INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Oligo/polysialic acid structure represents a group of glycan chains consisting of N-acetylneuraminic acid (Neu5Ac),¹ N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN) (1, 2). 28-Linked oligo/poly-Neu5Gc structure was first found in polysialoglycoprotein (PSGP) isolated from the unfertilized eggs of rainbow trout (Oncorhynchus mykiss) (3). Following this discovery, 28-linked poly-Neu5Ac structure was shown to occur in various animal glycoproteins from insect to human by using immunochemical and enzymatic probes specific to 28-linked oligo/poly-Neu5Ac glycotopes (4-11). Recently, we demonstrated that oligo/poly-Sia chains on PSGP isolated from Salvelinus fish eggs exhibit a remarkable degree of diversity in their building blocks arising from the different substitution at C-5, i.e. Neu5Ac, Neu5Gc, and KDN, and in the presence of either O-acetyl or O-lactyl substitution (2). These structural diversities in oligo/poly-Sia may be potentially relevant to the functional diversity that may be required for multiple cellular recognition processes on the cell surface in biological events, such as fertilization and early embryogenesis (2, 12).

Rather extensive debates have long been going on regarding the biological significance of the structural diversity in Sia residues that are expressed in species-specific, tissue-specific, developmental stage-specific, and tumor-specific manners (1, 13-17). We were motivated to confirm the diversity in oligo/poly-Sia structure not only in teleost fishes, but also in mammals because the components of Sia in most animal tissues consist of not only Neu5Ac and Neu5Gc (18), but also KDN (19). The amount of oligo/poly-Sia expressed on mammalian cells was anticipated to be so tiny that establishment of highly sensitive immunochemical probes and chemical methods was of first priority. As shown by previous studies (20, 21), development of anti-oligo/poly-Sia antibodies appeared to be difficult because of their structural similarity to endogenous glycolipids and glycoproteins present in neural and extraneural tissues (1, 22, 23), and the precise determination of the immunospecificity of the anti-oligo/poly-Sia antibodies was troublesome because immobilization of the oligo/poly-Sia chains on plastic plates, membranes, and TLC plates was difficult (24).

In this study, we developed a new monoclonal antibody, mAb.2-4B, specific to oligo/poly-Neu5Gc by immunization of MRL/MpjUmm-lpr autoimmune mice with dipalmitoylphosphatidylethanolamine (PE)-conjugated oligo/poly-Neu5Gc and determined its immunospecificity using these PE-conjugated oligo/poly-Sia chains for solidification on the plastic surface (24). Subsequently, using this antibody, the presence of oligo/poly-Neu5Gc structure on glycoproteins derived from mammalian tissues was suggested. To confirm this, chemical detection was carried out by the new fluorometric high performance liquid chromatography (HPLC) method (19, 25, 26) in conjunction with periodate oxidation (C₇/C₉ analysis) and the mild acid hydrolysis/TLC method (2, 27). The Neu5Gc28Neu5Gc sequence was identified in glycopeptides derived from pig spleen. These results, together with those obtained in the previous studies using mAb.kdn8kdn (28-30), specific to oligo/poly-KDN, show that the diversity in poly-Sia structure is observed not only in fish egg glycoproteins, but also in mammal-derived glycoproteins.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- High molecular mass PSGPs, O. mykiss PSGP containing only 28-linked oligo/poly-Neu5Gc structure and Salvelinus namaycush PSGP containing exclusively 28-linked oligo/poly-Neu5Ac structure, were isolated from the unfertilized eggs of rainbow trout (O. mykiss) and lake trout (S. namaycush), respectively, as described previously (2). KDN-rich glycoprotein containing 28-linked KDN chains was isolated from the ovarian fluid of rainbow trout as described previously (31). Clostridium perfringens exosialidase and Arthrobacter ureafaciens exosialidase were purchased from Boehringer Mannheim (Mannheim, Germany) and Nacalai (Kyoto, Japan), respectively. Peptide:N-glycanase F was obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Colominic acid in sodium salt form and PE were purchased from Sigma. Affinity-purified peroxidase-conjugated goat anti-mouse IgM + IgG antibody was obtained from American Qualex. Alkaline phosphatase-conjugated goat anti-mouse IgM antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt and nitro blue tetrazolium chloride were purchased from Life Technologies, Inc. Prestained molecular mass markers were obtained from Bio-Rad. 1,2-Diamino-4,5-methylenedioxybenzene (DMB) was a product of Dojindo Laboratories (Kumamoto, Japan).

Chemical Analysis-- Neu5Ac and Neu5Gc were quantitated by the resorcinol method (32) and the thiobarbituric acid method (33, 34). KDN was quantitated by the thiobarbituric acid method as described (35).

