From the Biology Department, The Johns Hopkins University, Baltimore, Maryland 21218
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ABSTRACT |
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Recent studies from many laboratories revealed
remarkable structural, distributional, and functional diversities of
oligo/polysialic acids (OSA/PSA) that exist in organisms ranging from
bacteria to man. These diversities are further complicated by the fact that OSA/PSA spontaneously form lactones under even mildly acidic conditions. By using high performance anion-exchange chromatography (HPAEC) with nitrate eluents, we found that lactonization of
Recent studies on oligo/polysialic
acids1
(OSA/PSA)2 have revealed
remarkable structural (1-3), tissue/cell distributional (4),
functional (5-7), and evolutionary diversities ranging from bacteria
to man (8, 9). Structural diversities of OSA/PSA are further
complicated by their ease of lactonization. The existence of lactones
in OSA/PSA was suggested first many years ago (10). It was suspected
that lactonization could significantly alter the physicochemical and
biological properties of OSA/PSA such as charge density, conformation,
and antigenicity (11, 12). Both It has been suggested by NMR and substitution studies (11) that
lactonization of oligo/poly( Materials--
Quaternary methylamine anion exchanger is from
Millipore Waters Chromatography Division (Milford, MA). Sephacryl S-300
and Sephadex G-25 resins are from Sigma. Bio-Gel P-2 resin is from Bio-Rad. Oligo(
To remove the contaminating free oligo/poly-Neu5Gc chains (resulted
from auto-hydrolysis) as well as peptides free of oligo/poly-Neu5Gc from PSGP, 10 mg of the PSGP was fractionated on a Sephacryl S-300 column (1 × 118 cm) equilibrated and eluted with 10 mM phosphate buffer (pH 7.6). The effluent was monitored by
A215 nm, and the absorption peaks were checked
with HPAEC by injecting small aliquots of the fractions before and
after partial hydrolysis with 0.1 M HCl at 80 °C for 15 min. Fractions containing intact PSGP showed the diagnostic peak
patterns of oligo/poly-Neu5Gc by HPAEC (27) only after partial
hydrolysis. Such fractions were therefore pooled and desalted on a
Sephadex G-25 column (0.7 × 19 cm) equilibrated and eluted with
water, freeze-dried, and stored at Chromatographic Conditions--
Dionex (Sunnyvale, CA) Bio-LC
was used with the CarboPac PA-1 and PA-100 columns in combination with
a pulsed amperometric detector (PAD-2) using the SC-PAD-2 detector cell
with a gold working electrode and a silver/AgCl reference electrode.
The detector sensitivity was set at 1 µA. Potential and time settings
of the detector were as follows: E1 = +0.05 V
(t1 = 0.42 s), E2 = +0.65 V (t2 = 0.18 s),
E3 =
For the separation of the lactones, neutral nitrate gradients were
produced by mixing 0.5 M NaNO3 and water.
Freshly prepared 0.5 M NaNO3, typically of pH
6.0-6.5, was used directly for HPAEC. However, the pH of the solution
stored in the reservoir of the eluent degas module (Dionex) must be
tested to confirm that a proper pH is
maintained.3 To ensure
detector response and to maintain a stable base line, a postcolumn
pneumatic controller (Dionex) driven by pressured nitrogen gas was used
to add a solution of 0.5 M NaNO3 in 1 M NaOH at 0.5 ml/min to the postcolumn eluent before it
enters the detector. When it was desirable to have lactone rings
opened, alkaline elutions were used that contained a constant flow of 0.1 M NaOH in addition to a gradient generated with 0.5 M NaNO3. All eluents were sparged and pressured
under helium using the Dionex eluent degas module.
Lactonization under Acidic Conditions--
Lactonization of
Lactonization in strong acid was performed by treating the samples with
1 M HCl at ambient temperature for 2 h or at 4 °C overnight. The samples were diluted 100 times with water before injection. When 250 mg of colominic acid (Na+-form) was
dissolved in 1 ml of 1 M HCl and kept at ambient
temperature for 2 h, a white, milky suspension of extensively
lactonized PSA was formed, which will be referred to as
"polylactone." Polylactone was separated by centrifugation and
washed with 1 ml of distilled de-ionized water three times by repeated
suspension and centrifugation. After freeze-drying, the polylactone
samples were stored at Lactonization by Dialysis against Water--
Colominic acid (100 mg, Na+-form) was dissolved in 1 ml of water and dialyzed
against 1 liter of distilled de-ionized water at 4 °C for 3 days in
a dialysis tubing (molecular mass cut-off = 1 kDa) with a daily change
of water. After dialysis, the sample was lyophilized and stored at
Dialysis of pentamer and hexamer of oligo-Neu5Ac was performed on a
micro scale.4 Briefly, 50 µl each of the samples (Na+-form) at 1 mg/ml was
transferred into a 250-µl polypropylene screw-top microvial (Sun
Brokers, Wilmington, NC). A cut piece of dialysis membrane
(molecular mass cut-off = 1 kDa) was placed on top of the vial and
screwed tight with an open-top cap. The vial was immersed upside-down
in 500 ml of distilled de-ionized water and dialyzed with stirring at
4 °C for 1 day. After dialysis, the samples were diluted with water
before analysis by HPAEC.
