Dependence of the Bi-functional Nature of a Sialyltransferase from Neisseria meningitidis on a Single Amino Acid Substitution*

Warren W. WakarchukDagger, David Watson, Frank St. Michael, Jianjun Li, Yuyang Wu, Jean-Robert Brisson, N. Martin Young, and Michel Gilbert

From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada, K1A 0R6

Received for publication, December 14, 2000, and in revised form, January 17, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha -linked galactose from a Pk epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633). Using a synthetic Pk-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both alpha -2,3 and alpha -2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the alpha -2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha -2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha -2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the alpha -2,6-sialyltransferase activity in vitro as well as in vivo.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mucosal pathogens in the genera Neisseria, Campylobacter, and Haemophilus all possess a major cell surface lipooligosaccharide (LOS)1 containing N-acetylneuraminic acid (Neu5Ac) (2-4). These carbohydrate structures are not a static feature of the bacterium's outer surface but are produced with significant variation depending on the genetic makeup of the strain and the environment in which it is growing (5-7). This phenomenon is thought to play an important role in the pathogenesis of these organisms (8) (9), relating as it does to the structural similarity of the terminal oligosaccharide portion of the LOS to those found on human glycolipids. In Neisseria a major structure is a terminal alpha -2,3-sialyllacto-N-neotetraose (Fig. 1), in Haemophilus influenzae strain RM118, the LOS structures include alpha -2,3-sialyllactose (7), and in the NTHi 375 strain there is a simple disialic acid structure (perhaps related to the ganglioside GD3) (4). In Campylobacter jejuni there are structures similar to various gangliosides, from GD3 to GT1a (10). Such oligosaccharides provide these pathogens with a means of evading the host immune response through molecular mimicry as well as providing ligands for binding to receptors on human cells (11), and they are therefore potent virulence factors (12).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.   Outer core oligosaccharides from N. meningitidis MC58(L3) and 126E(L1). The genes for the glycosyltransferases used for each addition have been identified, and their names are shown above each sugar residue (14, 18, 23).

The genetic analysis of the biosynthesis of LOS has shown that variation in the oligosaccharide portion of the LOS can arise in several ways. In H. influenzae, some LOS biosynthesis genes including glycosyltransferases are inactivated through a mechanism involving a variable number of tandem tetranucleotide repeats, which produces a phase-variable LOS phenotype (13). A related phase variation mechanism is used in Neisseria meningitidis, Neisseria gonorrhoeae, and C. jejuni, where changes in the length of polynucleotide tracts in certain glycosyltransferase genes leads to gene inactivation, which in turn allows alternate oligosaccharides to be produced (14-16). In addition to these mechanisms, a new source of structural diversity exists in C. jejuni, where the sialyltransferase Cst-II from the OH4384 strain has been shown to be bi-functional, using two different acceptor oligosaccharides and has been shown to be mono-functional in the 0:19 serostrain (17). The existence of these different mechanisms of structural variation makes it impossible to predict the LOS structure based on the gene complement alone.

The N. meningitidis alpha -2,3-sialyltransferase, Lst, has a relaxed acceptor specificity, being able to use synthetic acceptors that present terminal N-acetyllactosamine, lactose, or galactose (18). Furthermore, the Lst from N. meningitidis strains MC58(L3) and 126E(L1) can also use a terminal alpha -D-galactose to make an alpha -2,3-sialyl-Pk epitope in vitro with a synthetic acceptor molecule (19). We have previously demonstrated that the LOS of the N. meningitidis 126E(L1) was also sialylated but at the O6 position of the terminal alpha -D-galactose (1). To our knowledge, this structure has not been reported in any mammalian glycolipid (nor in any other LOS); hence, we cannot conclude that it is used as a form of molecular mimicry as is the case with the sialyllacto-N-neotetraose found in the strains with the L3 immunotype. The L1 immunotype has been isolated from disease outbreaks (20), and therefore, this unique LOS structure may play a role in meningococcal disease. We therefore sought to determine the enzyme responsible for its biosynthesis.