Preparation of Oligo/poly-Neu5Ac, Oligo/poly-Neu5Gc, Oligo/poly-KDN, and a Series of Oligo/poly-Neu5Gc Chains with Defined Degrees of Polymerization (DPs)-- These oligo/poly-Sia chains were prepared as described previously (24). For preparation of the series of 28-linked oligo/poly-Neu5Gc chains with known DPs, ~5 mg of oligo/poly-Neu5Gc fraction obtained from O. mykiss PSGP were applied to a Mono-Q HR5/5 anion-exchange column (0.5 × 5 cm; Pharmacia, Uppsala, Sweden) in an Irika HPLC system. The sample was loaded onto the column and eluted with 5 mM Tris-HCl (pH 8.0) followed by an NaCl gradient (0-320 mM) in 5 mM Tris-HCl (pH 8.0). The flow rate was 500 µl/min, and fractions were collected every minute. Oligomers with DPs ranging from 1 to 9 thus obtained were separately desalted by chromatography on a Sephadex G-25 column (1.7 × 140 cm; eluted with 5% ethanol) and lyophilized.

Synthesis of Oligo/poly-Sia-PE-- Lipidation was carried out essentially according to the method of Stoll et al. (Ref. 36; see Refs. 24 and 37), which is based on reductive amination. Oligo/poly-Neu5Gc with DP = 1-13, on average 6 (1.0 mg of Sia), in 100 µl of water was incubated at 60 °C for 2 h with PE (4.5 mg) dissolved in 900 µl of a mixture of chloroform/methanol (1:2, v/v). One milligram of sodium cyanoborohydride in 100 µl of methanol was added and further incubated at 60 °C for 16 h. The solution was applied to a DEAE-Toyopearl 650 M anion-exchange column and eluted with chloroform, methanol, and 2 M sodium acetate (30:60:8, v/v/v). The eluent was desalted by chromatography on a Sephadex G-50 column (1.2 × 100 cm; eluted with water) and lyophilized. For synthesis of a series of PE-conjugated oligo/poly-Neu5Gc chains with known DPs, 0.02 µmol of each oligo/poly-Neu5Gc was dissolved in 10 µl of water and lipidated as described previously (24).

Immunization of Mice and Production of Hybridoma Cells-- MRL/MpjUmmCrj-lpr autoimmune mice (Charles River, Yokohama, Japan) were immunized by intravenous injections at days 0, 4, 7, 11, and 21 of 1.5 µg of Sia from oligo/poly-Neu5Gc-PE noncovalently adsorbed to 50 µg of acid-treated Salmonella minnesota in 10 mM sodium phosphate buffer (pH 7.2) containing 0.15 M NaCl (PBS) according to the procedure reported by Galanos et al. (Ref. 38; see Ref. 39). On day 24, a spleen was excised, and the cells were dissociated. Spleen cells (3 × 10⁸) were fused with 4.1 × 10⁷ P3-X63 Ag8.U1 (P3U1) mouse myeloma cells (39) according to the procedure of Kohler and Milstein (40). The fused cells were suspended in Dulbecco's modified essential medium (Life Technologies, Inc.) containing 20% fetal bovine serum (lot 8409, Filtron, Brooklyn, Australia), 500 µM hypoxanthine, 20 µM aminopterin, and 800 µM thymidine (Sigma) and inoculated onto 96-well polystyrene plates (Nunc, Roskidle, Denmark). The hybridoma cells were screened by measuring titers of the supernatant of the cells with oligo/poly-Neu5Gc-PE and oligo/poly-Neu5Ac-PE. Antibody titers were monitored by the solid-phase enzyme-linked immunosorbent assay (ELISA) as described previously (24). The oligo/poly-Neu5Gc-PE-positive and oligo/poly-Neu5Ac-PE-negative hybridoma cells were cloned twice by limiting dilution, and a clone named 2-4B was obtained.

Purification of Mouse Anti-oligo/poly-Neu5Gc Antibody (mAb.2-4B)-- Anti-oligo/poly-Neu5Gc-PE antibody-producing clone 2-4B cells were first cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum and then in serum-free medium (Cosmedium-001, Cosmo Bio Co., Tokyo, Japan). The serum-free medium of the monoclone was collected and centrifuged at 5,700 × g for 10 min at room temperature. The immunoglobulin was precipitated from the supernatant solution with 50% saturated ammonium sulfate. The precipitate was collected by centrifugation at 5700 × g for 10 min, dissolved, dialyzed against PBS, and subjected to chromatography on a Sephacryl S-300 column (1.6 × 107 cm; eluted with PBS) (2). The immunoglobulin fractions were collected and stored at 80 °C until use. The immunoglobulin class was determined by a monoclonal typing kit (Amersham, Tokyo, Japan). The monoclonal antibody thus prepared was designated mAb.2-4B.

