Source at Brookhaven National
Laboratories under cryogenic conditions using a wavelength of 1.15 Å.
Data sets for P234S both in the presence and absence of acceptor were solved using native GTB (Protein Data Bank accession code 1LZ7) as a
starting model for rigid body refinement using the program CNS (19).
Data collection and refinement statistics are presented in Table
II.
The reported structures of GTA and GTB are virtually identical
except for the four critical amino acid residues (4). These enzymes
represent a paradigm for structure-function relationships since changes
in only four amino acids alter enzyme specificity. Of these residues,
Arg/Gly-176 is far from the active site, Gly/Ser-235 appears in close
vicinity to the acceptor binding site, and Leu/Met-266 and Gly/Ala-268
are both within the donor recognition pocket. The P234S mutation is
striking in that this one mutation of a single residue in GTB results
in the near abolishment of B donor (UDP-Gal) transfer and a large
increase in A donor (UDP-GalNAc) transfer activity.
The results of kinetic analysis with UDP-Gal and UDP-GalNAc for wild
type GTB, P234S GTB, and wild type GTA are presented in Table I. For
the P234S GTB mutant, the kcat for UDP-Gal has decreased from the 5.1 s The crystal structure of the mutant enzyme provides a structural basis
for the reversal in enzyme donor preference and weakened acceptor
binding. Fig. 1 shows the position of
residue 234 in P234S and wild type GTB relative to two of the residues
that normally serve to differentiate between the donor sugars. It is
clear that in the mutant enzyme the replacement of proline by serine
creates a void at the proline C-1-3
galactosyltransferase (GTB) that transfers galactose from UDP-Gal to
type 1 or type 2,
Fuc1
2
Gal-R (H)-terminating acceptors.
Similarly the A antigen is produced by an
1-3
N-acetylgalactosaminyltransferase that transfers
N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human
1-3 N-acetylgalactosaminyltransferase and GTB are
highly homologous enzymes differing in only four of 354 amino acids
(R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction
studies have shown that the latter two of these amino acids are
responsible for the difference in donor specificity, while the other
residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B
cell surface structures. It had codons corresponding to GTB with a
single point mutation that replaced the conserved amino acid proline
234 with serine. Active enzyme expressed from a synthetic gene
corresponding to GTB with a P234S mutation shows a dramatic and
complete reversal of donor specificity. Although this enzyme contains
all four "critical" amino acids associated with the production of
blood group B antigen, it preferentially utilizes the blood group A
donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The
crystal structure of the mutant reveals the basis for the shift in
donor specificity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-2Gal
- R
(H) acceptor substrates (1, 2). The A-synthesizing
1-3
N-acetylgalactosaminyltransferase (GTA,1 EC 2.4.1.40,
glycoprotein-fucosylgalactoside
-N-acetylgalactosaminyltransferase) transfers
GalNAc from UDP-GalNAc to H-terminating acceptors producing the A
antigen GalNAc
1-3[Fuc
1-2]Gal
-R. The B-synthesizing
1-3 galactosyltransferase (GTB, EC 2.4.1.37,
glycoprotein-fucosylgalactoside
-galactosyltransferase)
utilizes UDP-Gal as its donor producing Gal
1-3[Fuc
1-2]Gal
-R. GTA and GTB are highly homologous
enzymes, differing at only four amino acids of 354 (3). Changing these four amino acids, R176G, G235S, L266M, and G268A, alters the
specificity of transfer from that of GTA to GTB. X-ray diffraction
studies of crystals of GTA and GTB and their complexes with H-acceptor and UDP have revealed the basis for donor and acceptor specificity (4).
Both residues 266 and 268 are involved in recognition of the donor
monosaccharide with Leu/Met-266 being primarily responsible for
the discrimination between GalNAc and Gal. Donor specificity is not
absolute since small levels of crossover reactions have been observed
where GTA is able to use UDP-Gal to synthesize B antigens at about
0.4% the rate of UDP-GalNAc transfer (5, 6). Similarly GTB can slowly
synthesize the A antigen, and A antigen structures have been observed
on normal group B red blood cells (7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
lac (pCW
was a gift of F. W. Dahlquist (14), and pCW
lac was a gift of W. Wakarchuk), transformed into Escherichia coli BL21
(Novagen), and characterized by DNA sequence analysis.
lac.
1-2Gal
-O(CH2)7CH3 (6,
18). Assays were carried out at 37 °C in a total volume of 12 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 and acceptor were
used, and the amount of substrate consumed was less than 15% to ensure
linear initial reaction rates. Data were analyzed as described
previously (6, 13) for a general two-substrate reaction using Equation 1, .0
where [A] and [B] represent the
concentration of acceptor and donor, respectively.
