Oligo/polysialic acid (oligo/polySia) is a collective name for linear oligo/polymers of sialic acid. Oligo/polySia chains constitute a structurally unique group of carbohydrate residues of glyco-conjugates found in living organisms that range evolutionary diversity from bacteria to human. In animal, natural occurrence of oligo/polySia has so far been reported in limited types of cells and is often dependent on onco- and ontodevelopmental stages in sharp contrast to more abundant and widespread distribution of monomeric sialic acid residues that occur on almost all types of cells. Studies have shown that polySia is a biologically important glycotope, being an oncodevelopmental antigen of human and having potential roles in cell growth, differentiation, fertilization, and neuropathogenicity (Troy, 1992).
Figure 1. Separation of [alpha]2-9-linked oligo/polyNeu5Ac on a CarboPac PA-100 column. Elution with 1M NaNO3: 2% for 0-3 min, 10% at 10 min, 16% at 40 min, and 26% at 80 min. (a) A solution of [alpha]2-9-linked polyNeu5Ac (50 µg) in 0.1 M acetic acid (100 µl) was heated for 15 min at 60°C. After hydrolysis pH of the mixture was adjusted to ~13 by adding 50 µl of 0.5 M NaOH and kept at room temperature at least 1 h before injection. A 100 µl portion (equivalent to 33 µg Neu5Ac) was injected. (b) Mild acid hydrolysate of [alpha]2-9-linked polyNeu5Ac (11 µg NeuAc) prepared as (a) was coinjected with a mixture of mono- and [alpha]2-8-linked oligoNeu5Ac (n = 2-7, each equivalent to 100 ng Neu5Ac). Peaks are labeled with DP.
In bacteria linkage isomers of [alpha]2-8- and [alpha]2-9-linked polyNeu5Ac were known in capsules of some strains as homopolymers while in others as copolymers of each linkage type (Troy, 1992). In animal our recent studies on fish eggs and sea urchin eggs and sperm revealed extensively diverse polySia structures: [alpha]2-8-linked oligo/polyNeu5Gc as the first example of polySia glycotope in eukaryote (Inoue and Iwasaki, 1978, 1980); [alpha]2-8-linked copolymers of Neu5Ac and Neu5Gc (Sato et al., 1993); [alpha]2-8-linked oligo/polyKDN (Kanamori et al., 1990); [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc (Kitazume et al., 1994a, 1996; Kitazume-Kawaguchi et al., 1997); and capping of oligo/polySia by KDN (Nadano et al., 1986), by 8-O-sulfated Neu5Ac (Ijuin et al., 1996), and by 9-O-sulfated Neu5Gc (Kitazume et al., 1996; Kitazume-Kawaguchi et al., 1997). At present [alpha]2-8-linked polyNeu5Ac is the only chemically established polySia glycotope in mammals, including human, although [alpha]2-9-linked Neu5Ac dimer was reported in human embryonal carcinoma cells (Fukuda et al., 1985). However, wide occurrence of [alpha]2-8-linked oligo/polyKDN was shown in mammalian cells and tissues by immunochemical methods (Kanamori et al., 1994; Qu et al., 1996; Ziak et al., 1996). More recently, occurrence of [alpha]2-8-linked oligoNeu5Gc in pig spleen and Wistar rat tissues was shown by using newly developed monoclonal antibody, and the results were partially supported by chemical analysis (Sato et al., 1998). All these results are indicative of occurrence of diverse polySia structures in mammals including human.