Antibody Binding Assay-- Antibody binding to various oligo/poly-Sia-PE chains, a series of oligo-Neu5Gc-PE chains (DP = 1-9), O. mykiss PSGP, and S. namaycush PSGP was determined using the ELISA method (24). A 96-well Aminoplate (Sumitomo Bakelite, Tokyo, Japan) was used. For the ELISA test, oligo/poly-Sia-PE and oligo-Neu5Gc-PE chains were serially diluted in ethanol (1.6-50 ng of Sia/well and 19-75 pmol of Neu5Gc/well, respectively) on the plate. mAb.2-4B was used at 1-200 µg/ml and was dissolved in PBS containing 1% bovine serum albumin (BSA). The ELISA procedure for O. mykiss PSGP and S. namaycush PSGP was as follows. The wells were coated with 50 µl of glycoproteins diluted serially in PBS (31-1000 ng of Sia/well) by incubation at 37 °C for 1 h. The wells were then blocked with 1% BSA/PBS at 37 °C for 2 h and incubated with mAb.2-4B (2.5 µg/well) at 4 °C overnight. Antibody binding was detected using peroxidase-conjugated goat anti-mouse IgG + IgM antibody as described previously (24).

Antibody Binding Assay for Acid-treated and Exosialidase-digested Oligo/poly-Neu5Gc-PE-- The wells of the 96-well Aminoplate were coated with oligo/poly-Neu5Gc-PE (7.5-125 ng of Sia/well) by incubation at 37 °C for 2 h and were washed three times with PBS. To each well were added 100 µl of 0.1 M HCl or 50 mM sodium acetate buffer (pH 4.8) with or without 2 microunits of A. ureafaciens exosialidase or 100 µl of PBS, and incubation was carried out at 37 °C for 20 h. The wells were rinsed three times with PBS and blocked with 1% BSA/PBS by incubation at 37 °C for 2 h. Fifty microliters of mAb.2-4B (2.5 µg/well) were added and incubated at 4 °C overnight. Antibody binding was carried out as described above.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- Pig spleen and various Wistar rat tissues were homogenized on ice in PBS containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1% aprotinin. The homogenates (5 mg of protein/ml) dissolved in Laemmli buffer (see Ref. 41) were placed at 65 °C for 15 min. After ~50-100 µg of protein were electrophoresed per lane on 3-15% gradient gels (10) or 10% polyacrylamide gels, immunoblotting on nitrocellulose or polyvinylidene fluoride membrane was performed (41) using a semidry blotting apparatus. Briefly, after transfer, the membrane was incubated in 0.1 M NaOH at 37 °C for 30 min, blocked for 1 h with PBS containing 1% BSA and 0.05% Tween 20, and then incubated with mAb.2-4B (50-100 µg/ml diluted with the same solution) at 4 °C overnight. The membrane was serially washed with PBS containing 0.05% Tween 20 and with Tris-buffered saline (0.1 M Tris-HCl (pH 7.5) and 0.15 M NaCl) containing 0.05% Tween 20 and then incubated with the secondary antibody, alkaline phosphatase-conjugated anti-mouse IgM antibody (50 µg/ml diluted with Tris-buffered saline containing 1% BSA and 0.05% Tween 20), at 37 °C for 45 min. The membrane was serially washed with Tris-buffered saline containing 0.05% Tween 20, Tris-buffered saline, and 0.1 M Tris-HCl (pH 9.5) containing 50 mM MgCl₂ and 150 mM NaCl. The membrane was developed with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (165 µg/ml) and nitro blue tetrazolium chloride (330 µg/ml) in 0.1 M Tris-HCl (pH 9.5) containing 50 mM MgCl₂ and 150 mM NaCl.

Exosialidase and Peptide:N-Glycanase F Treatments of the Blotting Membrane-- Tissue homogenates were electrophoresed and transferred to the nitrocellulose or polyvinylidene fluoride membrane as described above. After alkali treatment of the transblotted membrane, the membrane was treated with C. perfringens exosialidase (0.1 unit/ml) in 50 mM sodium acetate buffer (pH 5.0) containing 1% BSA at 37 °C for 18 h for rat tissue homogenates or with A. ureafaciens exosialidase (2.5 units/ml) in 50 mM sodium acetate buffer (pH 5.5) containing 1% BSA at 37 °C for 24 h or peptide:N-glycanase F (5 units/ml) in 250 mM phosphate buffer (pH 8.25) at 37 °C for 24 h for pig spleen homogenates.

Purification of Oligo-Neu5Gc-containing Glycopeptide(s) from Pig Spleen-- Eight-hundred grams of pig spleen (Shibaura Zouki, Tokyo, Japan) were homogenized in 2.4 liter of cold acetone and filtered. The acetone powder was delipidated with chloroform/methanol extraction (42). After washing with ethanol, the delipidated acetone powder (300 g) was incubated with actinase E (2.0 g; Kokusan Kagaku, Tokyo, Japan) in 2 liter of 0.1 M Tris-HCl (pH 8.0) containing 10 mM CaCl₂ and 0.5% Nonidet P-40 at 37 °C for 3 days (17). The solution was centrifuged at 5,700 × g for 20 min. The supernatant was then mixed with 0.5 volume of 90% phenol and centrifuged at 2100 × g for 15 min. The aqueous phase was removed, and the lower phase was mixed with 0.5 volume of 0.1 M Tris-HCl (pH 8.0) and centrifuged. The aqueous phase thus obtained was dialyzed against water and evaporated to 300 ml (2). The concentrated solution was then mixed with 600 ml of cold ethanol at 80 °C for 1 h and centrifuged at 5700 × g for 15 min. The supernatant was subjected to chromatography on a DEAE-Sephadex A-25 anion-exchange column (2.1 × 42 cm) with a linear gradient of NaCl (0-1.0 M) in 10 mM Tris-HCl (pH 8.0). Fraction A-IV (see Fig. 4) was further purified by chromatography on a Sephacryl S-100 column (1.2 × 103 cm; eluted with 0.1 M NaCl) and desalted by passage through a Sephadex G-25 column (1.2 × 98 cm; eluted with 5% ethanol).

