Identification of oligo-N-glycolylneuraminic acid residues in mammal-derived glycoproteins by a newly developed immunochemical reagent and biochemical methods.

The occurrence of the alpha2-->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.

acid (KDN) (1,2). ␣238-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, ␣238-linked poly-Neu5Ac structure was shown to occur in various animal glycoproteins from insect to human by using immunochemical and enzymatic probes specific to ␣238-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)(14)(15)(16)(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 (C 7 /C 9 analysis) and the mild acid hydrolysis/TLC method (2,27). The Neu5Gc␣238Neu5Gc 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.
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 ␣238-linked oligo/poly-Neu5Gc chains with known DPs, ϳ5 mg of oligo/poly-Neu5Gc fraction obtained from O. mykiss PSGP were applied to a Mono-Q HR5/5 anion-exchange column (0.5 ϫ 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 l/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 ϫ 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 ϭ 1-13, on average 6 (1.0 mg of Sia), in 100 l of water was incubated at 60°C for 2 h with PE (4.5 mg) dissolved in 900 l of a mixture of chloroform/methanol (1:2, v/v). One milligram of sodium cyanoborohydride in 100 l 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 ϫ 100 cm; eluted with water) and lyophilized. For synthesis of a series of PE-conjugated oligo/ poly-Neu5Gc chains with known DPs, 0.02 mol of each oligo/poly-Neu5Gc was dissolved in 10 l of water and lipidated as described previously (24).
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 ϫ 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 ϫ g for 10 min, dissolved, dialyzed against PBS, and subjected to chromatography on a Sephacryl S-300 column (1.6 ϫ 107 cm; eluted with PBS) (2). The immunoglobulin fractions were collected and stored at Ϫ80°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 ϭ 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 g/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 l 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 g/well) at 4°C overnight. Antibody binding was detected using peroxidase-conjugated goat anti-mouse IgG ϩ 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 l 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 l 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 g/well) were added and incubated at 4°C overnight. Antibody binding was carried out as described above.
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 CaCl 2 and 0.5% Nonidet P-40 at 37°C for 3 days (17). The solution was centrifuged at 5,700 ϫ g for 20 min. The supernatant was then mixed with 0.5 volume of 90% phenol and centrifuged at 2100 ϫ 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 Detection of ␣238-Linked Oligo-Sia by the Periodate Oxidation/ Fluorometric HPLC Method (C 7 /C 9 Analysis)-Samples (ϳ1 g of Sia) were dissolved in 25 l of 40 mM sodium acetate buffer (pH 5.5) and after addition of 2 l of 0.25 M NaIO 4 left at 0°C for 3 h in the dark. Then, 5 l of 3% ethylene glycol and 32 l of 0.5 M NaBH 4 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 C 7 analog of Neu5Ac (5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid; C 7 -(Neu5Ac)) or Neu5Gc (5-hydroxylacetamido-3,5-dideoxy-L-arabino-2heptulosonic acid; C 9 (Neu5Gc)), whereas internal residues in ␣238linked oligo/poly-Sia chains remained intact: Neu5Ac, C 9 (Neu5Ac); or Neu5Gc, C 9 (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 l of 10 mM trifluoroacetic acid and incubated at 50°C for 2 h after addition of 20 l of the DMB solution. The reaction mixture (2-20 l) 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, ϫ 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 C 7 and C 9 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 C 7 (Neu5Gc)-DMB and C 9 (Neu5Gc)-DMB and OF-gp glycopeptide derived from trout ovarian fluid glycoproteins (44) for C 7 (Neu5Ac)-DMB and C 9 (Neu5Ac)-DMB.
Identification of ␣238-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 ϫ 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 ϫ 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% NH 4 OH, and water (6:1:2.5, v/v/v) for 12 h; and visualized by the resorcinol reagent (2). 600-MHz 1 H 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-MHЈ. A-IV-MHЈ and authentic Neu5Gc␣238Neu5Gc prepared from O. mykiss PSGP (2) were subjected to 600-MHz 1 H NMR spectral measurements with a Bruker AMX-600 spectrometer. Sample preparation and conditions for measurements were previously described (2).