Although the structural diversity of polySia has been identified by chemical methods that include isolation and purification of the compounds and structural studies using instrumental analyses such as 1H-NMR spectroscopy and mass spectrometry, studies of biosynthesis, function, and metabolism of polySia chains depend on immunochemical methods for their detection and identification. However, immunochemical methods need specific antibody for each structural type of polySia group and are not able to detect new type of polySia structure. Moreover, these methods require enzymes specifically reactive to each antigenic glycotope to confirm the specific binding of antibodies to antigens. We have already shown that diversity of polySia is not limited to the structure of building unit sialic acid but is extended to modification of sialic acid residues by substitution of their hydroxyl group(s) with acetyl and sulfate, interresidue linkage types, and the degree of polymerization (DP). Efforts to determine DP-dependent spec-ificities of antibodies against [alpha]2-8-linked oligo/polyNeu5Ac and oligo/polyKDN, and to develop monoclonal antibodies that recognize oligo/polyNeuGc, have recently been made (Sato et al., 1995, 1998). However, at present we have neither antibodies nor enzymes that recognize [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc. Another disadvantage of immunochemical methods is they do not give quantitative results that are indispensable to the studies on biosynthesis and metabolism of polySia chains that have often been shown to be developmentally dependent. Thus, in view of both biological significance and structural diversity of oligo/polySia glycotopes, development of highly sensitive and reliable analytical method for detection and identification is urgent. To study (1) biosynthetic mechanism, (2) biological function, and (3) metabolism, the method should provide sufficiently accurate information on the actual chain length of polySia chains because both biological and chemical properties of these unique structural elements are highly dependent on their chain length (Manzi et al., 1994). We have already reported that a capillary electrophoretic method partly fulfills these requirements (Cheng et al., 1998). Recently, two different chromatographic methods were developed, i.e., (1) highly efficient and specific separation of oligo/polymers of diverse types of sialic acid by high performance anion-exchange chromatography (HPAEC) with pulsed amperometric detector (Zhang et al., 1997), and (2) detection and identification of fmol levels of oligosialic acids by fluorometric high performance anion-exchange chromatography (Sato et al., 1998). This paper describes more extensive studies of these chromatographic methods aiming at application to further diverse types of oligo/polySia chains including those with different interresidue linkage types, capped by sulfated Neu5Gc, and expressed on functional glycoproteins that are available only in small amounts. Studies on the function of bioactive molecules, mechanism of biosynthesis, and metabolism of functional polySia chains by using the present techniques will be reported elsewhere.
Figure 2. Separation of [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc on a CarboPac PA-100 column. Elution with 1M NaNO3: 2% for 0-3 min, 10% at 10 min, and 25% at 55-60 min. (a) PolySia-gp (Fraction 1sb, 5 µg Neu5Gc) was hydrolyzed in 50 mM sodium acetate buffer (pH 4.8, 200 µl) for 2 h at 80°C, and then the mixture was adjusted to pH ~ 13 with 50 µl of 0.5 M NaOH. A 50 µl portion (equivalent to 1 µg Neu5Gc) was injected. Peaks are labeled with DP and S denotes 9-O-sulfate substitution at the nonreducing terminal Neu5Gc residues. (b) Retention time vs. DP of [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc (open circles), and [alpha]2-8-linked oligo/polyNeu5Gc (solid circles). Separation and analysis of [alpha]2-8-linked and [alpha]2-9-linked polyNeu5Ac by HPAEC-PED
Resolution of [alpha]2-8-linked polyNeu5Ac on a CarboPac PA-100 column was remarkable as already reported (Zhang et al., 1997). Here we show the separation of [alpha]2-9-linked oligo/polyNeu5Ac generated by mild acid hydrolysis of a highly polymerized sample of Neu5Ac ([alpha]2-9-linked polyNeu5Ac) under similar conditions as described previously (Figure
