Acceptor specificity of Salmonella GDP-Man:αLRha1→3αDGal-PP-Und β1→4-mannosyltransferase: A simplified assay based on unnatural acceptors [16]

Full text of DOI:10.1021/ja982285+

12986
J. Am. Chem. Soc. 1998, 120, 12986-12987
Scheme 1. Salmonella Group E1 O Antigen Biosynthesis
Acceptor Specificity of Salmonella
GDP-Man:rLRha1f3rDGal-PP-Und
â1f4-Mannosyltransferase: A Simplified Assay
Based on Unnatural Acceptors
(upper) and the Corresponding Unnatural ManTâ4 Acceptors
(lower)a
Yongxin Zhao, John B. Biggins, and Jon S. Thorson*
Laboratory for Biosynthetic Chemistry
Molecular Pharmacology & Therapeutics Program
Memorial Sloan-Kettering Cancer Center and
the Sloan-Kettering DiVision
Graduate School of Medical Sciences
Cornell UniVersity, 1275 York AVenue, Box 309
New York, New York 10021
ReceiVed June 30, 1998
The first unique mannosyl transfer in the biosynthesis of the
common core of most Asn-linked eukaryotic glycoproteins is
catalyzed by GDP-R-D-Man:GlcNAcâ1f4GlcNAcâ1f4-PP-Dol
â1f4-mannosyltransferase.1,2 Unfortunately, due to low expres-
sion levels and enzyme lability, the chemical mechanism for this
key reaction remains undetermined. Interestingly, dolichol-
dependent biosynthesis shares striking similarities with bacterial
undecaprenol-dependent glycosylation.3 This relationship, in
conjunction with the superior biochemical and genetic manipu-
latability of bacterial systems, renders prokaryotic mannosyl-
transferases attractive as accessible models for their eukaryotic
counterparts. One such model mannosyltransferase, GDP-R-D-
Man:RRha1f3RGal-PP-Und â1f4-mannosyltransferase (ManTâ4)
is involved in the biosynthesis of the Salmonella group E1
O-antigen repeat unit (2, Scheme 1).4 The biosynthesis of the
natural acceptor for this enzyme, RRha1f3RGal-PP-Und (1), is
initiated by enzyme-catalyzed galactosyl-1-phosphate transfer
from UDP-Gal to undecaprenol phosphate,5 with subsequent loss
of UMP, followed by enzyme-catalyzed rhamnosyl transfer from
TDP-Rha and loss of TDP.6 Of the reagents required for the
biosynthesis of 1, UDP-R-D-Gal:P-Und R-galactose-1-phosphoryl
transferase (Gal-1-PT), TDP-â-L-Rha:Gal-PP-Und 1f3-rham-
nosyltransferase (RhaTR3) and TDP-â-L-Rha are not commercially
available.7 Thus, we report the synthesis of simplified analogues
of 1 (Scheme 1) and compare their abilities to function as ManTâ4
acceptors. In addition to providing a new method for the practical
synthesis of Gram negative cell wall antigens,8 this work reveals
a new quantitative ManTâ4 assay and begins to map acceptor
requirements for this intriguing enzyme.
a Gal-1-PT/UDP-Gal, RhaTR3/TDP-Rha. (b) ManTâ4/GDP-Man. (c)
AgOTf, 1,1,3,3-tetramethylurea, 4-(4-nitrophenyl)-1-butanol, CH2Cl2, 0
°C. (d) NaOMe, MeOH, 20 min. (e) AgOTf, 2,6-di-tert-butylpyridine,
CH2Cl2, -40 °C. (f) Ac2O, pyridine. (g) DMDO, 0 °C, CH2Cl2; 4-(4-
nitrophenyl)-1-butanol, ZnCl2, -78 °C f 20 °C, THF. (h) TBAF, THF.
) 9:1), from bromide 410 and 4-(4-nitrophenyl)-1-butanol, fol-
lowed by Ze´mplen deacylation efficiently provided 6 (85%).