Detection of 28-Linked Oligo-Sia by the Periodate Oxidation/Fluorometric HPLC Method (C₇/C₉ Analysis)-- Samples (~1 µg of Sia) were dissolved in 25 µl of 40 mM sodium acetate buffer (pH 5.5) and after addition of 2 µl of 0.25 M NaIO₄ left at 0 °C for 3 h in the dark. Then, 5 µl of 3% ethylene glycol and 32 µl of 0.5 M NaBH₄ dissolved in 0.2 M sodium borate buffer (pH 8.0) were added successively and left at 0 °C overnight. During these procedures, nonreducing terminal Neu5Ac or Neu5Gc residues were oxidized to give rise to the C₇ analog of Neu5Ac (5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid; C₇- (Neu5Ac)) or Neu5Gc (5-hydroxylacetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid; C₉(Neu5Gc)), whereas internal residues in 28-linked oligo/poly-Sia chains remained intact: Neu5Ac, C₉(Neu5Ac); or Neu5Gc, C₉(Neu5Gc) (25, 43, 44). The resultant sample was hydrolyzed in 0.1 M trifluoroacetic acid at 80 °C for 1 h and dried up. A 7 mM solution of DMB was freshly prepared by dissolving DMB dihydrochloride in 50 mM trifluoroacetic acid containing 0.75 M 2-mercaptoethanol and 18 mM sodium hydrosulfite. The dried sample was dissolved in 20 µl of 10 mM trifluoroacetic acid and incubated at 50 °C for 2 h after addition of 20 µl of the DMB solution. The reaction mixture (2-20 µl) was directly analyzed by a Jasco LC-900 HPLC system equipped with a Jasco FP-920 fluorescence detector (wavelengths for excitation set at 373 nm and emission at 448 nm), operating isocratically at 1.0 ml/min at a column temperature of 26 °C. A TSK-gel ODS-120T (250, inner diameter, × 4.6 mm) was used. Methanol/acetonitrile/water (7:9:84, v/v/v) was used as eluent (19, 26). Retention times and response factors on HPLC for the C₇ and C₉ analogs of Neu5Gc and Neu5Ac were determined by the concomitant derivatization of the following compounds: fraction S6 derived from chum salmon egg PSGP (45) for C₇(Neu5Gc)-DMB and C₉(Neu5Gc)-DMB and OF-gp glycopeptide derived from trout ovarian fluid glycoproteins (44) for C₇(Neu5Ac)-DMB and C₉(Neu5Ac)-DMB.

Identification of 28-Linked Neu5Gc Oligomer by the Mild Acid Hydrolysis/TLC Method-- Fraction A-IV (see Fig. 6a) was incubated with 50 mM sodium acetate buffer (pH 4.8) at 37 °C for 48 h and subjected to chromatography on a Sephacryl S-100 column (1.2 × 113 cm; eluted with 0.1 M NaCl). The free oligosialic acid fraction was collected and desalted by passage through a Sephadex G-25 column (1.2 × 108 cm; eluted with 5% ethanol). One microgram of sialic acid was spotted on a TLC plate (Silica Gel 60, Merck); developed in 1-propanol, 25% NH₄OH, and water (6:1:2.5, v/v/v) for 12 h; and visualized by the resorcinol reagent (2).

600-MHz ¹H NMR Spectroscopy-- The free sialic acid fraction was subjected to preparative TLC on a Silica Gel 60 plate as previously reported (2). The dimeric sialic acid was further purified by passage through a Sephadex G-25 column and denoted A-IV-MH. A-IV-MH and authentic Neu5Gc28Neu5Gc prepared from O. mykiss PSGP (2) were subjected to 600-MHz ¹H NMR spectral measurements with a Bruker AMX-600 spectrometer. Sample preparation and conditions for measurements were previously described (2).

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Preparation of a Monoclonal Antibody (mAb.2-4B) Specific to 28-Linked Oligo-Neu5Gc-- Three MRL/MpjUmmCrj-lpr autoimmune mice were immunized with oligo/poly-Neu5Gc-PE for 24 days as described under "Experimental Procedures," and splenocytes prepared from one of the mice were fused with the P3U1 cells.

