Acid-catalyzed Lactonization of alpha 2,8-Linked Oligo/Polysialic Acids Studied by High Performance Anion-exchange Chromatography*

Ye Zhang and Yuan C. LeeDagger

From the Biology Department, The Johns Hopkins University, Baltimore, Maryland 21218

    ABSTRACT
Top
Abstract
Introduction
References

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 alpha 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(alpha 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

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 alpha 2,8- and alpha 2,9-linked oligo/poly-Neu5Ac lactonized rapidly under acidic conditions (11, 12). Similar phenomenon was observed in gangliosides containing alpha 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(alpha 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.

It has been suggested by NMR and substitution studies (11) that lactonization of oligo/poly(alpha 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 alpha 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. (Neu5Acalpha 2,8)3-(1':9)-lactone (A), (Neu5Acalpha 2,8)3-(1":9')-lactone (B), and (Neu5Acalpha 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 alpha 2,8-linked Neu5Ac trimer. The positions of lactone rings are marked with A and B.


    EXPERIMENTAL PROCEDURES

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(alpha 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(alpha 2,8-Neu5Gc), Oligo(alpha 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").

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 -20 °C.

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 = -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.

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 alpha 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.

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 -20 °C.

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 -20 °C. Freeze-drying and subsequent storage of the dried sample at -20 °C did not change the lactonization pattern (data not shown).

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 -20 °C for 6 months to 1 year and analyzed by HPAEC.

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.

    RESULTS

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).


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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.

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(alpha 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.

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; (Neu5Acalpha 2, 8)3-(1':9)-lactone (A) and (Neu5Acalpha 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).

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 (Neu5Acalpha 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.

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 -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.

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 (Neu5Acalpha 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).

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 alpha 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.

Lactonization and Hydrolysis of Oligo/Poly(alpha 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(alpha 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 Neu5Gcalpha 2,8-Neu5Gc linkage, while still allowing the preferential cleavage of the Neu5Gcalpha 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

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. (Neu5Acalpha 2,8)3-(1':9)-lactone and (Neu5Acalpha 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 (Neu5Acalpha 2,8)3 during chromatography using neutral nitrate eluent.

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 alpha 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.

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 Neu5Acalpha 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. HSO3-8Neu5Acalpha 2,8-Neu5Acalpha 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.

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 alpha 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(alpha 2,8-Neu5Gc) chains from PSGP by inducing polylactonization prior to acid hydrolysis.

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).

    ACKNOWLEDGEMENTS

We thank Drs. S. Inoue and Y. Inoue for providing us with oligo(alpha 2,8-Neu5Gc), oligo(alpha 2,8-KDN), and PSGP and Drs. Y. Ohta and Y. Tsukada for sialidase, purified oligo(alpha 2,8-Neu5Ac), and colominic acid. We also thank Dr. R. T. Lee for reviewing the manuscript and providing helpful discussions.

    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. 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.

    ABBREVIATIONS

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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