Lactonization after Freeze-Storage in Phosphate Buffer--
OSA
of DP2-6 were purified on a quaternary methylamine column as described
earlier and desalted by passing through a Bio-Gel P-2 column
equilibrated and eluted with 10 mM phosphate buffer (pH
7.5). The sample solutions were frozen and stored at Enzymatic Digestion of Lactonized Samples--
Neuraminidase
digestion was performed at 4 °C to minimize possible opening or
rearrangement of the lactone rings as well as auto-hydrolysis of
glycosidic linkages. Dialyzed OSA and colominic acid were diluted with
80 mM sodium acetate buffer (pH 4.8) to a final
concentration of 40 mM sodium acetate and digested with a
neuraminidase (20-80 milliunits of enzyme per µg of substrate) at
4 °C for different periods and directly injected to the CarboPac PA-1 column. Following the analysis, the column was cleaned by eluting
with 0.2 M NaOH at 0.2 ml/min overnight to remove any neuraminidase that might have accumulated under neutral eluting condition.
Acid Hydrolysis of Lactonized Samples--
The stability of
colominic acid polylactones in acid was tested by hydrolyzing
lactonized (experimental) and freshly NaOH-treated (control) samples.
For experimental samples, to a mixture of 57 µl of 1 M
HCl and 10 µl of 1 M NaOH in a 0.5-ml screw-capped
polypropylene microcentrifuge tube (USA/Scientific Plastics, Ocala, FL)
was added 400 µl of 3 mg/ml polylactone. For control samples, 10 µl of 1 M NaOH and 400 µl of 3 mg/ml polylactone were mixed
and kept at ambient temperature for 5 min to open all lactone rings. To the mixture was then added 57 µl of 1 M HCl. The
experimental and control, both in capped tubes with a final
concentration of 0.1 M HCl, were heated at 80 °C in a
glycerol bath in a heating block. After different times, the tubes were
cooled on ice, and a calculated amount of NaOH was added to neutralize
the solutions. Aliquots of the neutralized samples were analyzed with
HPAEC using an alkaline sodium nitrate as eluent.
Acid hydrolysis of purified PSGP was carried out in a similar manner.
For the experimental, 20 µl of 10 mg/ml PSGP and 10 µl of 0.1 M NaOH were mixed and kept at ambient temperature for 10 min to remove any possible O-acetylation that may prevent
lactonization. To the mixture was added 10 µl of 4 M HCl
and left at ambient temperature for 2 h (to induce lactonization)
followed by dilution with water to a final concentration of 0.1 M HCl. For the control, equivalent amounts of
solutions were used except that the NaOH and HCl were pre-mixed
and diluted first before adding to the PSGP. The samples were then
heated at 80 °C for 15 min before they were neutralized and analyzed
by HPAEC.
Lactonization of Colominic Acid--
We have shown that colominic
acid can be separated into a series of peaks by HPAEC using a neutral
nitrate eluent (27). Under neutral elution conditions, retention of
OSA/PSA on the CarboPac PA-1 column is mainly determined by the number
of ionized carboxyl groups. Lactonization reduces the number of
negative charges and should cause the elution time to decrease. Indeed, we observed that the peak distribution pattern of OSA/PSA changed upon
lactonization. However, fragmentation of OSA/PSA due to glycosidic bond
cleavage can also change the peak distribution pattern. To distinguish
between the two possible causes, we treated the sample with NaOH (final
concentration of 20 mM over and above neutralization of
acids) immediately before injection or carried out the elution using an
alkaline nitrate eluent. Brief exposure to alkali such as these opened
lactone rings but retained glycosidic linkages.
Representative chromatograms from a series of periodic injections of a
colominic acid sample kept at 4 °C in 20 mM HCl solution are shown in Fig. 2. The original peak
distribution pattern of the sample (Fig. 2A) changed within
40 min in 20 mM HCl (Fig. 2B). The peak height
of high DP homologues decreased dramatically, and new peaks (which are
unlabeled) appeared between the two adjacent low DP homologue peaks
(which are labeled with DP). Continued incubation resulted in further
decrease of the high DP peaks, and the pattern of new peaks between the
two adjacent low DP homologues became more complex (Fig.
2C). However, in the subsequent analysis (Fig.
2D) in which the same sample after 10 h incubation was
treated with NaOH prior to injection, all the original high DP peaks
were restored concomitant with disappearance of the new peaks found between the two adjacent low DP homologues. The peak distribution patterns of Fig. 2, A and D, are nearly the same
except that in Fig. 2D monomer and dimer peaks increased
somewhat, apparently due to limited glycosidic cleavage. These results
suggest that there was no significant degradation of the high DP
homologues under the acidic condition used here. Therefore, the
apparent diminishing of high DP homologues in Fig. 2, B and
C, was mostly caused by lactonization and not by hydrolysis.
The lactonized species not only appeared as the alkali-sensitive new
peaks between two adjacent low-DP homologues (Fig. 2, B and
C) but also fused together, forming a broad raised base line
(Fig. 2C) similar to the pattern shown by capillary
electrophoresis (16).