Figure 3. Separation of [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc capped with 9-O-sulfated Neu5Gc on a CarboPac PA-100 column. (a) A mixture of 9-O-sulfated Neu5Gc[alpha]2->(->5-Oglycolyl-Neu5Gc[alpha]2->)n (n = 0-3, each equivalent to 50-250 ng Neu5Gc). Elution with 1 M NaNO3: 2% for 0-3 min, 10% at 10 min, and 15% at 25 min. Peak 1 represents Neu5Gc monomer, externally added, whereas peaks 2-4 represent nonsulfated [alpha]2-5-Oglycolyl-linked (Neu5Gc)n, n = 2-4, originally present in thesolution of 9-O-sulfated Neu5Gc-capped oligo-Neu5Gc. (b) Detection and identification of 9-O-sulfated Neu5Gc[alpha]2->(->5-Oglycolyl-Neu5 Gc[alpha]2->)n in the mild acid hydrolysate of ESP-Sia isolated from sea urchin egg cell surface complex. ESP-Sia (20 µg Neu5Gc) was hydrolyzed in 10 mM trifluoroacetic acid for 15 min at 60°C and then the mixture was adjusted to pH ~ 12 with 0.5 M NaOH and filtered with Centricon-10 (3000 r.p.m. at 4°C). A portion of the filtrate (2 µg Neu5Gc) was injected. Elution with 1 M NaNO3: 2% for 0-3 min, 10% at 10 min, and 25% at 55 min, and 30% at 60 min. (c) Detection and identification of 9-O-sulfated Neu5Gc as a capping residue of polySia-gp isolated from sea urchin jelly. A fraction of polySia-gp (Fraction 2P, 2.7 µg Neu5Gc) was hydrolyzed in 50 mM sodium acetate buffer (pH 4.8) for 2 h at 80°C, and analyzed immediately after neutralization with NaOH to prevent hydrolysis of sulfate ester under alkaline conditions. Injected amount: 1.1 µg Neu5Gc. NaNO3 gradient was same as Figure 2a. Peaks are labeled with DP and S denotes 9-O-sulfate substitution at nonreducing terminal Neu5Gc. Separation and analysis of oligo/polyNeu5Gc having [alpha]2-5-Oglycolyl linkages by HPAEC-PED
In 1994 we reported identification of a new type of polySia chain unusually linked through the 5-Oglycolyl group of Neu5Gc in sialic acid-rich glycoproteins (polySia-gp) isolated from the jelly coat of sea urchin eggs (Kitazume et al., 1994a). Our earlier study showed that DP of the oligo/polySia chains to be from 4 to more than 40 (average ~20) based on anion exchange chromatography of sialooligosaccharide alditols liberated by alkaline borohydride treatment of the parent glycoprotein although the resolution between each oligomer was insufficient. Result of separation and detection of [alpha]2-5-Oglycolyl-oligo/polyNeu5Gc liberated under very mild acid hydrolysis of a fraction of polySia-gp (1sb) by HPAEC-PED was recorded in Figure Separation and analysis of 9-O-sulfated Neu5Gc-capped oligoNeu5Gc chains by HPAEC-PED
We reported the occurrence of 9-O-sulfated Neu5Gc-capped [alpha]2-5-Oglycolyl-linked oligo/poly-Neu5Gc chains in sea urchin egg cell surface glycoprotein (Kitazume et al., 1996). Our study also showed that sulfated oligoSia chains were on the sea urchin egg receptor for sperm and sulfate residue is important in binding of the receptor to sperm (Kitazume-Kawaguchi et al., 1997). Separation of 9-O-sulfated Neu5Gc and 9-O-sulfatedNeu5Gc-capped [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc, i.e., Neu5Gc9SO4[alpha]2->(->5-Oglycolyl-Neu5Gc[alpha]2->)n (n = 1 - 4) was recorded in Figure Evaluation and limitation of the HPAEC-PED methods in application to biological material
The results of this study, when combined with the previously published one (Zhang et al., 1997), showed that analysis of oligo/polySia chains by HPAEC-PED on a CarboPac PA-100 column was applicable to almost all types of oligo/polySia chains known to occur in living organisms. Compared with the capillary electrophoretic method (Cheng et al., 1998), resolution of polymers of high DP was remarkable with HPAEC-PED. Difference in retention time between the different series of homologous oligo/polySia chains with the same DP was sufficient for identification purpose. This was applied not only to the series with different sialic acid building blocks but also the linkage isomers of the same sialic acid component. Because the resolution of polySia by CarboPac PA-100 column was excellent, we can estimate the real DP of long polySia that is known to be expressed in certain glycoproteins such as embryonic N-CAM and human neuroblastoma cells (Livingston et al., 1988; Troy, 1992).
When HPAEC-PED method is applied to biologically functional oligo/polySia compounds that are available only in minute amount and/or that are difficult to purify, we found that sensitivity of this method is insufficient. For instance, when the same colominic acid sample was analyzed after mild acid-hydrolysis, injection of 180 ng Neu5Ac was sufficient to detect decamer of Neu5Ac. However, for detection of the peaks of polymers with DP 40, 50, and 70, injection amounts of 5, 10, and 30 µg Neu5Ac were necessary. Obviously improvement of prehydrolysis methods (Zhang and Lee, 1999) may partly solve the problem.
Table I.
Another problem is that amperometric response is not highly selective to sialic acid components. When the samples contained a large proportion of peptide components many unidentifiable peaks appear in low salt region and overlap oligoSia peaks, and for these samples purification is necessary before injection.