Characterization of mAb.2-4B-- The class of mAb.2-4B was determined to be IgM. A number of papers reported that the class of antibodies raised against glycolipids was dominantly IgM, but not IgG (see, for example, Refs. 46 and 47). Fig. 1 shows the results of binding of mAb.2-4B with oligo/poly-Neu5Gc-PE on an ELISA plate. As little as 3 ng (as Sia) of oligo/poly-Neu5Gc-PE was detectable when 20-200 µg/ml mAb.2-4B was used. As shown in Fig. 2a, mAb.2-4B reacted only with oligo/poly-Neu5Gc-PE. No reaction was observed with oligo/poly-Neu5Ac-PE, oligo/poly-KDN-PE, or PE, even at higher concentrations of these neoglycolipids (50 ng of Sia/well). Acid treatment (0.1 M HCl, 37 °C, 20 h) or exosialidase digestion (A. ureafaciens, 2.5 microunits, 37 °C, 20 h) of oligo/poly-Neu5Gc-PE resulted in complete loss of binding (Fig. 2b), indicating the requirement of oligo/poly-Neu5Gc structure for binding of mAb.2-4B.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Reactivities of mAb.2-4B with oligo/poly-Neu5Gc-PE as determined by ELISA. Wells were coated with oligo/poly-Neu5Gc-PE at varying amounts (3-50 ng/well) and incubated with diluted mAb.2-4B at protein concentrations of 200 (), 100 (), 50 (), 20 (), 10 (), and 5 () µg/ml.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Characterization of mAb.2-4B. a, shown is the reactivity of mAb.2-4B (2.5 µg/well) with oligo/poly-Neu5Gc-PE (), oligo/poly-Neu5Ac-PE (), oligo/poly-KDN-PE (), and PE () on wells coated at 1.5-50 ng of Sia/well or their equivalent amount of lipid. b, plastic wells were coated with oligo-Neu5Gc-PE (7.5-125 ng of Sia/well) and treated with PBS (), 0.1 M HCl (), and 2.5 microunits of exosialidase/well () at 37 °C for 20 h. Each well was assayed for binding with mAb.2-4B (2.5 µg/well) as described under "Experimental Procedures." c, shown is the reactivity of mAb.2-4B (2.5 µg/well) with a set of oligo-Neu5Gc-PE chains with DP = 1-9 (75 pmol/well). d, shown is the reaction of mAb.2-4B (2.5 µg/well) with O. mykiss PSGP (PSGP(Om); ) containing exclusively 28-linked oligo/poly-Neu5Gc and S. namaycush PSGP (PSGP(Sn); ) containing exclusively 28-linked oligo/poly-Neu5Ac on the well coated with various amounts (31-1000 ng of Sia/well).

Immunochemical Detection of Oligo/poly-Neu5Gc Structure in Pig Spleen Using mAb.2-4B-- Immunochemical detection of oligo/poly-Neu5Gc structure was performed with pig spleen homogenates. Homogenates were run on a 10% polyacrylamide gel and transferred to the polyvinylidene fluoride membrane. After alkali treatment, the membranes were treated with exosialidase or peptide:N-glycanase F and stained with mAb.2-4B (Fig. 3). Two bands of 50 and 52 kDa completely disappeared after the treatment with exosialidase or peptide:N-glycanase F. These results strongly indicate that the glycoproteins of 50 and 52 kDa contained N-linked glycan chain(s) with oligo/poly-Neu5Gc structure with DP  2. 

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of pig spleen homogenates using mAb.2-4B. Homogenates were run on 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Lane 1, membrane incubated with mAb.2-4B and visualized as described under "Experimental Procedures"; lane 2, exosialidase-treated membrane; lane 3, peptide:N-glycanase F-treated membrane.

Preparation of Glycopeptides from Pig Spleen-- The delipidated acetone powder (300 g) prepared from 800 g of pig spleen was exhaustively digested with actinase E (19). The soluble glycopeptide fraction obtained on phenol and the subsequent ethanol precipitation was subjected to DEAE-Sephadex A-25 chromatography (Fig. 4). The sialic acid-containing fractions were divided into four pooled fractions, A-I, A-II, A-III, and A-IV, with yields of Sia of 11, 11, 23, and 2.7 mg, respectively. Fraction A-IV was further subjected to gel filtration on Sephacryl S-100 because oligo-Sia structure was found to be enriched in this fraction as shown below. Fraction A-IV gave a single peak with a molecular mass of ~30 kDa (see Fig. 6a).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   DEAE-Sephadex A-25 chromatography of glycopeptide fractions derived from pig spleen. The column (2.1 × 41 cm) was eluted with 0-0.8 M NaCl in 10 mM Tris-HCl (pH 8.0). The elution profile was monitored by absorbance at 280 nm () and by the resorcinol method (A580; ) for sialic acid. The broken line represents a gradient curve of NaCl concentration. Fractions A-I, A-II, A-III, and A-IV were pooled as indicated by the bar.