By comparing samples with and without NaOH treatment, significant
lactonization of OSA/PSA was also observed under conditions that were
frequently used for acid hydrolysis. For example, at pH 4.8, a
lactonization pattern similar to that of Fig. 2B was observed at both 37 and 55 °C (not shown). At 80 °C in 0.1 M HCl, which is a typical hydrolytic condition for
de-sialylation of glycoconjugates, instead of fragmentation of OSA/PSA
chains revealed by the NaOH-treated hydrolysate, extensive
lactonization occurred as indicated by broad peaks around monomer,
dimer and trimer, respectively (not shown).
Since lactonization requires protonation of the carboxyl group, we
tested whether colominic acid in free acid form can spontaneously form
lactones. A colominic acid (Na+-form) solution was passed
through a Dowex 50 (H+-form) column and examined by HPAEC.
A pattern similar to that of Fig. 2C was observed (not
shown), indicating that lactonization proceeded quickly and
extensively. This result was consistent with that obtained by IR
spectroscopy (11, 12). Interestingly, mere dialysis of the colominic
acid sample (Na+-form) against water also induced limited
and selective lactonization, which gave a pattern (not shown) similar
to that of Fig. 2B.
Lactonization of OSA at pH 3.2--
To ascertain if the shifting
of individual peaks is due to lactonization and also to assign proper
DP values to these peaks, we studied lactonization of purified
individual oligo(
As a specific example, Neu5Ac pentamer (Fig. 3D) formed
three groups of lactonized species, designated as 5L3,
5L2, and 5L1 (see Footnote 2). We interpret
that 5L3, 5L2, and 5L1 are
pentamers with different numbers of lactone rings since they eluted in
close proximity to non-lactonized dimer, trimer, and tetramer
(indicating that they have 2, 3, and 4 residual carboxyl groups, see
"Discussion"), respectively. Within each L peak there
are lactone ring position isomers, which is reflected by the broad and
skewed nature of the L peaks. Such assignments were supported by data
obtained under higher chromatographic resolution, which was
accomplished by using a more moderate gradient or isocratic elution.
For example, the two lactone ring position isomers (see Fig. 1;
(Neu5Ac
We also quantified each species at timed intervals, on the basis that
the molar response factors of lactonized species are equal to or close
to those of their parent non-lactonized species (see
"Discussion"). Quantitative analysis of the average lactonization rate at potential lactonizable sites on each OSA revealed that OSA with
higher DP lactonized more rapidly and had higher percentage of
potential sites being lactonized (not shown).
Lactonization of OSA in 1 M HCl--
Fig.
4 shows the lactonization patterns of
oligo-Neu5Acs of DP2-6 after incubation in 1 M HCl at
4 °C overnight. Treating these samples with NaOH reversed all the
lactone (L-labeled) peaks to their non-lactonized
precursors (see dash-lined chromatograms in Fig. 4).
Compared with the patterns shown in Fig. 3, lactonization was more
extensive, and more than half of dimers eluted as an alkali-sensitive
peak that had the same elution time as monomer, which indicated that
the lactonized dimers contained one lactone ring per molecule (labeled
as 2L1). Lactone peaks (i.e. 3L2,
4L3, 5L4, and 6L5) having
approximately the same elution time with monomer also appeared as the
major species in each of other OSA, suggesting that a significant
fraction of these molecules underwent complete lactonization, which
involved all carboxyl groups except that on the reducing-terminal
residue (see the (Neu5Ac The Initial Stage and Enzymatic Digestion--
The above results
show that lactonization of OSA/PSA can occur in discrete stages (see
"Discussion"). The most interesting stage is the "initial
stage," which is manifested by a limited and selective lactonization
pattern (Fig. 2B). This stage can be reached also by simply
dialyzing the sample against water or by prolonged storage in 10 mM phosphate buffer (pH 7.5) at
To pinpoint the lactone ring positions of these species, we digested
the sample with sialidase. Since the carboxyl group on sialic acid is
essential for substrate recognition by neuraminidase (30) and the
enzymatic digestion of OSA/PSA proceeds solely from the non-reducing
terminus (27), the digestion will stop at the lactonized sialic acid
residues to yield new OSA/PSA species with a lactone ring at the
non-reducing terminus. After digestion of the dialyzed pentamer and
hexamer with a large excess of sialidase (Fig. 5, A2 and
B2), most of the non-lactonized pentamer and hexamer were
degraded into monomer. However, enzymatic digestion also generated new
peaks that were resistant to further digestion but sensitive to NaOH.
There were two (Fig. 5A2) and three (Fig. 5B2) such new peaks generated from dialyzed pentamer and hexamer,
respectively. Comparing each of the new peaks with monolactonized OSA
of a certain DP, we found that they share the similar elution times
whether pretreated with NaOH or not (not shown). Therefore, these new peaks represent monolactones of shorter lengths, which were assigned and labeled accordingly in Fig. 5, A2 and B2. An
analogous pattern was also seen from
(Neu5Ac Polylactone and Its Resistance to Acid Hydrolysis--
Treating
colominic acid with 1 M HCl formed an insoluble suspension
of lactonized sample which we termed polylactone in analogy to the
glacial acetic acid-treated colominic acid (31). Our MALDI-time of
flight mass spectrometry data suggested that polylactone contains
predominantly fully lactonized PSA (data not shown). Compared with the
starting material (Fig. 6A),
the polylactone (Fig. 6B) is considerably enriched in higher
DP polymers (OSA being soluble and removed). When this polylactone was
treated at 80 °C in 0.1 M HCl, a large portion of higher
DP polymers was preserved even after heating for 90 min (Fig.
