2 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; and 3 Institute of Biochemical Sciences, National Taiwan University, Taipei, 115 Taiwan
Received on July 22, 2003; revised on October 3, 2003; accepted on October 3, 2003
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Abstract |
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Key words: capillary electrophoresis / hydrolysis / lactonization / oligosialic acid / polysialic acids
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Introduction |
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Recently we have developed a highly sensitive and efficient method for the analysis of oligosialic acids and their lactone species by capillary electrophoresis (CE; Cheng et al., 1998, 1999a
). By using this method, the pathway of formation of the fully lactonized
(2
8)-linked trimer and tetramer in glacial acetic acid, the delactonization of fully lactonized trimer and tetramer in basic solution, and simultaneous hydrolysis/lactonization of trimer and tetramer in acidic aqueous solution have been elucidated (Cheng et al., 1999b
, 2000
; Yu et al., 2001
). In this article, we discuss the controlled hydrolysis of
(2
8)/
(2
9) PSA and the lactonization of
(2
8)/
(2
9) alternatively linked trimers and tetramers in acidic condition based on the analysis by CE.
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Results |
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After alkaline treatment and desalting by gel filtration, the sample of Figure 5b was analyzed by CE with the coinjection of the authentic samples. Three peaks shown in Figure 5c were identified: monomer, (2
8) dimer, and
(2
8)/
(2
9) trimer. Thus the generation of
(2
8) dimer, from the cleavage of 2a by sialidase and alkaline treatment of a lactonized dimer (peak 2 of Figure 5c), confirms that the lactonization positions of the two monolactones are solely in the
(2
8) linkage. Also, the generation of 1 in Figure 5c shows that the formation of the dilactone (peak E of Figure 5a) comes only or at least largely from 1 (see Discussion) and, finally, the two trimer isomers with partial separation in CE analysis can be identified.
Identification of monolactones from two tetramers
Two tetramers (3, 4) were also obtained by the controlled hydrolysis of (2
8)/
(2
9) PSA and purified by a Mono Q column. As described, compound 3 with
(2
8)/
(2
9)/
(2
8) linkages is in much higher quantity than 4 with
(2
9)/
(2
8)/
(2
9) linkages, and they could not be separated in the traditional chromatography. The lactonization of 3 and 4 is more complicated than that of 1 and 2. As shown in Figure 6, four monolactone species and three dilactone species were formed from 3 and 4 in glacial acetic acid. In an attempt to identify each peak shown in the CE profiles, two facts that have been demonstrated in the previous reports were applied: (1) Lactonization in the
(2
8) linkage is easier than that in the
(2
9) linkage, and (2) due to the charge repulsion, the carboxyl groups in the middle position are much more easily lactonized than those in both ends. Based on these facts, compound 4a with the
(2
8) linkage in the middle position should be the first monolactone formed from 3 and 4, corresponding to peak B in Figure 6. Three other monolactones were identified by the hydrolysis of sialidase.
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Discussion |
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Analysis of lactonized species of oligomers by CE
Zhang and Lee (1999) have used high-performance anion-exchange chromatography (HPAEC) to analyze lactonized and nonlactonized OSA of DP2-6. The separation by HPAEC mainly depends on the negative charges of molecules. Fully lactonized species from dimer to hexamer have approximately the same elution times as the monomer. Multiple forms of partially lactonized species with the same negative charges for each OSA could not be well separated by HPAEC. The separation by CE depends not only on the charges of the molecules but also on the mass of the molecules. Because of high resolution in separation, CE can separate all of the partially lactonized species of OSA having slightly different pKa of the carboxyl groups, even though they have the same charges and masses. In the separation by CE and the hydrolysis of sialidase, all the nonlactonized and lactonized species produced by lactonization and hydrolysis could be identified. Therefore the pathway of lactonization could be clearly drawn.