In this paper we demonstrate the that the Lst of 126E(L1) is also responsible for the addition of the Neu5Ac at O6 of the alpha -galactose in the 126E(L1) LOS. Additionally, the bi-functional nature of the enzyme from the 126E(L1) can be introduced into the mono-functional MC58(L3) enzyme by site-directed mutagenesis of a single residue.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains and Plasmids-- The following strains of N. meningitidis were used: immunotype L1 strain 126E (NRCC 4010); immunotype L3 strain MC58 (NRCC 4728). The cloning vector pCW was used for expression of the lst genes and has been described previously (21). Plasmids were propagated in Escherichia coli strain AD202 (CGSC 7297).

Preparation and Analysis of Lipooligosaccharide-- N. meningitidis 126E(L1) was grown and harvested, and the LOS was extracted by the hot phenol-water method as previously described (1), except that the plates were supplemented with 25 µg/ml CMP-Neu5Ac. Small scale isolations were performed as described previously (22). Capillary electrophoresis-mass spectrometry of LOS was performed as previously described (1, 22). Methylation analysis to determine linkage positions between residues was performed as previously described (1).

Measurement of Sialyltransferase Activity-- For detection of activity in N. meningitidis strains, cells were scraped off freshly grown Columbia blood agar plates that had been supplemented with 25 µg of CMP-Neu5Ac/ml. The cell pellet was then extracted with 0.2% Triton X-100 in 20 mM Tris HCl, pH 8.0. The cell-free extract was then assayed as previously described (19) with acceptor molecules derived from aminophenylglycosides labeled either with FCHASE or FEX fluorophors. Analysis of all of the site-directed mutants was performed with a construct that contained only the transferase gene. The recombinant proteins were assayed in sonicated extracts. The thin layer chromatography conditions for separation of the alpha -2,3/2,6-sialylated Pk-FCHASE were: isopropanol, n-butyl alcohol, 0.1 M HCl (2:1:1) or ethyl acetate, methanol, water, acetic acid (4:2:1:0.1). High performance TLC plates (Whatman) were used for the separations.

Genetic Manipulations of the 126E(L1) and MC58(L3) alpha -2,3-Sialyltransferase-- The construction of the allelic replacement was performed essentially as described previously (23). Briefly, the kanamycin resistance gene from pUC-4K was liberated from the plasmid as a SalI fragment. The 5'-upstream region and 3'-downstream regions of the alpha -2,3-sialyltransferase structural gene were amplified from N. meningitidis 126E(L1) by polymerase chain reaction with Pwo polymerase as described by the manufacturer (Roche Molecular Biochemicals). The primers for the 126E(L1) upstream-flanking sequence (5' end) of the coding regions were: 5'-GGGGGGGAATTCCATATTTTGGCGGCTTTGTTCGCGC-3' and 5'-GGCGGTGGGCATATGATCCCTAAAACTCCATTCCGACAAATTG-3'. The region is 355 base pairs. The primers for the L1 downstream-flanking sequence (3' end) of the coding sequence were 5'-GGGGGGGTCGACTCGCATAGCAAATCAAAATAGAAAATGG-3' and 5'-GGGGGGAAGCTTCCGCGCACTGCCCGCCGTTTGGTCGG-3'. The region is 350 base pairs. The coding sequence from MC58 L3 was amplified with the following primers: 5'-CTTAGGAGGTCATATGGGCTTGAAAAAGGCTTGTTTGACC-3' and 5'-GGGGGGGTCGACTTAATTTTTATCGTCAAATGTCAAAATC-3'. The fragment was 1140 base pairs. The plasmid for the gene replacement was assembled in the following steps. pUC19 was digested with EcoRI and HindIII and purified for use as the cloning vector. A four-fragment ligation was performed with the 5'-flanking sequence of 126E(L1)-lst (EcoRI/NdeI-digested), the MC58(L3)-lst structural gene (NdeI/SalI-digested), the 3'-flanking sequence of 126E(L1)-lst (SalI/HindIII), and the pUC19 vector (EcoRI/HindIII). Once this construct was verified, we then inserted a KanR marker between the structural gene and the 3'- flanking sequence. The recombinant plasmids were introduced into N. meningitidis by electroporation.

The gene disruption vector was constructed in a different fashion. The sialyltransferase gene (NdeI-SalI fragment as described above) was restricted with Sau3A-I to generate three fragments. The middle 22-base pair fragment was discarded. The two larger fragments were ligated with the kanamycin resistance marker that had been liberated from pUC-4K with BamHI. These fragments were then ligated into pUC19 which had been digested with EcoRI and SalI. These plasmids were then introduced into the N. meningitidis strain by electroporation.