6D). However, if it was first treated with NaOH prior to
heating, the majority of higher DP polymers degraded into monomer and
OSA (Fig. 6C). Quite clearly, lactonization can prevent
acid-catalyzed cleavage of the Lactonization and Hydrolysis of Oligo/Poly( HPAEC Chromatography
Elution Position Versus Number of Negative Charges--
Although
capillary electrophoresis could also differentiate lactonized and
non-lactonized oligo-Neu5Acs of DP3-5 (26, 33), it is not effective
for polymers (16). HPAEC under our conditions has a much higher
resolution and a more even peak distribution over a much wider DP range
(27). We also take advantage of the ease of interpretation of the data
from HPAEC, because negative charges on the molecules are the main
determining factor for retention time under the neutral elution
conditions. Therefore, each non-lactonized OSA/PSA peak can be
unequivocally assigned with its DP based on the extrapolation from the
elution positions of a few OSA with known DP of 1-6 as standards (27).
The validation of such an extrapolation was confirmed by MALDI-time of
flight mass spectrometry analysis of a pooled PSA sample in a peak of
DP-14 eluted from the CarboPac PA-100 column (data not shown).
Moreover, molecules with the same number of carboxyl groups elute close
together as a group, and within such a group, the lactonized species
always elute slightly earlier than the non-lactonized species (Fig. 3). In general, among a group of lactonized species with the same number of
carboxyl groups, those with larger DP elute earlier. This information
is helpful for the assignment of lactonized species and can be
explained by the difference in the charge-to-mass ratios, which was
supported by our observations that lactobionic acid eluted earlier than
either galactonic acid or gluconic acid under neutral nitrate elution conditions.
Lactone Ring Opening and Detector Response--
OSA/PSA
lactones are very sensitive to alkali. Assay of ester function using
hydroxylamine and ferric chloride (34) showed that the lactone rings
opened within 30 s when exposed to 20 mM NaOH.
Moreover, the opaque suspension of polylactone became clear immediately
after adding NaOH. This explains the absence of lactone peaks when the
lactonized samples were chromatographed under alkaline conditions.
Rapidity of de-lactonization also affects the PAD response. It is most
likely that before the lactone species enter the PAD detector during
HPAEC with neutral eluent, most of the lactone rings are opened upon
post-column addition of NaOH. Therefore, the molar response factors of
lactonized species should be equal to those of their non-lactonized
precursors. The tests with the two isolated isomeric lactones,
i.e. (Neu5Ac The Three Stages of Lactonization
Lactonization of OSA/PSA occurs at three distinct stages. The
initial stage can be attained by mild conditions such as dialysis, long
term freezing in phosphate buffer, or mild acid treatment around pH 5. This stage is precursory to further lactonization under more acidic
conditions (Fig. 2). Dialysis-induced lactonization can be explained by
the loss of Na+ ions through dialysis, since dialysis of
colominic acid (Na+-form) against 0.9% NaCl did not
generate any lactone. The middle stage is attained under mild acidic
conditions of pH 2-3 or by Dowex 50 (H+-form) treatment
and characterized by formation of multiple lactone species. Generally
speaking, for a given oligo-Neu5Ac at pH 3.2, the maximum number of
lactone rings formed equals its DP minus 2 (Fig. 3). This rule is also
applicable to Selectivity of Lactonization
Fig. 5 revealed that lactonization at the initial stage happened
between two internal Neu5Ac residues and not between the two Neu5Ac
residues at either terminus (shown as two circles connected with
a zigzag line in Fig. 5C). We rationalized
that there are repulsing forces between the neighboring negatively
charged carboxyl groups, and in non-lactonized OSA/PSA, the repulsing
forces exerted on any internal carboxyl group come from both sides so
that orientation of the carboxyl group is confined. However, the
repulsing force exerted on the carboxyl group at either reducing or
non-reducing terminus comes from one side only so that it tends to push
the terminal carboxyl group away from the backbone, resulting in a conformational difference between the Neu5Ac Implications and Perspectives
Our studies serve to caution that care must be used in
preparation, storage, and analysis of OSA/PSA samples. To prevent
lactonization as well as cleavage of glycosidic linkages, OSA/PSA
should be prepared as Na+-form and should be either
freeze-dried or stored frozen in water (not in phosphate buffer).
During the isolation of individual OSA from the hydrolysate of
colominic acid, lactonization may compromise the purity of the isolated
sample. We recommend adjusting the pH of the sample solution to 12 for
a few minutes to open lactone rings prior to such a separation.