Selective lactonization in (2
8)/
(2
9) alternatively linked OSA
For OSA, lactonization occurs more easily in the (2
8) linkage than in the
(2
9) linkage. For the lactonization of two trimers, the dilactone is generated solely from
(2
8)/
(2
9) trimer, not from
(2
9)/
(2
8) trimer. This clearly indicates that the internal carboxyl group of
(2
8)/
(2
9) trimer can be lactonized in the
(2
9) linkage, whereas the external carboxyl group at the nonreducing end of
(2
9)/
(2
8) trimer has little chance to be lactonized in the
(2
9) linkage. In principle, all of the positions of
(2
8) linkages in
(2
8)/
(2
9) alternatively linked OSA could be lactonized regardless of external or internal carboxyl groups involved. As for the site of
(2
9) linkage, only internal carboxyl groups involved can be lactonized. This principle can explain why the two monolactones, 4b and 4c, and two dilactones, 4e and 4f, of two tetramers could not be found. Lactonization in reducing the end occurs more easily than in the nonreducing end. It is evident, as shown in Figure 6, that 3b is further lactonized to 3e but not to 3f and that 4a is further converted to 4d, not to 4e.
In analysis by CE, negative charges and masses of the molecules are the main factors for mobility. Molecules with the same number of carboxyl groups and DP should have close mobilities as a group. Therefore it is possible that some compounds whose generation we have ruled out may be formed in tiny or minor amount and may merge into the peaks of the major products in the analysis by CE.
Hydrolysis of (2
8)/
(2
9) alternatively linked OSA/PSA by sialidase
Although neuraminidase from Vibrio cholerae showed a much higher hydrolytic rate on the linkage of (2
8) PSA than that of
(2
9) PSA, there is not much difference in cleaving both
(2
8) and
(2
9) linkages in the
(2
8)/
(2
9) alternatively linked PSA. That is the reason we cannot use neuraminidase to differentiate two trimers (1 and 2) and two tetramers (3 and 4). In the previous report, both
(2
8) PSA and
(2
8)/
(2
9) alternatively linked PSA were found to be susceptible to neuraminidase cleavage, whereas
(2
9) PSA was resistant. The
(2
9) linkage in both
(2
9) PSA and
(2
8)/
(2
9) alternatively linked PSA showed different accessibility by neuraminidase. However, the antibody produced from the injection of
(2
8)/
(2
9) alternatively linked PSA as antigen could precipitate
(2
9) PSA but failed to react with
(2
8) PSA. This implied that the conformation of
(2
8)/
(2
9) alternatively linked PSA is similar to that of
(2
9) PSA but not to that of
(2
8) PSA. These findings revealed that the chemical properties of
(2
8)/
(2
9) alternatively linked PSA are similar partly to that of
(2
9) PSA and partly to that of
(2
8) PSA.
Conclusion
Both lactonization and hydrolysis in OSA/PSAs are catalyzed by acid in aqueous solution. To simplify the analysis of products, we carry out lactonization in glacial acetic acid to avoid hydrolytic reactions. In our studies, lactonization in glacial acetic acid was faster than in 0.1 N acetic acid, but all lactonized products in both systems were the same (unpublished data). Lactonization can significantly alter the charge density and conformation of OSA/PSA. Therefore, it has been reported that lactonization of PSA in vivo might have biological influence in epitopic change, interaction between cell membranes and metabolism of PSA. Natural formation of lactones in ganglioside has been detected. However, it remains unclear whether lactonization in vivo occurs only by acid-catalyzed chemical reaction or is controlled by specific enzymes. Our investigation of acid-catalyzed lactonization of (2
8)/
(2
9) alternatively linked OSA will not only provide useful information in the identification and analysis of the isomers but also shed light on our understanding of related processes in vivo for the three different linkages of PSA.
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Materials and methods |
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Chromatographic conditions
CE was performed on a Beckman CE P/ACE system 2100 (Palo Alto, CA) using fused silica capillaries (117 cm x 75 µm ID) and applying 20 kV at 25°C. Phosphate buffer (50 mM, pH 8.0) was used as the running buffer. The spectra were monitored by UV absorption at 200 nm. Samples were injected into the capillaries by high-pressure nitrogen (20 psi) for 3 s. The capillaries were regenerated by washing with double-distilled water for 3 min and then 0.1 N NaOH for 5 min.