Results and discussion
a, b
c
n
2
3
4
5
6
7
9
10
11-13
(->8Neu5Ac[alpha]2->)n
95
80
72
67
21
48
-
43c
25d
(->8Neu5Gc[alpha]2->)n
85
74
69
60
-
43
27
24
(->5-Oglycolyl-Neu5Gc[alpha]2->)n
100
84
61
45
28
Evaluation of a HPLC-FD method of oligo/polySia analysis
DMB is now widely used as a highly sensitive fluogenic reagent reactive with sialic acid in identification and differential quantitation of KDN, Neu5Ac and Neu5Gc separated on a reverse phase HPLC column (Hara et al., 1987; Inoue et al., 1996). The detection limit of 100 fmol (30 pg) of sialic acid was attainable in reverse phase chromatography (Hara et al., 1987). Recently, application of DMB-derivatization of oligoSia followed by anion-exchange HPLC was also reported (Sato et al., 1998). The report showed that the reducing terminal sialic acid in oligoSia chain was tagged with DMB under conditions used for derivatization of sialic acid monomers and DMB-oligoSia chains were separated and quantitated by fluorometric anion-exchange HPLC with a detection limit of 13 fmol of Neu5Ac[alpha]2-8Neu5Ac. In the present study, we applied the method for analysis of higher oligomers of sialic acid. The major problem when the DMB-method is applied to oligo/polySia analysis is that derivatization requires acidic medium (pH 2-3) and needs at least 2 h at 50-60°C for the maximum yield. We established conditions that gave most satisfactory results for the analysis of oligo/polySia chains by incubating samples with the DMB reagent without prehydrolysis. Each oligo/polySia samples, (Sia)n, gave ladders of homologous series of DMB-(Sia)n, n = 1, 2, 3, - n, on the chromatogram. The yields of DMB-(Sia)n, i.e., the DMB-tagged parent oligo/polySia were determined for model compounds under optimized conditions and recorded as percentage of total DMB-(Sia)n, n = 2, 3, - n, in Table I. The yield was gradually decreased with increasing DP starting with almost 100% for dimers but still attains about 25% at decamers for [alpha]2-8-linked polySia. For [alpha]2-5-Oglycolyl-linked oligomers of Neu5Gc, the yield dropped down to 28% at hexamer even if we used milder conditions than those used for [alpha]2-8-linked polysialic acids. The elution profiles for biological materials containing various types of oligo/polySia chains are given in Figure
a, b
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c, d
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Figure 4. Separation of DMB-oligo/polySia by anion-exchange chromatography on a MonoQ HR 5/5 column and detection by fluorometry. Samples were derivatized under conditions specified for each compound. All derivatized sample solutions were adjusted to pH~13 with NaOH and kept at room temperature at least for 1 h before injection to cleave ester linkages formed. Elution with NaCl in 10 mM Tris-HCl (pH 8.0): 0 M, for 0-10 min, first linear gradient 0-0.14 M, between 10-20 min; second linear gradient 0.14-0.57 M, between 20-70 min. (a) Colominic acid. Sample (10 µg Neu5Ac) was derivatized in the medium containing 20 mM trifluorocetic acid for 2 h at 50°C. Injected amount: 1 µg Neu5Ac. (b) Rainbow trout PSGP (a glycoprotein containing [alpha]2-8-linked oligo/polyNeu5Gc). Sample (420 ng NeuGc) was derivatized as (a). Injected amount: 140 ng Neu5Gc. (c) PolySia-gp (Fraction 1sb) from sea urchin jelly. Sample (2.6 µg NeuGc) was derivatized in the medium containing 5 mM trifluoroacetic acid for 2 h at 50°C. Injected amount: 650 ng Neu5Gc. (d) ESP-Sia from sea urchin egg cell surface complex. Sample (1.1 µg Neu5Gc) was derivatized as (c). Injected amount: 450 ng Neu5Gc. Peaks are labeled with DP and S denotes 9-O-sulfate substitution at nonreducing terminal Neu5Gc.
For derivatization of [alpha]2-8-linked polySia chains the reaction medium containing 20 mM trifluoro-acetic acid gave good results (Figure
High-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED)
A DX 500 ion chromatography system (Dionex, Sunnyvale, CA) was used with a CarboPac PA-100 column. An ED40 electrochem-ical detector was operated at integrated amperometry mode with a repeating potential sequence E1 = 0.05 V, t1 = 0.4 S: E2 = 0.75 V, t2 = 0.2S; E3 = -0.15 V, t3 = 0.4 S, and integration period between 0.2-0.4 S from the start as recommended by the manufacturer (Rohrer et al., 1998). We use the term pulsed electrochemical detection (PED), which encompasses all subtechniques generated by the instrument used (LaCourse, 1997), although the method used in this paper was basically same as that generated by pulsed amperometric detector (PAD). Samples in 100 µl amounts were injected manually or by using a Spectra System AS300 autosampler (Thermo Separation Products, San Jose, CA). Eluents were the mixture of water (A), 0.2 M NaOH (B), and 1 M NaNO3 (C), with initial conditions of A/B/C = 48/50/2. Concentration of NaOH was always kept at 0.1 M and oligo/polySia compounds were eluted at a flow rate of 1 ml/min with concentration gradient of NaNO3 as described in the figure captions (Zhang et al., 1997).