Synthesis of the disaccharide analogue 12 was initiated with
Koenigs-Knorr coupling of glycal 711 and 4 to give 8 (69% R,
R/â ) 10:1).12 The corresponding glycal 9 was epoxidized using
3,3-dimethyldioxirane13 to provide the 1,2-anhydro derivative,
which furnished crystalline 10 (43% R, R/â ) 1:9) in the presence
of zinc chloride and 4-(4-nitrophenyl)-1-butanol.14 Deprotection
provided the potential mannosyltransferase substrate 12 (84% R,
R/â ) 12:1). To test the potential acceptors, 3, 6, and 12 were
individually incubated with GDP-R-D-Man or GDP-R-D-[U-14C]-
Man and extracts from a ManTâ4 overexpressing strain,15 and the
reaction progress was monitored by HPLC.16
Assays of ManTâ4 extracts with 3 or 6 revealed no observed
ManTâ4-catalyzed glycosylation. However, assays containing 12
(retention time of 13.90 min.) unveiled the time-dependent
formation of a new product with a retention time of 12.99 min
(Figure 1a). Molecular weight determination of the new product
was consistent with mannosylation of 12,17 and assays containing
12 in the presence of GDP-[U-14C]Man demonstrated the time-
dependent incorporation of [U-14C]Man into the product peak
The first analogue, p-nitrophenyl-â-L-Rha (3), is commercially
available, whereas Koenigs-Knorr9 synthesis of 5 (64% R, R/â
* To whom correspondence should be addressed. E-mail: jthorson@
sbnmr1.ski.mskcc.org. Fax: (212) 717-3066.
(1) Abbreviations: Asn, asparagine; Dol, dolichol; Gal, D-galactose; GDP,
guanosine diphosphate; Glc, D-glucose; GlcNAc, 2-N-acetyl-D-glucose; Man,
D-mannose; Rha, L-rhamnose (6-deoxy-L-mannose); TDP, thymidine diphos-
phate; UDP, uridine diphosphate; UMP, uridine monophosphate; Und,
undecaprenol.
(2) (a) Hubbard, S. C.; Ivatt, R. J. Annu. ReV. Biochem. 1981, 50, 555-
583. (b) Kornfeld, R.; Kornfeld, S. Annu. ReV. Biochem. 1985, 54, 631-664.
(c) Manzella, S. M.; Hooper, L. V.; Baenziger, J. U. J. Biol. Chem. 1996,
271, 12117-12120. (d) O’Connor, S. E.; Imperiali, B. Chem. Biol. 1996, 3,
803-812.
(3) Bugg, T. D. H.; Brandish, P. E. FEMS Microbiol. Lett. 1994, 119, 255-
262.
(9) Banoub, J.; Bundle, D. R. Can. J. Chem. 1979, 57, 2091-2097.
(10) (a) Finan, P. A.; Warren, C. D. J. Chem. Soc. 1962, 2823-2824. (b)
Farkas, I.; Menyhart, M.; Bognar, R.; Gross, H. Chem. Ber. 1965, 98, 1419-
1426.
(11) (a) Gordon, D. M.; Danishefsky, S. J. J. Am. Chem. Soc. 1992, 114,
659-663. (b) Gervay, J.; Peterson, J. M.; Oriyama, T.; Danishefsky, S. J. J.
Org. Chem. 1993, 58, 5465-5468.
(12) The use of 6-O-triisopropylsilyl greatly influenced regioselectivity,
and the use of di-tert-butylpyridine gave slightly higher yields than either
2,4,6-collidine (50-55%) or 1,1,3,3-tetramethylurea (40%). Peracetylated
rhamnal or pertriisopropylsilylated rhamnal failed to give a stable epoxide
product with DMDO. Our rationale to synthesize 12, as opposed to the
4-nitrophenyl disaccharide (not tested), was to provide both a hydrophobic
handle reminiscent of 1 and stability to chromophore hydrolysis under alkaline
assay conditions.
(4) (a) Liu, D.; Haase, A. M.; Lindqvist, L.; Lindberg, A. A.; Reeves, P.
R. J. Bacteriol. 1993, 175, 3408-3413. (b) Wang, L.; Romana, L. R.; Reeves,
P. R. Genetics 1992, 130, 429-443.
(5) Wang, L.; Reeves, P. R. J. Bacteriol. 1994, 176, 4348-4356.
(6) (a) Raetz, C. R. H. Annu. ReV. Biochem. 1990, 59, 129-170. (b) Jiang,
X.-M.; Neal, B.; Santiago, F.; Lee, S. J.; Romana, L. K.; Reeves, P. R. Mol.