Preparation of (2
8)/
(2
9) alternatively linked PSA
The preparation of (2
8)/
(2
9) alternatively linked PSA was based on the previous report (González-Clemente et al., 1990
; Gotschlich et al., 1969
; Vann and Freese, 1994
). A culture of E. coli K92 was inoculated into 2 L medium and allowed to grow at 37°C. Release of the capsular polysaccharide in the medium was maximal (450 µg/ml ) in the stationary phase of growth for 76 h. The culture was separated to cell and medium by centrifugation (7000 x g at 4°C). The supernatant was then diluted twofold with 0.2% Cetavlon (hexadecyltrimethylammonium bromide) and allowed to stand for at least 2 h. The Cetavlon complex containing polysaccharide was harvested by centrifugation (8000 rpm, 15 min). The pellet containing polysaccharide was dissolved in 1 M CaCl2. The paste was then extracted by homogenizing vigorously in a Sonicator (Sonics 2210, Branson). The viscous mixture was slowly adjusted to 25% ethanol stirred for 1 h at 4°C, and centrifuged to remove cell debris and precipitated nucleic acid and protein. The supernatant was adjusted to 80% ethanol, and the crude polysaccharide fraction was recovered by centrifugation (16,000 x g, 4°C).
The pellet was dissolved in 10% sodium acetate and stirred vigorously with an equal volume of cold buffered phenol for 30 min in an ice bath. The phases were separated by centrifugation at 10,000 rpm. The aqueous layer was removed, adjusted to 25% ethanol, allowed to stand in the cold for 1 h, and then centrifuged at 10,000 rpm. Polysaccharide was recovered from the supernatant by adjusting to 80% ethanol and then centrifuging after standing overnight at 4°C. It was then processed through at least one additional cycle of phenol extraction and ethanol precipitation until the interface was minimal. The final ethanol precipitate was dissolved in 10% sodium acetate and dialyzed extensively against water. The dialyzate was centrifuged at 100,000 x g for 2 h to remove lipopolysaccharide. The supernatant was then lyophilized and checked by NMR and CE.
Preparation of (2
8)/
(2
9) alternatively linked OSA
Alpha-(2 8)/
(2
9) alternatively linked PSA was incubated in 0.01 N acetic acid at 40°C for 1 day and evaporated to give a dry residue by lyophilization. Next, 0.1 N NaOH was added to hydrolyze the lactonized mixture at room temperature for 20 min. The hydrolyzed mixture was fractionated by means of a Mono Q HR 5/5 column (Pharmacia, Uppsala, Sweden). The column was applied with a gradient of 0.06 N to 0.5 N NaCl for 60 min and developed at an average flow of 0.5 ml/min. Chromatography was monitored by UV absorption at 214 nm. Fractions were collected, desalted by Superdex PE 7.5/300 (Pharmacia), eluted by distilled water, and identified by CE.
Lactonization in glacial acetic acid
Alpha-(2 8)/
(2
9) alternatively linked OSA (25 µg) was treated with glacial acetic acid (500 µl) at room temperature for different time intervals and then dried by speed vacuum to remove excess acetic acid. Dried samples were dissolved in distilled water (5 µl) and analyzed by CE.
Neuraminidase hydrolysis
OSAs were lactonized in glacial acetic acid for 10 min, treated with 0.5 mU Arthrobacter ureafaciens neuraminidase (Nacalai Tesque, Japan) in 20 µl of 0.1 M ammonia acetate at room temperature, and analyzed directly by CE at different time intervals.
Fast atom bombardment mass spectrometry
Fast atom bombardment mass spectra of the sample were obtained on an Autospec OA-TOF mass spectrometer (Micromass, Manchester, UK) fitted with a cesium ion gun operated at 30 kV. Five milligrams of hydrolysate was dissolved in 5% acetic acid for loading onto the probe tip coated with monothioglycerol as matrix.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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