Identification of 28-Linked Oligo-Neu5Gc Structure in Glycopeptides from Pig Spleen by C₇/C₉ Analysis-- For chemical detection of 28-linked oligo-Sia, the periodate oxidation/fluorometric HPLC method was applied for the glycopeptide fractions A-I through A-IV. The nonreducing terminal Sia residues were oxidized to the C₇ analog of Sia upon reaction, whereas internal 8-O-substituted Sia residues were resistant to oxidization, thus remaining as the C₉ compound of Sia. The results are shown in Fig. 5 and Table I. Fractions A-I and A-II had no oligosialic acid structure because no C₉ derivative of Neu5Gc or Neu5Ac was detected (Fig. 5). The C₉ derivatives of Neu5Gc and Neu5Ac were found in fractions A-III and A-IV, suggesting the presence of homo- and/or hetero-oligomeric structure of Neu5Gc and Neu5Ac, such as Neu5Gc28Neu5Gc2, Neu5Gc28Neu5Ac2, Neu5Ac28Neu5Gc2, and Neu5Ac28Neu5Ac2. The molar ratio of C₉ to C₇ derivatives was higher in fraction A-IV (0.07~0.09) than in fraction A-III (~0.01).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   HPLC of DMB derivatives obtained by periodate oxidation/DMB derivatization of fractions A-I through A-IV. Samples of fraction A-I through A-IV (1 µg of Sia) were analyzed. Peaks 1, 1, 2, and 2 represent C7(Neu5Gc)-DMB, C9(Neu5Gc)-DMB, C7(Neu5Ac)-DMB, and C9(Neu5Ac)-DMB, respectively. HPLC was performed as described under "Experimental Procedures." Elution profiles were fluorometrically monitored as a function of retention time.

Identification of Di-Sia Structure (Neu5Gc28Neu5Gc) in Fraction A-IV by Mild Acid Hydrolysis/TLC-- Fraction A-IV (1.5 mg of Sia) was treated with 50 mM sodium acetate buffer (pH 4.8) at 37 °C for 2 days, and the free oligo-Sia fraction (A-IV-MH) was separated from major glycopeptide fractions by Sephacryl S-100 chromatography (Fig. 6b). After desalting, A-IV-MH was analyzed by TLC (Fig. 7). The band in lane 3 marked by the arrowhead was identical in mobility to the authentic sample of the dimer, Neu5Gc28Neu5Gc (Fig. 7), indicating that fraction A-IV has 28-linked oligo-Neu5Gc structure with DP  2. To confirm this band as Neu5Gc28Neu5Gc, the material eluted from the band was further purified for ¹H NMR measurement by preparative TLC and denoted A-IV-MH.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Sephacryl S-100 chromatography of fraction A-IV before (a) and after (b) mild acid hydrolysis. The elution profile was monitored by the thiobarbituric acid method (A549) for sialic acid. Fraction A-IV obtained by DEAE-Sephadex A-25 chromatography (see Fig. 4) was subjected to Sephacryl S-100 gel filtration (1.2 × 103-cm column), yielding a single peak of sialoglycopeptide under ~30 kDa in a. The peak fraction was renamed A-IV and subjected to mild acid hydrolysis (pH 4.8, 2 days, 37 °C). The free oligosialic acid fraction in b was pooled and denoted A-IV-MH. The elution position of authentic Neu5Gc dimer is indicated by the arrowhead.

View larger version (48K): [in this window] [in a new window]   Fig. 7.   TLC analysis of the free oligosialic acid fraction (A-IV-MH) derived from fraction A-IV by pH 4.8-catalyzed hydrolysis at 37 °C for 2 days. About 1 µg of A-IV-MH was spotted on a Silica Gel 60 plate and developed in 1-propanol, 25% NH4OH, and water (6:1:2.5, v/v/v) for 12 h. The bands were visualized by heating the plate at 100 °C for 30 min after spraying with the resorcinol reagent. As standards, the partial acid hydrolysates of colominic acid (28-linked oligo/poly-Neu5Ac) and O. mykiss PSGP (28-linked oligo/poly-Neu5Gc)), supplemented with Neu5Ac and Neu5Gc, respectively, were run in lanes 1 and 2. Lane 3, A-IV-MH. The numbers in lanes 1 and 2 represent the corresponding DPs of oligo/poly-Sia. O, origin.

¹H NMR Measurement of A-IV-MH-- 600-MHz ¹H NMR spectra of authentic Neu5Gc28Neu5Gc obtained from partial acid hydrolysis of O. mykiss PSGP and A-IV-MH described above were determined in D₂O at 25 °C. Proton signals were assigned as listed in Table II, and the chemical shifts for both the authentic dimer and A-IV-MH were found to be identical. Therefore, it can be concluded that Neu5Gc28Neu5Gc2 structure exists in the pig spleen glycopeptide fractions.