Acid-catalyzed cleavage of The demonstrated ease of lactonization of OSA/PSA may have biological
significance. It was reported that subtle changes in the charge and
hydration states of OSA/PSA could influence the interactions between
cell membranes (38). Lactonization may incur similar influence. The
mild acidic conditions used for inducing lactonization do exist in the
endosomes and lysosomes of the cells. Lactonization of internalized
OSA/PSA in these organelles may affect their metabolism since
lactonized OSA/PSA are resistant to neuraminidase digestion.
Lactonization of OSA/PSA on cell-surface glycoproteins such as N-CAM
may also occur under pathological conditions such as ischemia,
infection, and malignancy that can cause pH to drop. This may
consequently affect cell adhesion and intercellular communication.
Natural occurrence of ganglioside lactones has been already detected
(17-19) and was suggested to have important biological roles (20). The
mild acidic conditions that can induce lactonization of ganglioside
(13, 14, 39) can certainly induce lactonization of OSA/PSA on
glycoproteins. Based on our results, spontaneous lactonization would
most likely stop at the initial stage under the mild acidic conditions
likely to be encountered in a living organism. It can be even
speculated that a "lactone synthetase" may exist that can drive the
lactonization of OSA/PSA beyond the initial stage and a "lactonase"
that catalyzes the reverse. Even at the initial stage, it is still
possible to cause significant impacts on the biological activities of
OSA/PSA by altering the conformation and orientation of the OSA/PSA
chains. Such possibility was supported by the observation that only 2% lactonization of the Neu5Ac residues in colominic acid or less than
20% lactonization (perhaps equivalent to our initial stage) in B
polysaccharide caused a significant loss of antigenicity (12). Recent
studies revealed that some types of cells and tissues had little or no
expression of PSA detected by monoclonal antibodies, although the same
cells and tissues had intense expression of the mRNA transcripts of
either one or both of the two polysialyltransferases, i.e.
PST and STX (4, 40). It is interesting to see whether the negaive
detection by antibodies was caused by the epitopic changes on PSA upon lactonization.
It has been reported that conjugates of OSA/PSA with certain enzymes
can protect these enzymes from inactivation by proteinases, while
retaining most of the enzymatic activities, and can also reduce the
rate of clearance from circulation (41, 42). Induced lactonization of
OSA/PSA on such conjugates may provide further adjustment of
pharmacological parameters and may also reduce the possibility of
generating immune responses in the host (12).
2,8-linked OSA/PSA (oligo/poly-Neu5Ac, oligo/poly-Neu5Gc and
oligo/poly-KDN) proceeds readily, and the lactonization process
displays three discrete stages. The initial stage is characterized by
limited lactonization occurring between two internal sialic acid
residues, reflected by a regular pattern of lactone peaks
interdigitated with non-lactonized peaks on HPAEC. In the middle stage,
multiple lactonized species are formed from a molecule with a given
degree of polymerization (DP), in which the maximum number of lactone rings formed equals DP minus 2. At the final stage, completely lactonized species become the major components, resulting in drastic changes in the physicochemical properties of the sample.
Interestingly, the smallest lactonizable OSA are tetramer, trimer, and
dimer at the initial, middle, and final stages, respectively. At any of
the stages, OSA/PSA of higher DP lactonize more rapidly, but all the
lactone rings rapidly open up when exposed to mild alkali. Lactonized
OSA/PSA are resistant to both enzyme- and acid-catalyzed glycosidic
bond cleavage. The latter fact was utilized to obtain more high DP
oligo/poly(
2,8-Neu5Gc) chains from a polysialoglycoprotein. Our
results should be useful in preparation, storage, and analysis of
OSA/PSA. Possible biological significance and bioengineering potentials
of lactonization are discussed.
INTRODUCTION
Top
Abstract
Introduction
References
2,8- and
2,9-linked
oligo/poly-Neu5Ac lactonized rapidly under acidic conditions (11, 12).
Similar phenomenon was observed in gangliosides containing
2,8-linked Neu5Ac dimer (13, 14). We have also demonstrated that
passing a solution of sodium salt of colominic acid (a mixture of
oligo/poly(
2,8-Neu5Ac) homologues) through a Dowex 50 (H+-form) column or simply dialyzing it against water can
induce lactonization (15). Failure to completely de-lactonize OSA/PSA mixtures after partial hydrolysis under acidic conditions severely limited their separation by capillary electrophoresis (16). Similarly,
during the separation of OSA from hydrolysate of colominic acid on an
anion-exchange column, we observed that each OSA peak of an expected
degree of polymerization (DP) contained a small amount of OSA with a
higher than expected DP (e.g. the OSA of DP5 in the expected
DP4 peak). Co-existence of OSA of two different DP was also detected in
samples obtained elsewhere. Apparently, loss of negative charges upon
lactonization of OSA caused them to elute at the positions of lower DP.
The natural occurrence of sialic acid lactones in glycolipids also
underscores the significance of lactonization in vivo
(17-20). However, it is not clear whether lactonization in
vivo occurs spontaneously by merely acid-catalyzed chemical
reactions or is actively controlled by enzymatic processes, although
lactonization could be indirectly regulated by
O-substitutions and de-O-substitutions on sialic
acids, which are suggested to be tightly controlled in living systems
(21-23). Therefore, systematic investigation on acid-catalyzed
lactonization of OSA/PSA will not only provide useful information for
proper sample handling in vitro but also shed light on our
understanding of related processes in vivo.