KA is the Michaelis constant for acceptor, KB is the Michaelis constant for donor, and
Kia is the dissociation constant for acceptor
(Table I). The high Km
values for the P234S mutant with UDP-Gal precluded complete
two-substrate analysis therefore the Km for UDP-Gal
was determined at 15 mM acceptor and the
Km for acceptor was determined at 500 µM UDP-Gal donor.
(Eq. 1)
Effects of Pro-234 to Ser mutation on the catalytic properties of GTB
Data collection and refinement statistics for P234S GTB mutant grown in
the absence and presence of H-antigen
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 of wild type GTB to 0.24 s
1. This is comparable to 0.088 s
1, the
kcat of the wild type GTA cross-reaction
utilizing UDP-Gal as a donor. While the binding of UDP-Gal has been
affected with an increase in the Km for UDP-Gal from
27 µM of wild type GTB to 106 µM for the
mutant, a much larger effect is seen on acceptor binding. There is a
50-fold increase in acceptor Km compared with that
of wild type GTB. For P234S, kcat for UDP-GalNAc is 14.4 s
1, comparable to the kcat
of wild type GTA (17.5 s
1). The binding of UDP-GalNAc to
the mutant has been affected somewhat with a Km of
167 µM and Kib of 49 µM. Acceptor binding to P234S has been dramatically
affected with over a 300-fold increase in both Km
and Kia.
position. This atom normally makes
van der Waals contact with Met-266 in wild type GTB, and its
removal enables Met-266 to alter its side chain orientation. This
conformational change provides sufficient space to accommodate the
N-acetyl group of UDP-GalNAc and also creates an unfavorable
void and lack of enzyme-substrate complementarity in the donor-binding
pocket for UDP-Gal, analogous to the void observed in GTA recognition
of UDP-Gal (4).
View larger version (26K):
[in a new window]
Fig. 1.
Superposition of the structures of native GTB
(blue) and P234S (orange). The
substitution of P234S changes the conformation of Met-266, which allows
the preferential binding of the blood group A donor UDP-GalNAc (modeled
in yellow). A water molecule (cyan) observed to
be hydrogen bonded to Ser-234 in the unliganded mutant structure
must be displaced upon binding of the H-antigen acceptor
(white), which accounts for the increased
Km for acceptor binding observed for the P234S
mutant.
Acceptor binding is also affected in P234S. The movement of the Met-266 side chain results in the loss of its van der Waals contacts with the acceptor moiety. Also the mutation of P234S creates a new van der Waals contact between the serine hydroxyl group and the C-6 methyl group of the fucose ring of the acceptor. Significantly the mutation to serine causes the ordering of a water molecule about Ser-234, which must be displaced in order for acceptor to bind. The effect of these changes is to weaken the binding of acceptor for reaction with either UDP-Gal or UDP-GalNAc donors. In addition, the point mutation at residue 234 occurs in the immediate vicinity of the critical residue Ser-235 that is thought to act on the acceptor by forcing the aliphatic tail to adopt different conformations between GTA and GTB. We have previously speculated that this residue may be responsible for selecting the glycoconjugate structures possessing the H-antigen acceptor (4). It is therefore plausible that changes introduced at the neighboring residue 234 would influence acceptor binding and ultimately the behavior of the mutant. Further, in the production of GTA/GTB hybrid enzymes the placement of a Ser residue (as in GTB) instead of a Gly residue (as in GTA) at position 235 results in weaker acceptor binding (13). The presence of tandem serine residues may further destabilize acceptor binding via steric and local charge distribution effects.
There are two examples reported of glycosyltransferases with altered
donor specificity for a single amino acid replacement. In
1-4-galactosyltransferase, mutation of tyrosine 289 to leucine gave
an enzyme that is capable of transferring GalNAc at a rate comparable
to Gal (20). The x-ray structure of the mutant showed that there was
sufficient room for the enzyme to accommodate the N-acetyl
group of the alternate donor. For
1-3-glucuronosyltransferase, mutation of histidine 308 to arginine produces an enzyme that can
efficiently utilize UDP-Glc, UDP-Man, and UDP-GlcNAc as well as
UDP-GlcA donor (21). However, unlike these examples of broadened donor
specificity, the P234S mutation in glycosyltransferase B results in a
complete reversal of donor saccharide recognition. The reason for the
difference in behavior of cloned P234S, which produces predominantly
blood group A structures, and the phenotype of red blood cells of
individuals with this mutation that react with both anti-A and anti-B
reagents is not clear. The cloned enzyme is a truncated, soluble form,
whereas full-length membrane-associated enzyme biosynthesizes cell
surface structures. Cell surface blood group structures are also found
on glycoproteins, whereas a lipid-like acceptor was used to
characterize the cloned enzyme.