Site-directed Mutagenesis-- The MC58(L3)-lst allele was mutagenized using the U-DNA method of Kunkel (24). All mutant genes were completely sequenced to ensure no other mutations were introduced.

Capillary Zone Electrophoresis-Electrospray Mass Spectrometry-- A crystal model 310 CE instrument (AYI Unicam, Madison, WI) was coupled to an API 3000 mass spectrometer (PerkinElmer Life Sciences/Sciex, Concord, Canada) via a microionspray interface. A sheath solution (isopropanol, methanol, 2:1) was delivered at a flow rate of 1 µl/min to a low dead volume tee (250 µm internal diameter, Chromatographic Specialties, Brockville, Canada). The separations were obtained on a 90-cm-length bare-fused silica capillary using 30 mM morpholine in deionized water, pH 9.0, containing 5% methanol. A voltage of 25 kV was typically applied for the separation. The outlet of the capillary was tapered to ~15 µm internal diameter using a laser puller (Sutter Instruments, Novato, CA). Mass spectra were acquired with dwell times of 3.0 ms/step of 1 m/z unit in full-mass scan mode. 20 nl of sample was typically injected by using 150 mbar for a duration of 0.1 min.

NMR Spectroscopy-- NMR experiments were performed on a Bruker AMX-600 and Varian INOVA 600 NMR spectrometers. Experiments on Pk-FCHASE compound were done using an inverse broadband detection probe at 27 °C with the mono-deuterated water resonance at 4.756 ppm. Experiments on alpha -2,6-sialyl-Pk-FCHASE compound were done using a 5-mm Z gradient triple resonance probe at 25 °C with the mono-deuterated water resonance at 4.774 ppm. NMR samples were prepared by dissolving 0.1-0.3 mg of material in 600 µl of D2O after freeze-drying. All NMR experiments were performed as previously described (17). For the proton chemical shift reference, the methyl resonance of internal or external acetone was set at 2.225 ppm (1H). For the 13C chemical shift reference, the methyl resonance of internal or external acetone was set at 31.07 ppm relative to external dioxane at 67.40 ppm.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection of alpha -2,6-Sialyltransferase Activity-- When we analyzed reactions performed with the 126E(L1)-Lst and the Pk-FCHASE acceptor, we noticed a second product peak (slightly faster migrating) in the CE electropherograms (Fig. 2), which was sensitive to sialidase treatment (data not shown). This second product is only seen with Pk-FCHASE acceptor. The major product (~90% of the product) had the same migration time as alpha -2,3-Neu5Ac-Pk-FCHASE that we had previously described (19). We were able to resolve these two products by TLC, but we could only see a second product in reactions performed with the 126E(L1)-Lst and not MC58(L3)-Lst. We were able to obtain enough of the unique secondary product using preparative TLC, and this material was subjected to analysis by NMR spectroscopy (see below).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Capillary electrophoresis analysis of MC58(L3)-Lst and 126E(L1)-Lst. Sialyltransferase reactions with both versions of the Lst enzyme were analyzed by capillary electrophoresis. The top electropherogram shows the products from the MC58(L3)-Lst reaction (solid line), and the lower electropherogram shows the products from the 126E(L1)-Lst reaction (dashed line). Peak A is the starting material Pk-FCHASE, peak B is the alpha -2,3-Neu5Ac-containing product, and peak C is the alpha -2,6-Neu5Ac-containing product. RFU, relative fluorescence units.

NMR Analysis-- To assess the linkage specificity of the 126E(L1)-Lst, the unique product formed with Pk-FCHASE as an acceptor was analyzed by NMR spectroscopy on nanomole amounts. We have previously published the NMR spectrum for alpha -2,3-Neu5Ac-Pk made with the MC58(L3)-Lst (19), and we found the NMR spectrum of the major sialylated product made with 126E(L1)-Lst was identical to our previously published one. We present here only the spectra from a nonsialylated acceptor, Pk-FCHASE = compound I, and the unique product formed in a reaction with the 126E(L1)-Lst, alpha -2,6-sialyl-Pk-FEX = compound II. The comparison of these two spectra clearly shows the site of sialylation of the terminal alpha -galactose in the acceptor. At the time the preparative sialylation reaction was performed, we were using the FEX aglycon, but we have seen that during storage that the FCHASE aglycon is more stable. The nonsialylated Pk-FCHASE spectra was collected on material made at a later time.