High performance liquid chromatography with fluorescent detection (HPLC-FD)
A Hewlett Packard HPLC system series 1100 with a fluorescence detector 1046A (set at 373 nm for excitation and 448 nm for emission) was used with a MonoQ HR 5/5 column (0.5 × 5 cm, Pharmacia, Sweden). Samples were eluted with 10 mM TrisHCl (pH 8.0) containing 0-0.7 M NaCl at a flow rate of 0.5 ml/min.
Oligo/polySia samples
Colominic acid (Na-salt, molecular weight range 10,000-12,000, DP ~ 35) was a product of Nacalai tesque (Kyoto, Japan). [alpha]2-9-Linked polyNeu5Ac (Bhattacharjee et al., 1975) was a gift from Dr. Harold Jennings, National Research Council of Canada, Canada. Sialic acid-rich glycoprotein (polySia-gp) containing [alpha]2-5-Oglycolyl-linked poly/Neu5Gc was isolated and fractionated from jelly of Hemicentrotus pulcherrimus by a method modified from the original (Kitazume et al., 1994a). Oligosialic acids with known degree of polymerization (DP) were prepared by controlled acid hydrolysis of the polymers and separated by preparative anion-exchange chromatography on DEAE-Sephadex A-25 by the method described previously (Nomoto et al., 1982). Sea urchin egg cell surface glycoprotein (ESP-Sia) and 9-O-sulfated oligoNeu5Gc samples prepared from ESP-Sia (Kitazume et al., 1996) were kindly given by Dr. Shinobu Kitazume-Kawaguchi (RIKEN, Saitama, Japan). Isolation of rainbow trout egg PSGP (polysialoglycoprotein) was as described previously (Nomoto et al., 1982).
Mild acid hydrolysis and derivatization
Unless otherwise stated, oligo/polySia samples analyzed by HPAEC-PED were subjected to prehydrolysis under mild conditions set for each analysis and described in the figure captions. All hydrolysates were adjusted to alkaline pH with 0.5 N NaOH before injection to cleave intramolecular ester linkages formed during acid hydrolysis (Cheng et al., 1998). For fluorometric HPLC analysis, samples were derivatized with DMB (1,2-diamino-4,5-methylenedioxybenzene, Dojinbo, Japan) under acidic conditions described for each series of oligo/polySia samples without prehydrolysis. Sample solutions were adjusted to alkaline pH (pH ~ 13) with NaOH after derivatization. Concentration of other reagents in the derivatization mixture was as described previously (Hara et al., 1987; Inoue et al., 1996).
We are indebted to Dr. Y.C.Lee (The Johns Hopkins University) for valuable information for HPAEC-PED analysis. For the terminology of PED, we thank Dr. W.R.LaCourse (University of Maryland, Baltimore County) for suggestion. This research was supported in part by NSC Grant 87-2311-B-001-122, the Grant from Academia Sinica, and National Health Research Institutes Grant DOH88-HR-805.
Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; KDN, 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid; Sia, sialic acid; HPLC, high-performance liquid chromatography; HPAEC, high-performance anion-exchange chromatography; PED, pulsed electrochemical detection; DP, degree of polymerization; DMB, 1,2-diamino-4,5-methylenedioxybenzene; FD, fluorescence detector; PSGP, [alpha]2-8-linked oligo/polyNeu5Gc chain-containing glycoprotein isolated from salmonid fish eggs; polySia-gp, [alpha]2-5-Oglycolyl-linked oligo/polyNeu5Gc chain-containing glycoprotein isolated from the jelly coat of sea urchin eggs; ESP-Sia, [alpha]2-5-Oglycolyl-linked oligoNeu5Gc chain-containing glycoprotein isolated from the sea urchin egg cell surface complex.
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