2,8-Neu5Ac) occurs between two adjacent
sialic acid residues, the carboxyl group of one residue esterifying the
9-hydroxyl group of the residue at the reducing side to form a
6-membered ring (Fig. 1). The same
lactone ring was also observed between the two
2,8-linked sialic
acid residues in GD3 and GD1b ganglioside lactones (24, 25). If this is the only type of lactone ring, there will be three possible lactone species from a trimer, i.e.
(Neu5Ac
2,8)3-(1':9)-lactone (A), (Neu5Ac
2,8)3-(1":9')-lactone (B), and (Neu5Ac
2,
8)3-(1':9, 1":9')-di-lactone (AB) (see Fig. 1). It is
obvious that the possible patterns of lactonization will become
progressively complicated as DP increases. The lack of detailed
understanding of the lactonization most likely is due to unavailability
of effective methodology. Spectrometric methods such as IR (11, 12),
NMR (11, 12), and CD (26) usually reveal only the averaged properties
such as the ratio of lactonized and non-lactonized species, whereas
more detailed and specific information such as the positions of the
lactone rings and the distribution of different lactone species is
difficult to obtain. We have developed a highly sensitive and efficient method for analysis of OSA/PSA using high performance anion-exchange chromatography (HPAEC) and pulsed amperometric detection (PAD) by
utilizing either neutral or alkaline conditions for separation (27). By
using this method, we systematically studied acid-catalyzed lactonization of OSA/PSA as reported in this paper.
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Fig. 1.
The three isomeric lactones of
2,8-linked Neu5Ac trimer. The positions of
lactone rings are marked with A and B.
EXPERIMENTAL PROCEDURES
2,8-Neu5Ac) of DP2-6, colominic acid
(Na+-form) as well as Arthrobacter ureafaciens
neuraminidase are gifts from Drs. Y. Tsukada and Y. Ohta, Kyoto
Research Laboratories, Marukin Shoyu Co., Uji, Japan. The enzyme
activity was defined and determined as reported (28).
Oligo(
2,8-Neu5Gc), Oligo(
2,8-KDN), and polysialoglycoprotein
(PSGP) from salmon eggs are gifts from Drs. S. and Y. Inoue, Academia
Sinica (Taipei, Taiwan). Oligo-Neu5Acs were also prepared as described
(29) with some modifications. Briefly, a quaternary methylamine
anion-exchange column (1 × 29 cm) was used in 10 mM
phosphate buffer (pH 7.5) with a linear gradient eluent from 0 to 0.4 M NaCl. Samples were adjusted to pH 12 for a few minutes
prior to the chromatography (see "Discussion").
20 °C.
0.10 V (t3 = 0.36 s). These settings are optimal for nitrate eluent and also
give excellent results when eluting with NaOH only or NaOH with sodium
acetate. A Spectra SYSTEM AS3000 autosampler (Thermo Separation
Products, San Jose, CA) was used for sample injection and maintenance
of sample temperature at 4 °C prior to injection. Data were
collected via Dionex ACI (advanced computer interface) and Dionex
AI-450 software.
2,8-linked OSA/PSA were induced with such mild acidic solutions as
40 mM sodium acetate (pH 4.8), 10 mM sodium
phosphate (pH 3.2), and 20 mM HCl. Lactonization at
4 °C, room temperature (23 °C), 37, 55, and 80 °C were tested.
Samples were also lactonized by passing through a Dowex 50 (H+-form) column at room temperature.
20 °C.
20 °C. Freeze-drying and subsequent storage of the dried sample at
20 °C did not change the lactonization pattern (data not shown).
20 °C for 6 months to 1 year and analyzed by HPAEC.
RESULTS
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Fig. 2.
Lactonization of OSA/PSA in a colominic acid
sample in 20 mM HCl at 4 °C. A CarboPac PA-1 column
was used. An eluent (0.5 M NaNO3) gradient was
produced as 2% for 0-2 min, 20% at 9 min, and 65% at 61 min.
Non-lactonized OSA/PSA peaks are labeled with DP. A, before
incubation; B and C, after incubation for 40 min,
8 h and 40 min, respectively; D, the NaOH-treated
sample after 10 h incubation.
2,8-Neu5Ac) of DP2-6. All incubations were at
4 °C to minimize glycosidic cleavage. A set of chromatograms after
10 h incubation at pH 3.2 is shown in Fig.
3. Each chromatogram (Fig. 3,
B-E) except for that of dimer (Fig. 3A) contains
three kinds of peaks as follows: 1) the latest eluting original OSA
peak; 2) the small peaks of monomer, dimer, and trimer, etc. (resulted
from hydrolysis); and 3) the broad and skewed peaks labeled with
L. When these samples were treated with NaOH, all the
L peaks disappeared, whereas the original OSA peaks were restored
nearly to their original height (not shown). Therefore, the L peaks are
lactonized species.
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Fig. 3.