Both compounds were soluble and gave sharp resonances with line widths of a few Hz since the H-1 anomeric doublets (J1,2 = 4 and 8 Hz) are well resolved (data not shown). For compound I, the mono-deuterated water resonance was very broad and overlapped with one of the anomeric signals. In Table I, the proton assignments were obtained from standard homonuclear experiments, correlated spectroscopy (COSY), total correlation spectroscopy (TOSCY), nuclear Overhauser effect spectroscopy (NOESY), one-dimensional NOESY, one-dimensional TOCSY, and the 13C assignments from a heteronuclear multiple quantum coherence (HMQC) experiment, which detects C-H correlations (17).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Proton NMR chemical shifts for the fluorescent glycosides synthesized using the cloned glycosyltransferases
Data are in ppm from heteronuclear multiple quantum coherence (HMQC) spectra. The error is ±0.02 ppm for 1H chemical shifts and ±0.2 ppm for 13C chemical shifts.

The linkage site on the aglycon was determined from a comparison of the 13C chemical shifts of the enzymatic product with those of the precursor to determine glycosidation shifts, as done previously (17). Although a different fluorescent tag was used, it only influenced the proton chemical shift of the Glc residue. For compound I, the proton and 13C chemical shifts were in accord with those reported for Gal-alpha 1,4-Gal-beta 1,4-Glc-beta -1-O-2-(trimethylsilyl)ethanol (25). Compound II was identified as Neu5Ac-alpha 2,6-Gal-alpha 1,4-Gal-beta 1,4-Glc-beta -1-FEX. From a comparison of the heteronuclear multiple quantum coherence spectra of compound I and II, it is obvious that the linkage site is at C-6 of alpha Gal due to the large downfield shift of -2.6 ppm for alpha Gal C-6 upon sialylation (Fig. 3). Large proton chemical shifts differences of 0.18 and -0.17 ppm were also observed for the H-6 and H-6' resonances of alpha Gal upon sialylation. An upfield shift of 1.3 ppm for the alpha Gal C-5 was also observed (Table I), typical of 2-6 sialyloligosaccharides (26). The 13C chemical shifts of compound II are similar to those reported for the NeuN-alpha 2,6-Gal-alpha 1,4-Gal-beta 1,4-Glc moiety present in the 126E(L1) LOS from N. meningitidis, except for the chemical shift of NeuN C-4, which is obviously wrong in the latter (1). In Table I and Fig. 3, the proton chemical shift differences for the Glc unit between compounds I and II are attributed to different fluorescent aglycons.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Identification of the glycosidation site from a comparison of the heteronuclear multiple quantum coherence spectra of precursor and product glycosides. The two spectra of the precursor and enzymatic product different by one sugar are overlaid. Only one contour is drawn for each spectrum. The two cross-peaks for the same common atom in the two compounds that do not overlap are joined by a solid line. The linkage site is identified from the large 13C downfield glycosidation shift, indicated by filled cross-peaks. The proton chemical shift axis is F2, and the 13C chemical shift axis is F1. Cross-peaks are labeled using the letter a for alpha Gal, b for beta -Gal, c for Glc, d for Neu5Ac, and by the atom number as in Table I for compound I and II.

Site-directed Mutagenesis of MC58(L3)-Lst-- A sequence comparison of the 126E(L1)-Lst (GenBankTM accession U60662) and MC58(L3)-Lst (GenBankTM accession U60660) proteins showed only 6 amino acid changes. We sequentially changed all of the residues in MC58(L3)-Lst that differed from 126E(L1)-Lst to the corresponding residue from 126E(L1)-Lst (E40D, R102W, S129A, G168I, T242A, K273N). Only the G168I mutation resulted in an enzyme with alpha -2,6-sialyltransferase activity. We then performed additional mutagenesis to put all possible amino acids at the position Gly-168 in MC58(L3)-Lst to determine whether other amino acids also permit the second enzyme activity. The results of these experiments were analyzed by first measuring the activity of the mutants on beta -N-acetyllactosamine-FCHASE, the preferred acceptor for the MC58(L3)-Lst, to ensure the mutants were functional. Both Lst enzymes make only a single product, the alpha -2,3-linked Neu5Ac, with beta -N-acetyllactosamine-FCHASE. Only one of the mutations resulted in an inactive enzyme, G168P, and this was not due to lack of protein production (data not shown). These mutants were then analyzed for activity on the Pk-FCHASE acceptor using equivalent amounts of enzyme as measured on the beta -N-acetyllactosamine-FCHASE acceptor. Only a few of these had significant alpha -2,6-sialyltransferase activity, and these data is shown in Fig. 4. The mutants G168I and G168L showed levels of alpha -2,6-sialyltransferase activity equivalent to the 126E(L1)-Lst. The G168V mutant showed 40% of the alpha -2,6 activity level of 126E(L1)-Lst, and the G168M mutant showed 25% of the level of alpha -2,6 activity of 126E(L1)-Lst.