View this table: [in this window] [in a new window]   Table II Proton chemical shifts of authentic Neu5Gc dimer and A-IV-MH (Neu5Gc(2)28Neu5Gc(1))

SDS-Polyacrylamide Gel Electrophoresis/Western Blotting of Rat Tissues-- To detect the oligo/poly-Neu5Gc epitope in various rat tissues, SDS-polyacrylamide gel electrophoresis/Western blot analysis was carried out using mAb.2-4B. mAb.2-4B-reactive components were present in all rat tissues examined (Fig. 8a). These reactive bands were not observed in the control experiments (Fig. 8c). Smear bands of 10-66 kDa observed for submaxillary gland and thymus and those of 38-66 kDa for lung, spleen, and pancreas disappeared after the exosialidase treatment of the membrane. Notably, the 36-kDa band observed for submaxillary gland, thymus, and lung; the 38-kDa band for heart and spleen; the 40-kDa band for lung; the 42-kDa band for heart; the 47- and 50-kDa bands for adrenal gland; the 54-kDa band for pancreas; the 71-kDa band for adrenal gland; and the 130-kDa band for thymus completely disappeared after the sialidase treatment. These results strongly suggest that oligo/poly-Neu5Gc structure is present in glycoproteins of various rat tissues. Several mAb.2-4B-positive bands persisted in their stainability even after the exosialidase treatment (Fig. 8b). The observed reactivities may possibly be attributed to those with oligo-Neu5Gc sequences that terminate with an exosialidase-resistant KDN residue or that are modified by alkali-resistant substitution, although specificity of mAb.2-4B for such structures is unknown.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Western blot analysis of Wistar rat tissue homogenates using mAb.2-4B. Homogenates were run on 3-15% polyacrylamide gels in the presence of SDS, and the protein bands were electrophoretically transferred to nitrocellulose membranes. The membranes were soaked in 0.1 M NaOH at 37 °C for 30 min to remove possible O-acyl groups on the Sia residues and subjected to the immunostaining procedures described under "Experimental Procedures." a, the membrane was immunostained without sialidase treatment. b, the membrane was treated with sialidase prior to immunostaining. c, the membrane was immunostained without using mAb.2-4B as a primary antibody. Lane 1, submaxillary gland; lane 2, thymus; lane 3, lung; lane 4, heart; lane 5, liver; lane 6, spleen; lane 7, pancreas; lane 8, adrenal gland. The asterisks indicate the sialidase-sensitive band as described under "Results." No staining was observed in any of these lanes when the membrane was not treated with the primary antibody.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

To investigate the diversity in oligo/poly-Sia structure not only in fish eggs, but also in mammalian tissues, a monoclonal antibody that is highly specific to 28-linked oligo/poly-Neu5Gc was developed using lipid-conjugated oligo/poly-Neu5Gc as an immunogen and the MRL autoimmune mouse as a host. Based on the ELISA method using lipidated oligo/poly-Sia, mAb.2-4B was shown to react only with 28-linked oligo/poly-Neu5Gc, but not with 28-linked oligo/poly-Neu5Ac or 28-linked oligo/poly-KDN. The DP of oligo/poly-Neu5Gc required for recognition by the antibody was 2.

The use of mAb.2-4B for immunochemical detection enabled us to detect the presence of oligo/poly-Neu5Gc chains in pig spleen glycoproteins. Western blot analysis revealed that several glycoprotein components were mAb.2-4B-positive in pig spleen homogenate (50- and 52-kDa glycoproteins). In these glycoproteins, oligo/poly-Neu5Gc was shown to reside on N-linked glycan chain(s) since the bands of these glycoproteins became mAb.2-4B-negative on peptide:N-glycanase F digestion. Some components contained mAb.2-4B-positive but sialidase-resistant structures even after alkali treatment of the membrane. These components may contain modified sialic acid residue(s) with alkali-resistant substituent or KDN-capping structure, i.e. KDN2(8Neu5Gc2)_n. The KDN capping of oligo-Neu5Gc chains is known to occur in fish egg PSGP (49) for protection of oligo-Neu5Gc chains from attacks by bacterial sialidases (50, 51) and is also considered to be a termination signal for elongation of oligo/poly-Sia chains (49).

The presence of 28-linked oligo-Neu5Gc structure in pig spleen glycopeptides was also confirmed by chemical and biochemical methods. Based on the fluorescence-assisted periodate C₇/C₉ analysis, the sialoglycopeptide fraction A-IV, which was eluted at higher NaCl concentrations on DEAE-Sephadex A-25 chromatography, was found to be rich in C₉ derivatives of Neu5Gc and Neu5Ac. The proportion of the internal Sia residues involved in the formation of oligo/poly-Sia structure to the total Sia residues in pig spleen was estimated to be 1.7%. A band identical in mobility to authentic Neu5Gc28Neu5Gc was found by the mild acid hydrolysis/TLC analysis of fraction A-IV. This band was unequivocally identified to be Neu5Gc28Neu5Gc by ¹H NMR measurement. No clear band due to the presence of Neu5Ac dimer or hybrid dimers of Neu5Ac and Neu5Gc was observed by TLC analysis, probably because these dimer structures were present less frequently as compared with di-Neu5Gc structure. Notably, no di-Neu5Ac was observed (Fig. 7, lane 3). Considering the occurrence of the comparable amount of internal Neu5Ac and Neu5Gc residues in fraction A-IV (Table I), this result suggests that most internal Neu5Ac residues may be involved in the formation of Neu5Gc28Neu5Ac structure, but not Neu5Ac28Neu5Ac structure. The failure to detect oligomers with DP  3 by TLC strongly suggests that the chain length of the oligosialyl chain in pig spleen glycoproteins is not large, but is most likely solely a dimer.