This study highlights the dramatic effect of a single amino change on
the catalytic reaction of a glycosyltransferase enzyme that ultimately
affects blood group structure. We have shown that residues adjacent to
the four conserved amino acids in GTA and GTB can dramatically
influence the specificity of A and B transferases by inducing
conformational changes that alter the specificity and kinetics of these
enzymes. The enzymology and structure of other point mutations at
Pro-234 of both GTA and GTB enzymes are currently under investigation.
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ACKNOWLEDGEMENTS |
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Assistance with DNA sequencing was provided
by the Molecular Biology Service Unit, Department of Biological
Sciences, University of Alberta. We thank F. W. Dahlquist for the
pCW vector, W. Wakarchuk for the pCWlac vector, and O. Hindsgaul for
the Fuc
1-2Gal
-O(CH2)7CH3 acceptor.
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FOOTNOTES |
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* This work was supported by the Natural Sciences and Engineering Research Council of Canada (to M. M. P.), the Canadian Institutes of Health Research (to S. V. E.), and the Etablissement Français du Sang and Ministère Français de la Recherche (Contract E.A. 3034 to A. B. and F. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
780-492-0377; E-mail: monica.palcic@ualberta.ca.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212002200
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ABBREVIATIONS |
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The abbreviations used are:
GTA, 1-3
N-acetylgalactosaminyltransferase;
GTB,
1-3
galactosyltransferase;
MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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1. | Watkins, W. M. (1980) Adv. Hum. Genet. 10, 379-385 |
2. | Palcic, M. M., Seto, N. O. L., and Hindsgaul, O. (2001) Transfus. Med. 11, 315-323[CrossRef][Medline] [Order article via Infotrieve] |
3. | Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990) Nature 345, 229-233[CrossRef][Medline] [Order article via Infotrieve] |
4. | Patenaude, S. I., Seto, N. O. L., Borisova, S. N., Szpacenko, A., Marcus, S. L., Palcic, M. M., and Evans, S. V. (2002) Nat. Struct. Biol. 9, 685-690[CrossRef][Medline] [Order article via Infotrieve] |
5. | Greenwell, P., Yates, A. D., and Watkins, W. M. (1986) Carbohydr. Res. 149, 149-170[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Seto, N. O. L.,
Palcic, M. M.,
Compston, C. A.,
Li, H.,
Bundle, D. R.,
and Narang, S. A.
(1997)
J. Biol. Chem.
272,
14133-14138 |
7. | Goldstein, J., Lenny, L., Davies, D., and Voak, D. (1989) Vox Sang. 57, 142-146[Medline] [Order article via Infotrieve] |
8. | Yamamoto, F., McNeill, P. D., Kominato, Y., Hakomori, S., Ishimoto, S., Nishida, S., Shima, M., and Fujimura, Y. (1993) Vox Sang. 64, 120-123[Medline] [Order article via Infotrieve] |
9. | Mifsud, N. A., Watt, J. M., Condon, J. A., Haddad, A. P., and Sparrow, R. L. (2000) Transfusion 40, 1276-1277[CrossRef][Medline] [Order article via Infotrieve] |
10. | Roubinet, F., Janvier, D., and Blancher, A. (2002) Transfusion 42, 239-246[CrossRef][Medline] [Order article via Infotrieve] |
11. | Yu, L. C., Lee, H. L., and Lin, M. (1999) Biochem. Biophys. Res. Commun. 262, 487-493[CrossRef][Medline] [Order article via Infotrieve] |
12. | Seto, N. O. L., Palcic, M. M., Hindsgaul, O., Bundle, D. R., and Narang, S. (1995) Eur. J. Biochem. 234, 323-328[Abstract] |
13. |
Seto, N. O. L.,
Compston, C. A.,
Evans, S. V.,
Bundle, D. R.,
Narang, S. A.,
and Palcic, M. M.
(1999)
Eur. J. Biochem.
259,
770-775 |
14. | Gegner, J. A., and Dahlquist, F. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 750-754[Abstract] |
15. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1997) Current Protocols in Molecular Biology , Vol. 1 , pp. 8.5.7-10, John Wiley and Sons, Inc., New York |
16. | Seto, N. O. L., Compston, C. A., Szpacenko, A., and Palcic, M. M. (2000) Carbohydr. Res. 324, 161-169[CrossRef][Medline] [Order article via Infotrieve] |
17. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
18. | Palcic, M. M., Heerze, L. D., Pierce, M., and Hindsgaul, O. (1988) Glycoconj. J. 5, 49-63 |
19. | Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Ramakrishnan, B.,
and Qasba, P. K.
(2002)
J. Biol. Chem.
277,
20833-20839 |
21. |
Ouzzine, M.,
Gulberti, S.,
Levoin, N.,
Netter, P.,
Magdalou, J.,
and Fournel-Gigleux, S.
(2002)
J. Biol. Chem.
277,
25439-25445 |