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 4.   Sialyltransferase activity of site-directed mutants of the MC58(L3)-Lst on Pk-FCHASE. The mutants were examined along with both the 126E(L1) and MC58(L3) controls. The top panel shows a thin layer chromatography analysis of all of the strains. The arrowheads indicate the migration of alpha -2,3-sialyl-Pk-FCHASE and alpha -2,6-sialyl-Pk-FCHASE. These reactions were also quantitated by capillary electrophoresis, and the percentage of the reaction product that is alpha -2,6-linked is plotted versus the mutation at position 168 of the MC58(L3) gene.

We also noted that certain mutants were less active than the wild type on Pk-FCHASE in general but basically unaffected for activity on beta -N-acetyllactosamine-FCHASE. These mutants were G168F, G168Y, and G168W, and using the standard assay described here, they showed a higher specificity for beta -N-acetyllactosamine-FCHASE than they did for Pk -FCHASE.

Production of the 126E(L1)-lst Knock-out-- An insertional inactivation mutant of the lst gene was made in the 126E(L1) strain, and then the LOS was analyzed by mass spectrometry. When the lst gene has been inactivated, no Neu5Ac could be detected in the LOS (data not shown). The mutation should not cause any polar effects as the next gene downstream of lst, a cytochrome c' homologue, is transcribed in the opposite direction and is not involved in sialic acid metabolism (18).

Allelic Exchange of the 126E(L1)-lst Gene with That from MC58(L3) and with the G168I Mutant Allele-- To assess the bi-functional nature of the lst gene from 126E(L1) in vivo, we constructed isogenic mutants that contained either the MC58(L3)-derived lst gene or the G168I mutation. After obtaining transformants, they were screened for sialyltransferase activity, and then the resident lst gene was amplified by polymerase chain reaction and sequenced to ensure the correct gene had been inserted. Both gene replacement mutants produced similar levels of sialyltransferase activity as measured with the in vitro assay on synthetic acceptors. The activity was similar to what we had seen in vitro; the MC58(L3)-derived enzyme did not make a significant amount of the alpha -2,6-linked Neu5Ac, whereas the G168I mutant showed the same ratio of alpha -2,3 to alpha -2,6 product (data not shown). LOS was prepared from these strains and was analyzed by CE-electrospray mass spectrometry (Fig. 5). The level of Neu5Ac incorporation in the 126E(L1) mutant strain with the MC58(L3)-lst gene was very low, but certainly detectable. The level of Neu5Ac incorporation from the G168I mutant gene was comparable with the wild-type 126E(L1) strain. Methylation analysis of these LOS samples (Table II) showed 6-substituted Gal, indicating that the Neu5Ac in both cases was alpha -2,6-linked and that no alpha -2,3-linked Neu5Ac was present.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Capillary zone electrophoresis-electrospray mass spectrometry analysis of mixtures of O-deacylated LOS from different strains: total ion electropherograms (m/z 600-1500) and reconstructed ion electropherograms for triply-deprotonated ions at m/z 862.0 (nonsialylated) and m/z 959.0 (sialylated). a, 126E(L1) wild type (WT); b, MC58(L3) × 126E(L1); c, MC58(L3)::G1681. Separation conditions: 20-nl injection of 1 mg/ml O-deacylated LOS, bare fused-silica (90 cm × 50 µm internal diameter, 190-µm outer diameter), 5% methanol in 30 mM morpholine, pH 9.0, +25 kV.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Methylation analysis of LOS
Bold values indicate the level of Gal-substituted galactose in the various strains of LOS preparations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The proteins involved in the addition of the sialic acid to the outer core oligosaccharide portion of the LOS from C. jejuni, H. influenzae, N. meningitidis, and N. gonorrhoeae have been identified (7, 17, 18). These proteins have a variety of acceptor specificities, and only the C. jejuni and H. influenzae enzymes share some sequence identity with each other. No structure/function analysis for any of these enzymes has so far been reported. Since there is no conserved sequence in the group of enzymes as a whole, it has been difficult to predict which residues might be involved in donor/acceptor recognition or catalysis.