Lactonization of individual oligo-Neu5Ac in
phosphate buffer (pH 3.2) at 4 °C. The results from OSA of
DP2-6 are shown in A-E, respectively. A
CarboPac PA-1 column was used. An eluent (0.5 M
NaNO3) gradient was produced as 2% for 0-2 min, 20% at 9 min, and 30% at 23 min. All samples were incubated for 10 h prior
to injection. Peaks labeled as 3L1, 4L1,
4L2, etc. are lactone species (see text). Non-lactonized
species are labeled with numbers representing their
DP.
2, 8)3-(1':9)-lactone (A) and (Neu5Ac
2,
8)3-(1":9')-lactone (B)) in the peak 3L1 (Fig.
3B) were isolated using isocratic elution. They both
reverted to the original trimer upon the NaOH treatment (data not
shown). Interestingly, Neu5Ac dimer did not form any lactone even when
kept at 4 °C for 3 days (data not shown).
2,8)3-(1':9, 1":9')-di-lactone
(AB) in Fig. 1). Similar to the results observed at pH 3.2, OSA with
higher DP lactonized more rapidly and extensively. For instance,
whereas all the pentamer and hexamer molecules lactonized, the original
non-lactonized molecules still existed in OSA with DP <5.
Surprisingly, degradation by acid hydrolysis was hardly detected in
oligo-Neu5Acs kept at 4 °C in 1 M HCl, suggesting that
formation of lactone rings protects against glycosidic cleavage (also
see other results shown later).
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Fig. 4.
Lactonization of individual oligo-Neu5Ac in 1 M HCl at 4 °C overnight. The results from OSA of
DP2-6 are shown in A-E, respectively. Chromatographic
conditions and peak-labeling principles were identical to those used in
Fig. 3. Control samples that had been treated under the identical
condition for lactonization and then treated with NaOH prior to
injection are shown as dash-lined chromatograms in each
panel.
20 °C. The
lactonization patterns in Fig. 5,
A1 and B1, show that dialyzed pentamer and
hexamer each formed one major lactone peak that eluted near the
non-lactonized tetramer and pentamer peaks, respectively, indicating
only mono-lactone was formed from each oligomer. Similar patterns were
observed in oligo-Neu5Acs of DP4-6 after long term storage at
20 °C but that dimer and trimer did not form any lactone.
NaBH4-reduced colominic acid can still be lactonized in a
similar fashion as non-reduced colominic acid (not shown), supporting
the notion that lactonization at the initial stage does not involve the
sialic acid residue at the reducing terminus.
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Fig. 5.
Neuraminidase digestion of dialyzed
oligo-Neu5Ac. Chromatographic conditions and peak-labeling
principles were identical to those used in Fig. 3. A1 and
B1, dialyzed pentamer and hexamer, respectively;
A2 and B2, after digestion of the dialyzed
pentamer and hexamer, respectively; C1, C2, and
C3, the proposed schemes for the generation of different
lactone species based on the results of enzymatic digestion of dialyzed
tetramer (not shown), pentamer (A), and hexamer
(B), respectively. L, lactonization by dialysis;
E, neuraminidase digestion. The circles represent
sialic acid residues among which the reducing termini are at the
right side. Sites of lactone rings are marked by
angles. Glycosidic linkages involving either the reducing or
the non-reducing terminus are shown by zigzag
lines.
2,8)4-monolactone upon sialidase digestion (not
shown). A rationalization of these data is diagrammed in Fig.
5C and will be discussed later. Sialidase digestion of
dialyzed colominic acid further confirmed that lactonization occurred
non-randomly, since the regularity of the peak distribution pattern of
the lactone species was preserved, although all the lactone peaks were
shifted to new elution positions (not shown).
2,8-glycosidic linkage.
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Fig. 6.
Removal of OSA in a colominic acid sample by
precipitation of polylactone and the resistance of polylactone to acid
hydrolysis. The CarboPac PA-100 column was eluted with the
alkaline nitrate eluents, which had a gradient change of 0.5 M NaNO3 as 4% for 0-2 min, 20% at 9 min, and
50% at 79 min. Peaks are labeled with DP. A, colominic acid
(40 µg) before treatment; B, polylactone (40 mg) prepared
from the same colominic acid sample; C and D, the
resistance of polylactone to acid hydrolysis. Hydrolysates (40 mg each)
of lactone ring opened control (C) and intact polylactone
(D) were analyzed after incubation in 0.1 M HCl
at 80 °C for 90 min as described under "Experimental
Procedures." The insets in C and D
are enlarged views of the 35-70-min region.