Furthermore, we also identified several mAb.2-4B-reactive glycoprotein components in various rat tissue homogenates. Fig. 8 shows a common occurrence of oligo/poly-Neu5Gc structure in various tissue glycoproteins. Although 28-linked di-Neu5Gc structure is known to occur in various mammalian gangliosides (46, 47), this is the first demonstration of the ubiquitous presence of oligo/poly-Neu5Gc-containing glycoproteins of mammalian origin. The DPs of these oligo/poly-Neu5Gc chains are presently unknown. However, they appear low, as found for pig spleen glycoproteins, because most mAb.2-4B-reactive bands were not so broad (Fig. 8) as usually observed for those of polysialylated neural cell adhesion molecules (4-11). Most intriguing is the elucidation of the biological functions of these oligo-Neu5Gc-containing glycoproteins, and it is therefore important to identify and characterize newly detected oligo-Neu5Gc-containing glycoproteins and to study if the expression of these glycoproteins is developmentally regulated. Some speculation on the biological functions of oligo-Neu5Gc can be allowed if one considers that oligo-Sia is now the common structural unit in both glycoproteins and gangliosides in mammals. Higher gangliosides such as G_D3, G_T3, G_D1c, and G_Q1b are considered to be involved in cell adhesion (52), differentiation (47, 53, 54), signal transduction (55), ADP-ribosylation (56), and specific oncodevelopmental markers (57, 58), where the sialic acid species of these gangliosides is, however, largely of the Neu5Ac type at present. Interestingly, (Neu5Gc)G_D1c has recently been identified as a marker for rat CD4⁺ T lymphocytes that produce interleukin-2 (47), where some di-Neu5Gc-specific functions were suggested, including regulation of differentiation of this type of T cells. As far as the biological importance of Neu5Gc is concerned, much discussion has been made regarding species-specific, tissue-specific, developmental stage-specific, and tumor-specific functions of this sialic acid species (1, 13-17). CD22, a sialic acid-binding lectin that is involved in B cell maturation and activation, is known to have a species-dependent preference in the recognition of sialic acid species. Mouse CD22 preferentially recognizes Neu5Gc26Gal14GlcNAc over the corresponding Neu5Ac version (59, 60), whereas human CD22 equally recognizes both forms of sialic acid (61). The differential specificity of these CD22 proteins directly indicates the importance of Neu5Gc residues in this cell adhesion process in mouse. Neu5Gc residues are also known not to occur so frequently in human glycoconjugates (18), and Hanganutziu-Deicher (HD) antigens are well known to be one of the oncofetal antigens in human (62). In this regard, it would be a strong possibility that oligo-Neu5Gc units could be identified as an oncofetal antigen in human using our mAb.2-4B antibody.

In summary, we show here for the first time that there exists a structural diversity in oligo/poly-Sia in mammalian glycoproteins other than fish egg glycoproteins. Recently, mAb.kdn8kdn (28), which specifically recognizes 28-linked oligo/poly-KDN structure (DP  2) (24), and deaminoneuraminase, which hydrolyzes only KDN ketosidic linkages (50, 51), were developed, and by combination of these sensitive and specific probes, the presence of oligo-KDN sequence was indicated in mammalian tissues (28, 29) and in some lung carcinoma cells (30). Furthermore, using a sensitive chemical method, KDN residues were confirmed unequivocally in mammalian tissues (19), although the chemical identification of oligo-KDN structure still remains to be elucidated. In Table III, the occurrence of 28-linked oligo/poly-Sia in mammalian glycoproteins and the immunospecificity of the presently available anti-28-linked oligo/poly-Sia antibodies are summarized. The significance of the diversity in sialic acid structure is now considered to reside in variations of the ligand determinants that are specifically recognized by cognate sialic acid-binding proteins such as selectin, CD22, sialoadhesin, a complement regulatory protein (H-protein), and influenza virus hemaggulutinins (14, 16, 17). Accordingly, the functional importance of the diversity in oligo/poly-Sia structure in mammalian tissue should be exemplified by identification of specific binding proteins that may be species-, tissue-, and developmental stage-specifically expressed on the cell surface.

View this table: [in this window] [in a new window]   Table III Occurrence of 28-linked oligo/poly-Sia structure in mammalian glycoproteins and available anti-28-linked oligo/poly-Sia antibodies with their chain length specificity