Sialic acid transfer to the LOS of N. meningitidis species has been shown to be performed by the product of the lst gene (18). The terminal oligosaccharide produced in most strains is a mimic of the human glycolipid oligosaccharide sialyllacto-N-neotetraose. An alternate sialylated LOS structure with the terminal oligosaccharide alpha -2,6-Neu5Ac-Pk has been shown to be produced in the 126E(L1) strain (1, 20), but the enzyme for producing this novel alpha -2,6-sialylated structure was not reported. We have now determined the enzyme responsible for the addition of the alpha -2,6-linked Neu5Ac in the 126E(L1) strain and have shown it to be a bi-functional version of the Lst enzyme that previously had been shown to transfer the alpha -2,3-linked Neu5Ac in N. meningitidis MC58(L3) strain.

The first reported multifunctional enzyme involved in lipopolysaccharide biosynthesis was the 3-deoxy-alpha -D-mannooctulosonic acid (KDO) from Chlamydia pneumoniae, which adds KDO to the lipid A core through a alpha -2,6 linkage to GlcNAc and then in an alpha -2,4 linkage to the first KDO residue, and then in an alpha -2,8 linkage to the second KDO (27). Recently a bi-functional sialyltransferase from C. jejuni was described where the enzyme could also use two very different acceptors, namely a beta -linked Gal or a alpha -2,3-linked Neu5Ac (17). The sialyltransferase enzyme from the 126E(L1) strain of N. meningitidis described in this paper is also bi-functional but uses the same terminal sugar acceptor for the reaction and makes an alternate linkage (alpha -2, 6) in addition to the more usual alpha -2,3-linkage. This activity is also found only with terminal Gal residues that are in an alpha -linkage with the previous galactose residue. We have never seen any second (alpha -2,6) product formed during in vitro reactions on beta -linked galactose residues. We detected the alpha -2,6 product produced during in vitro enzyme reactions, but only about 10% of the synthetic Pk-FCHASE acceptor is converted to the alpha -2,6 linkage product, whereas the majority of the product formed in vitro is the alpha -2,3-sialyl-Pk structure. The MC58(L3)-Lst enzyme does produce detectable alpha -2,6-linked Neu5Ac, but the amount of alpha -2,6-linked Neu5Ac was about 45-fold less than was made by 126E(L1)-Lst, and this could only be seen using the ultra-sensitive laser-induced fluorescence detector on the CE. The isomeric alpha -2,3-sialyl-Pk structure has not been detected in vivo in the N. meningitidis 126E(L1) strain.

The ability of the enzyme to make these two products in vitro may not be too surprising since side reactions are possible with a synthetic acceptor molecule, but what is most important to bear in mind is that in vivo, only the alpha -2,6-Neu5Ac linkage is formed in the 126E(L1) strain. In our examination of the enzyme, we were able to pinpoint a single residue in the MC58(L3)-Lst, Gly-168, which when mutated to Ile (the residue at position 168 in 126E(L1), permits the enzyme to form the same level of alpha -2,6-sialylated product either in vitro on the synthetic Pk acceptor or in vivo on the LOS. What was surprising, however, was the fact that in vivo the MC58(L3)-Lst could form a very small amount of sialylated LOS in the 126E(L1) strain and that this material was also alpha -2,6-linked. The amount of Neu5Ac in this 126E(L1) mutant (MC58(L3)-Lst) was too small to analyze by NMR, but methylation analysis showed around 6% of the wild type level of the 6-substituted Gal. This level of Neu5Ac incorporation is much higher than what was expected, given the 45-fold lower alpha -2,6-sialytransferase activity of the MC58(L3)-Lst enzyme measured in vitro. These data suggest that some factor in vivo completely blocks the O3 position of the terminal alpha -galactose and that something also must promote the formation of the alpha -2,6-linked product since this is the linkage formed exclusively in L1. At present we do not know if this factor is another protein that may bind either Lst or the LOS, the conformation of the LOS during biosynthesis, or another molecule present at the site of biosynthesis. Molecular modeling of the 126E(L1) LOS suggests the conformation of the terminal trisaccharide could produce a sterically hindered product where the O3 of the terminal alpha -Gal is effectively blocked by the phosphoethanolamine substituent on Hep-2 of the inner core.2