2,8-Neu5Gc) in
PSGP--
Attempting to utilize the resistance of lactonized OSA/PSA
to acid hydrolysis, we studied the maximum release of
oligo/poly(
2,8-Neu5Gc) chains from a PSGP under different hydrolytic
conditions by comparing the yield of high DP OSA/PSA expressed in terms
of peak area ratio of octamer to trimer (G8/G3) to normalize for
variations in peak areas between injections. Trimer was chosen as an
index to reflect the degradation of high DP OSA/PSA during hydrolysis,
because monomer might be derived from mono-sialylated glycans in PSGP and dimer might accumulate more than others due to its higher stability
in acid (27, 32). As expected from our earlier report (27), prolonged
incubation under a milder condition (0.1 M acetic acid,
55 °C for 2 h) produced the lowest yield of high DP OSA/PSA (G8/G3 = 0.042). A higher yield (G8/G3 = 0.067) was obtained
after a short incubation under a stronger hydrolytic condition (0.1 M HCl, 80 °C for 15 min). The highest yield (G8/G3 = 0.092) was obtained by pretreating the sample with 1 M
HCl at ambient temperature for 2 h to induce complete
lactonization prior to hydrolysis in 0.1 M HCl at 80 °C
for 15 min. This result suggested that pre-induced lactonization may
stabilize the Neu5Gc
2,8-Neu5Gc linkage, while still allowing the
preferential cleavage of the Neu5Gc
2,6-Gal linkage (near the
peptide) under strong hydrolytic conditions. However, the optimal
condition for release of PSA of much higher DP from glycoconjugates
needs to be investigated since the available sample we had was a PSGP
from which the largest PSA released under all the conditions we tested
was only DP-11.
DISCUSSION
2,8)3-(1':9)-lactone and
(Neu5Ac
2,8)3-(1":9')-lactone (see Fig. 1) confirmed that their molar response factors were equal and did not change whether or
not they received NaOH treatment prior to injection. Neither did they
convert to each other nor revert to the parent
(Neu5Ac
2,8)3 during chromatography using neutral nitrate eluent.
2,8-linked oligo-Neu5Gc and oligo-KDN series (not
shown). At the final stage (with pH <1), complete lactonization
occurred, forming polylactone from PSA. Interestingly, the smallest
lactonizable OSAs are tetramer, trimer, and dimer at the initial,
middle, and final stages, respectively.
2,8-Neu5Ac di-sialic acid unit at either terminus and the internal di-sialic acid unit. This
is in agreement with the NMR studies which showed the abovementioned conformational difference in native OSA/PSA (35) but not in derivatives
that have the carboxyl groups being reduced to primary alcohols (36).
The conformation of the di-sialic acid unit at either terminus may not
allow the carboxyl group to easily access the 9-hydroxyl group to form
a lactone ring. Alternatively, compared with the carboxyl groups near
or on the terminal position, the confined or "crowded" orientations
of the carboxyl groups on internal residues may be more inclined to be
protonated to reduce the charge repulsion, thus allowing the
preferential lactonization between internal residues. This
rationalization can explain the phenomenon that the lactone ring
between the di-sialic acid unit forms much more easily in the sulfated
glycolipid, i.e.
HSO3-8Neu5Ac
2,8-Neu5Ac
2,6-Glc-Cer, than in its
un-sulfated counterpart (37), since the repulsion from the sulfate
group makes the non-reducing terminal Neu5Ac residue behave like an
internal residue. An internal Neu5Ac residue may also behave like a
terminal residue, if its neighboring residue is lactonized. This can
explain both the observed difficulty to form two consecutive lactone
rings along the internal residues (Fig. 5) and the non-random pattern
of lactonization of colominic acid at the initial stage.
2,8-glycosidic linkages was reported to
occur preferably at the linkages between two internal sialic acid
residues (32). As mentioned earlier, the favorable sites for
lactonization are also located between two internal sialic acid
residues. Therefore, we can expect some competition between
lactonization and glycosidic cleavage under acidic conditions. The
glycosidic linkages would be protected if lactonization proceeds much faster, such as under 1 M HCl at lower temperatures
(Fig. 4). We applied this principle for releasing maximum amounts of high DP oligo/poly(
2,8-Neu5Gc) chains from PSGP by inducing
polylactonization prior to acid hydrolysis.
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ACKNOWLEDGEMENTS |
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We thank Drs. S. Inoue and Y. Inoue for
providing us with oligo(2,8-Neu5Gc), oligo(
2,8-KDN), and PSGP and
Drs. Y. Ohta and Y. Tsukada for sialidase, purified
oligo(
2,8-Neu5Ac), and colominic acid. We also thank Dr. R. T. Lee for reviewing the manuscript and providing helpful discussions.
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FOOTNOTES |
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* 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.: 410-516-7041;
Fax: 410-516-8716; E-mail: yclee{at}jhu.edu.
1 In this paper, we refer to those with DP equal to or higher than 10 as polymers since they can be differentiated from the homologues of DP <10 by antibodies, as reported elsewhere, and can be easily prepared by precipitating polylactone from water as shown in this paper.
3 The pH may rise occasionally because of contamination from the NaOH-containing reservoirs.
4 Details of this procedure will be published elsewhere.
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ABBREVIATIONS |
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The abbreviations used are: OSA, oligosialic acids; PSA, polysialic acids; HPAEC, high performance anion-exchange chromatography; PAD, pulsed amperometric detection/detector; DP, degree of polymerization; Neu5Ac, 5-N-acetylneuraminic acid; Neu5Gc, 5-N-glycolylneuraminic acid; KDN, 2-keto-3-deoxy-D-glycero-D-galactonononic acid; PSGP, polysialoglycoprotein; 5L1, 5L2, 5L3, etc., lactones of pentameric Neu5Ac with one, two, and three lactone rings per molecule, respectively; GD3, II3(NeuAc)2-LacCer; GD1b, II3(NeuAc)2-GgOse4Cer.
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REFERENCES |
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