Microbiol. 1991, 5, 695-713. (c) Schnaitman, C. A.; Kiens, J. P. Microbiol.
ReV. 1993, 57, 655-682.
(13) Halcomb, R. L.; Danishefsky, S. J. J. Am. Chem. Soc. 1989, 111,
6661-6666.
(14) Galactal coupling in the absence of rhamnose reproducibly furnished
[4-(nitrophenyl)-1-butyl]-â-D-galactopyranoside in >90% yield.
(7) Zhao, Y.; Thorson, J. S. J. Org. Chem. 1998, 63, 7568-7572.
(8) (a) Robbins, P. W.; Uchida, T. J. Biol. Chem. 1965, 240, 375-383.
(b) Luderitz, O.; Westphal, O. Ang. Chem., Int. Ed. Engl. 1966, 5, 198-210.
10.1021/ja982285+ CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/01/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 49, 1998 12987
for the first 30 min (Figure 1c).18 In addition, control reactions
containing 12, GDP-Man, and pET11a-BL21 extracts (identical
Escherichia coli extracts but lacking ManTâ4 expression) or 12
and ManTâ4 extracts (but lacking GDP-Man) showed no change
over 120 min (data not shown), indicating that the mannosylation
of 12 is clearly dependent upon the expression of both the
exogenous ManTâ4 and GDP-Man. Finally, the newly formed
trisaccharide could only be cleaved by commercially available
â-mannosidases, confirming the Man-Rha linkage as beta.19
The advantage of the strategy which we present over existing
routes to 3-O-R-L-rhamnopyranosyl-D-galactopyranosides20 is in
providing a 3-O-R-L-rhamnopyranosyl-D-galactal which can be
readily derivatized from the reducing end. We have exploited this
strategy to provide a simplified chromophoric substrate which
allows for the first ManTâ4 quantitative assay system. Cumula-
tively, the presented results are consistent with the ManTâ4-
catalyzed formation of trisaccharide 2 and therefore provide
further confirmation of the original wbaO gene assignment put
forth by Liu et al.4a The observed lack of mannosylation of 3
and 6 indicate that ManTâ4 requires at least part, if not all, of the
galactosyl moiety; however, acceptance of 12 indicates that
ManTâ4 does not require the Und-PP portion of the native acceptor
1.
Acknowledgment. We gratefully acknowledge Professor Peter R.
Reeves (Department of Microbiology, University of Sydney) for gra-
ciously providing plasmid pPR1330 and Dr. George Sukenick (NMR Core
Facility, Sloan-Kettering Institute) for NMR and mass spectral analyses.
J.S.T. is a Rita Allen Foundation Scholar. This work was supported in
part by the Cancer Center Support Grant (CA-08748) and a grant from
the Special Projects Committee of The Society of Memorial Sloan-
Kettering Cancer Center.
Supporting Information Available: Experimental details and char-
acterization data for 4-6, 8-12 (3 pages, print/PDF). See any current
masthead page for ordering information and Web access instructions.
JA982285+
(16) A typical assay consisted of 10 mM acceptor, 10 mM GDP-R-D-Man,
10 mM MgCl2, 25 µL crude extracts in a total volume of 50 µL. After an
appropriate incubation period at 37 °C, an equal volume of MeOH was added,
denatured proteins were removed by centrifugation, and 20 µL was analyzed
by reverse-phase chromatography (Microsorb-MV C-18, 4.6 × 250 mm, 75%
10 mM NaH2PO4, 25% CH3CN, pH 7.5, 0.75 mL min-1, λ ) 325 nm). The
ꢀ325 for 3 (7820 M-1 cm-1), 6 (2340 M-1 cm-1), and 12 (2310 M-1 cm-1
)
used for these assays were determined experimentally (75% 10 mM NaH2-
PO4, 25% CH3CN, pH 7.5). Radiolabeled assays in addition contained 3.3
µM GDP-R-D-[U-14C]Man and were monitored with a Packard 150TR flow
scintillation analyzer. Although the lipid moiety of 12 is inverted from that
of 1, 12 is the major synthetic product, and assays with the minor R-analogue
revealed a similar rate of ManTâ4-catalyzed mannosylation.