Consequently the Lst variants with very low alpha -2,6 activity would be inactive in L1 strains when LgtC is functional. Since a relatively subtle change (Gly to Ile/Leu) results in the alpha -2,6-sialyltransferase activity, we speculate that the 126E(L1) strain maintained this version of Lst because it was advantageous for its survival in the human host, and as this oligosaccharide structure has not been described in man, it may or may not be molecular mimicry. We do not yet know if the specificity of the 126E(L1)-Lst is in all L1 immunotype strains. Data by Griffiss et al. (20) would suggest the structure we described in strain 126E(L1) and the lst gene variant occurs in other L1 immunotype strains.

Our mutagenesis of position 168 in the MC58(L3)-Lst revealed that all amino acid side chains are tolerated there except for proline. Surprisingly, the activity level of all of the mutants was similar on the acceptor beta -N-acetyllactosamine-FCHASE, except G168P, which was not active at all likely due to distortion of the enzyme active site. The mutants G168Y, G168F, and G168W maintained activity on beta -N-acetyllactosamine-FCHASE but have very reduced activity on the Pk-FCHASE compared with the MC58(L3)-Lst or 126E(L1)-Lst. When this residue is Ile or Leu, it then directs the specificity of the reaction to allow the formation of the alpha -2,6 linkage as well as the 126E(L1)-Lst. The presence of the aromatic amino acid side chains reduced the activity of the enzyme toward the Pk-FCHASE acceptor significantly. With the CE-based assay, the overall activity of the G168W/G168F/G168Y mutants were 4-9-fold less active on this acceptor (data not shown), whereas their activity on beta -N-acetyllactosamine-FCHASE appeared very similar to the wild type MC58(L3)-Lst protein. This suggests that the steric hindrance from the large inflexible ring containing side chains specifically block the enzyme from utilizing the alpha -linked terminal Gal residue.

We postulate that the position 168 in this enzyme must be in a cavity that has enough space to accommodate both the alpha  and beta  anomers of the terminal Gal. It will be essential to obtain structural information about this enzyme that will show us what the active site looks like to understand how these two different acceptor conformations are accommodated such that the product of the 126E(L1)-Lst reaction is alpha -2,6-linked Neu5Ac, whereas the MC58(L3)-Lst produces alpha -2,3-linked Neu5Ac. Other factors in the synthesis of the 126E(L1)-LOS influence the Neu5Ac linkage formation so it will also be important to identify what these are to fully understand the formation of this novel glycolipid structure.

    ACKNOWLEDGEMENTS

We thank Melissa J. Schur for expert technical assistance in the capillary electrophoresis analysis of the sialyltransferase assays.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Immunochemistry Program, Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Dr., Ottawa, Ontario, Canada K1A 0R6. Fax: 613-941-1327; E-mail: warren.wakarchuk@nrc.ca.

Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M011293200

2 J. R. Brisson, unpublished data.

    ABBREVIATIONS

The abbreviations used are: LOS, lipooligosaccharide; CE, capillary electrophoresis; Neu5Ac, N-acetylneuraminic acid; FCHASE, 6-(5-fluoresceincarboxamido)hexanoic acid succimidyl ester.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633[Abstract]
2. Moran, A. P., Prendergast, M. M., and Appelmelk, B. J. (1996) FEMS Immunol. Med. Microbiol. 16, 105-115[CrossRef][Medline] [Order article via Infotrieve]
3. Kahler, C. M., and Stephens, D. S. (1998) Crit. Rev. Microbiol. 24, 281-334[Medline] [Order article via Infotrieve]
4. Hood, D. W., Makepeace, K., Deadman, M. E., Rest, R. F., Thibault, P., Martin, A., Richards, J. C., and Moxon, E. R. (1999) Mol. Microbiol. 33, 679-692[CrossRef][Medline] [Order article via Infotrieve]
5. Jennings, M. P., Srikhanta, Y. N., Moxon, E. R., Kramer, M., Poolman, J. T., Kuipers, B., and van der Ley, P. (1999) Microbiology 145, 3013-3021[Abstract/Free Full Text]
6. Burch, C. L., Danaher, R. J., and Stein, D. C. (1997) J. Bacteriol. 179, 982-986[Abstract]
7. Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., Mansson, M., Schweda, E. K. H., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350[CrossRef][Medline] [Order article via Infotrieve]
8. Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605[Abstract]
9. Weiser, J. N., and Pan, N. (1998) Mol. Microbiol. 30, 767-775[CrossRef][Medline] [Order article via Infotrieve]
10. Aspinall, G. O., Fujimoto, S., McDonald, A. G., Pang, H., Kurjanczyk, L. A., and Penner, J. L. (1994) Infect. Immun. 62, 2122-2125[Abstract]
11. Harvey, H. A., Porat, N., Campbell, C. A., Jennings, M., Gibson, B. W., Phillips, N. J., Apicella, M. A., and Blake, M. S. (2000) Mol. Microbiol. 36, 1059-1070[CrossRef][Medline] [Order article via Infotrieve]
12. Preston, A., Mandrell, R. E., Gibson, B. W., and Apicella, M. A. (1996) Crit. Rev. Microbiol. 22, 139-180[Medline] [Order article via Infotrieve]
13. Hood, D. W., Deadman, M. E., Jennings, M. P., Bisercic, M., Fleischmann, R. D., Venter, J. C., and Moxon, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11121-11125[Abstract/Free Full Text]
14. Jennings, M. P., Hood, D. W., Peak, I. R., Virji, M., and Moxon, E. R. (1995) Mol. Microbiol. 18, 729-740[Medline] [Order article via Infotrieve]
15. Kahler, C. M., Martin, L. E., Shih, G. C., Rahman, M. M., Carlson, R. W., and Stephens, D. S. (1998) Infect. Immun. 66, 5939-5947[Abstract/Free Full Text]
16. Linton, D., Gilbert, M., Hitchen, P. G., Dell, A., Morris, H. R., Wakarchuk, W. W., Gregson, N. A., and Wren, B. W. (2000) Mol. Microbiol. 37, 501-514[CrossRef][Medline] [Order article via Infotrieve]
17. Gilbert, M., Brisson, J. R., Karwaski, M. F., Michniewicz, J., Cunningham, A. M., Wu, Y., Young, N. M., and Wakarchuk, W. W. (2000) J. Biol. Chem. 275, 3896-3906[Abstract/Free Full Text]
18. Gilbert, M., Watson, D. C., Cunningham, A. M., Jennings, M. P., Young, N. M., and Wakarchuk, W. W. (1996) J. Biol. Chem. 271, 28271-28276[Abstract/Free Full Text]
19. Gilbert, M., Cunningham, A. M., Watson, D. C., Martin, A., Richards, J. C., and Wakarchuk, W. W. (1997) Eur. J. Biochem. 249, 187-194[Abstract]
20. Griffiss, J. M., Brandt, B. L., Saunders, N. B., and Zollinger, W. (2000) J. Biol. Chem. 275, 9716-9724[Abstract/Free Full Text]
21. Wakarchuk, W. W., Campbell, R. L., Sung, W. L., Davoodi, J., and Yaguchi, M. (1994) Protein Sci. 3, 467-475[Abstract/Free Full Text]
22. Li, J., Thibault, P., Martin, A., Richards, J. C., Wakarchuk, W. W., and van der Wilp, W. (1998) J. Chromatogr. A 817, 325-336[CrossRef][Medline] [Order article via Infotrieve]
23. Wakarchuk, W., Martin, A., Jennings, M. P., Moxon, E. R., and Richards, J. C. (1996) J. Biol. Chem. 271, 19166-19173[Abstract/Free Full Text]
24. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve]
25. Gronberg, G., Nilsson, U., Bock, K., and Magnusson, G. (1994) Carbohydr. Res. 257, 35-54[CrossRef][Medline] [Order article via Infotrieve]
26. Sabesan, S., and Paulson, J. C. (1986) J. Am. Chem. Soc. 108, 2068-2080
27. Lobau, S., Mamat, U., Brabetz, W., and Brade, H. (1995) Mol. Microbiol. 18, 391-399[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.