From the Department of Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan, the ¶ Department of Applied Biological Sciences, School of
Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan, and the
Institute of Biological Chemistry, Academia
Sinica, Nankang, Taipei 115, Taiwan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The occurrence of the 2
8-linked oligomeric
form of N-glycolylneuraminic acid (oligo-Neu5Gc) residues
in mammalian glycoproteins was unequivocally demonstrated using a newly
developed anti-oligo/poly-Neu5Gc monoclonal antibody as well as by
chemical and biochemical methods. First, the antibody, designated
mAb.2-4B, which specifically recognized oligo/poly-Neu5Gc with a
degree of polymerization of >2, was developed by establishing a
hybridoma cell line from P3U1 myeloma cells fused with splenocytes from
an MRL autoimmune mouse immunized with
dipalmitoylphosphatidylethanolamine-conjugated oligo/poly-Neu5Gc. Second, oligo-Neu5Gc was shown to occur in glycoproteins derived from
pig spleen by Western blot analysis using mAb.2-4B, which was also
confirmed by fluorometric high performance liquid chromatographic analysis of the product of periodate oxidation/reduction/acid hydrolysis of the purified glycopeptide fractions and by TLC and 600-MHz 1H NMR spectroscopic analysis of their mild acid
hydrolysates. Finally, the ubiquitous occurrence of oligo-Neu5Gc chains
as glycoproteinaceous components in Wistar rat tissue was
immunochemically indicated. This is the first example demonstrating the
diversity in oligo/poly-Sia structure in mammalian glycoproteins, where
only poly-N-acetylneuraminic acid is known to occur. Such
diversity in oligo/poly-Sia structure also implicates a diverged array
of biological functions of this glycan unit in glycoproteins.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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). 2
8-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,
2
8-linked poly-Neu5Ac structure was shown to occur in various
animal glycoproteins from insect to human by using immunochemical and
enzymatic probes specific to
2
8-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 Neu5Gc2
8Neu5Gc 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
High molecular mass PSGPs, O. mykiss
PSGP containing only 2
8-linked oligo/poly-Neu5Gc structure and
Salvelinus namaycush PSGP containing exclusively
2
8-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
2
8-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
2
8-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 2
8-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
2
8-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 2
8-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 Neu5Gc
2
8Neu5Gc 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of a Monoclonal Antibody (mAb.2-4B) Specific to
2
8-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.
|
|
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.
|
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).
|
Identification of 2
8-Linked Oligo-Neu5Gc Structure in
Glycopeptides from Pig Spleen by C7/C9
Analysis--
For chemical detection of
2
8-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 Neu5Gc
2
8Neu5Gc
2
,
Neu5Gc
2
8Neu5Ac
2
, Neu5Ac
2
8Neu5Gc
2
, and
Neu5Ac
2
8Neu5Ac
2
. 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).
|
|
Identification of Di-Sia Structure (Neu5Gc2
8Neu5Gc) 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, Neu5Gc
2
8Neu5Gc (Fig. 7), indicating that
fraction A-IV has
2
8-linked oligo-Neu5Gc structure with DP
2. To confirm this band as Neu5Gc
2
8Neu5Gc, the material eluted
from the band was further purified for 1H NMR measurement
by preparative TLC and denoted A-IV-MH
.
|
|
1H NMR Measurement of A-IV-MH--
600-MHz
1H NMR spectra of authentic Neu5Gc
2
8Neu5Gc 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 Neu5Gc
2
8Neu5Gc
2
structure exists in
the pig spleen glycopeptide fractions.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2
8-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
2
8-linked
oligo/poly-Neu5Gc, but not with
2
8-linked oligo/poly-Neu5Ac or
2
8-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
(8Neu5Gc
2)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 2
8-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 Neu5Gc
2
8Neu5Gc was found by the mild acid
hydrolysis/TLC analysis of fraction A-IV. This band was unequivocally
identified to be Neu5Gc
2
8Neu5Gc 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 Neu5Gc
2
8Neu5Ac structure,
but not Neu5Ac
2
8Neu5Ac 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 2
8-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 GD3, GT3,
GD1c, and GQ1b 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)GD1c 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
Neu5Gc
2
6Gal
1
4GlcNAc 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 2
8-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
2
8-linked oligo/poly-Sia in mammalian glycoproteins and the immunospecificity of the presently available anti-
2
8-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.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. K. Furukawa (Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan) for constant encouragement and useful discussions throughout this research. Two of us (C. S. and K. K.) thank Professor J. Roth and Dr. C. Zuber (University of Zürich, Zürich, Switzerland) for providing us with Wistar rat tissues. We also thank Dr. A. Suzuki (Tokyo Metropolitan Institute of Medical Science) for providing us with acid-treated S. minnesota, Professor Y. Nagai (Medical and Dental University of Tokyo) for the kind gift of P3U1 cells, and Dr. I. Ichiba and Professor M. Isobe (Nagoya University, Nagoya, Japan) for obtaining the 600-MHz 1H NMR spectra. Finally, two of us (Y. I. and S. I.) express sincere thanks to Professor Rick Troy (University of California, Davis, CA) for constant support and understanding of our research work on oligo- and polysialic acids from a very early stage.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant-in-aid NSC 86-2311-B-001-096 from the National Science Council of Taiwan and a Grant-in-aid from Academia Sinica (to Y. I.), Monbusho International Scientific Research Program Joint Research Grant-in-aid 08044253 (to K. K.), and a grant-in-aid for the promotion of science from the Ministry of Education, Science, and Culture of Japan (to C. S.).
§ Present address: Dept. of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan.
** To whom correspondence should be addressed. Fax: 886-2-788-9759; E-mail: syinoue{at}gate.sinica.edu.tw.
1
The abbreviations used are: Neu5Ac,
N-acetylneuraminic acid; Neu5Gc,
N-glycolylneuraminic acid; KDN, deaminoneuraminic acid (2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid); PSGP, polysialoglycoprotein; mAb, monoclonal antibody; PE,
dipalmitoylphosphatidylethanolamine; HPLC, high performance liquid
chromatography; DMB, 1,2-diamino-4,5-methylenedioxybenzene; DP, degree
of polymerization; PBS, phosphate-buffered saline; ELISA, enzyme-linked
immunosorbent assay; BSA, bovine serum albumin; oligo/poly-Neu5Gc,
homo-oligomer/polymer of 2
8-linked Neu5Gc; oligo/poly-Neu5Ac,
homo-oligomer/polymer of
2
8-linked Neu5Ac; oligo/poly-Sia,
oligomer/polymer of
2
8-linked sialic acid; oligo/poly-KDN, homo-oligomer/polymer of
2
8-linked KDN; GD3,
Sia
2
8Sia
2
3Gal
1
4 Glc
1
1Cer; GT3,
Sia
2
8Sia
2
8Sia
2
3Gal
1
4Glc
1
1Cer;
GD1c, Sia
2
8Sia
2
3Gal
1
3GalNAc
1
4Gal
1
4Glc
1
1Cer;
GQ1b,
Sia
2
8Sia
2
3Gal
1
3GalNAc
1
4(Sia
2
8Sia
2
3) Gal
1
4Glc
1
1Cer.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|