(17) A mixture of 1.0 mM 12, 1.2 mM GDP-Man, 10 mM MgCl2, 1.2 mg
ManTâ.4 50 mM Tris-Cl, 1 mM EDTA, pH 8.5 in a total volume of 750 µL
was incubated at 37 °C for 4 h, the reaction was stopped by adding an equal
volume of methanol, and denatured protein was removed by centrifugation
(16000g, 15 min). The supernatant (0.5 mL) was chromatographed on a
Licrosorb RP-18 (7 µm) semipreparative column with a mobile phase of 25%
CH3CN, 75% H2O, flow rate of 1.5 mL min-1. The trisaccharide fraction
(20-22 min) and disaccharide fraction (24-26 min) were collected, concen-
trated, and submitted for mass spectral analysis.
Figure 1. (a) Time dependence of an assay containing 12, GDP-Man,
and ManTâ4 extracts monitored at 325 nm. (b) Time dependence of an
assay containing 12, GDP-[U-14C]Man, and ManTâ4 extracts monitored
by flow scintillation counting. (c) Plots of the disappearance of 12 (O,
from Figure 1a) and GDP-Man (0, from Figure 1b) and the corresponding
formation of the trisaccharide product (b, from Figure 1a; 9, from Figure
1b).
(18) The difference of this rate versus ManTâ4-catalyzed mannosylation
of 1 (275 nM min-1) is partially attributed to technical difficulties associated
with the selective extraction of 2.7
(19) A mixture of 1.0 mM 12, 1.2 mM GDP-Man, 3.32 µM GDP-R-D-
[U-14C]Man, 10 mM MgCl2, 960 µg ManTâ.4 50 mM Tris-Cl, 1 mM EDTA,
pH 8.5 in a total volume of 500 µL was incubated at 37 °C for 4 h the reaction
stopped by adding an equal volume of methanol and denatured protein removed
by centrifugation (16000g, 15 min). The recovered supernatant was evaporated
and the corresponding dried reaction mixture dissolved in 180 µL of 200 mM
NaH2PO4, 1 mM EDTA pH 5.5 buffer and separated into three equal aliquots.
To the first was added 0.5 U of R-mannosidase (Sigma), to the second was
added 0.04 U of â-mannosidase (Sigma), and to the third buffer was added
as a control. After incubation at 37 °C for 4 h, the reactions were analyzed by
HPLC as previously described.16 Although this result confirms the postulated
ManTâ4 stereoselectivity,4 the confirmation of the postulated regioselectivity
is currently in progress.
(20) (a) Betaneli, V. I.; Ovchinnikov, M. V.; Backinowsky, L. V.;
Kochetkov, N. K. Carbohydr. Res. 1980, 84, 211-224. (b) Lipta´k, A.;
Szurmai, Z.; Nanasi, P. Carbohydr. Res. 1982, 99, 13-21. (c) Bundle, D. R.;
Eichler, E.; Gidney, M. A. J.; Meldal, M.; Ragauskas, A.; Sigurskjold, B.
W.; Sinnott, B.; Watson, D. C.; Yaguchi, M.; Young, N. M. Biochemistry
1994, 33, 5172-5182.
(Figure 1b). A plot of the peak area versus time from Figure 1a
or b revealed the time-dependent formation of the new product
(A325 ) 18.0 µM min-1; [14C] ) 15.0 µM min-1) and the decrease
of 12 (A325 ) 23.0 µM min-1; [14C] ) 13.0 µM min-1) are linear
(15) PCR amplified wbaO (encoding ManTâ4) from pPR13304b was cloned
into a T7-driven pET11a-BL21 system, grown to an OD600 ) 0.55 at 37 °C
with shaking (250 rpm), the growth temperature reduced to 20 °C, and protein
expression induced with 1.0 mM IPTG. Rifampicin (75 µg mL-1) was added
2 h after induction, and the cultures were allowed to grow for an additional
12-14 h. Cells were harvested (2000g, 15 min), resuspended in 50 mM Tris-
HCl, 1 mM EDTA, pH 8.5, and lysed by sonication. The cellular debris was
removed by centrifugation (2000g, 15 min), and the membrane fractions were
isolated from the supernatant by centrifugation (30000g, 1 h), resuspended in
50 mM Tris-HCl, 1 mM EDTA, pH 8.5, and stored at -80 °C until used.
SDS-PAGE revealed the desired ManTâ4 as approximately 70-80% of the
total membrane-associated protein.