Preparation of a Monoclonal Antibody (mAb.2-4B) Specific
to ␣238-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.
Hybridoma colonies were screened for antibodies by assaying the binding affinity toward oligo/poly-Neu5Gc-PE but not toward oligo/poly-Neu5Ac-PE, and finally, one of the clones was established after subcloning by limiting dilution. The antibody released from the clone was designated mAb.2-4B. mAb.2-4B was prepared by precipitation of the serum-free culture supernatant with 50% saturated ammonium sulfate and gel filtration on a Sephacryl S-300 column, yielding 37 mg of immunoglobulin/2 liters of the culture supernatant.
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 Ն 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 ϳ30 kDa (see Fig. 6a).
Identification of ␣238-Linked Oligo-Neu5Gc Structure in Glycopeptides from Pig Spleen by C 7 /C 9 Analysis-For chemical detection of ␣238-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 C 7 analog of Sia upon reaction, whereas internal 8-O-substituted Sia residues were resistant to oxidization, thus remaining as the C 9 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 C 9 derivative of Neu5Gc or Neu5Ac was detected (Fig. 5). The C 9 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␣238Neu5-Gc␣23, Neu5Gc␣238Neu5Ac␣23, Neu5Ac␣238Neu5Gc␣23, and Neu5Ac␣238Neu5Ac␣23. The molar ratio of C 9 to C 7 derivatives was higher in fraction A-IV (0.07ϳ0.09) than in fraction A-III (ϳ0.01).
Identification of Di-Sia Structure (Neu5Gc␣238Neu5Gc) 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␣238Neu5Gc (Fig. 7), indicating that fraction A-IV has ␣238-linked oligo-Neu5Gc structure with DP Ն 2. To confirm this band as Neu5Gc␣238Neu5Gc, the material eluted from the band was further purified for 1 H NMR measurement by preparative TLC

Oligo-Neu5Gc Unit in Mammalian Glycoproteins
and denoted A-IV-MHЈ. 1 H NMR Measurement of A-IV-MHЈ-600-MHz 1 H NMR spectra of authentic Neu5Gc␣238Neu5Gc obtained from partial acid hydrolysis of O. mykiss PSGP and A-IV-MHЈ described above were determined in D 2 O at 25°C. Proton signals were assigned as listed in Table II, and the chemical shifts for both the authentic dimer and A-IV-MHЈ were found to be identical. Therefore, it can be concluded that Neu5Gc␣238Neu5Gc␣23 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 ␣238-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 ␣238-linked oligo/poly-Neu5Gc, but not with ␣238-linked oligo/poly-Neu5Ac or ␣238-linked oligo/poly-KDN. The DP of oligo/poly-Neu5Gc required for recognition by the antibody was Ն2.
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 Nlinked 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. KDN␣23(8Neu5Gc␣2) n 3. 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 ␣238-linked oligo-Neu5Gc structure in pig spleen glycopeptides was also confirmed by chemical and biochemical methods. Based on the fluorescence-assisted periodate C 7 /C 9 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 C 9 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␣238Neu5Gc was found by the mild acid hydrolysis/TLC analysis of fraction A-IV. This band was unequivocally identified to be Neu5Gc␣23 8Neu5Gc by 1 H 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␣23 8Neu5Ac structure, but not Neu5Ac␣238Neu5Ac structure. The failure to detect oligomers with DP Ն 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 ␣238-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.2-4Breactive 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 ϩ 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)(14)(15)(16)(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␣23 6Gal␤134GlcNAc 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 ␣238-linked oligo/poly-KDN structure (DP Ն 2) (24), and deaminoneuraminase, which hydrolyzes only KDN ketosidic linkages (50, 51), were developed, 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.  N-CAMs a of avians, amphibians, and mammals; postnatal tissues; human tumors; sodium channel of electroplax and rat brain Oligo/poly-Neu5Gc mAb.2-4B (DP Ն 2) Pig spleen and rat tissue glycoproteins Oligo/poly-KDN mAb.kdn8kdn (DP Ն 2) Rat and pig tissues; human lung carcinoma cells Oligo/poly-Neu5Ac, Neu5Gc Not available Pig spleen glycoproteins a N-CAMs, neural cell adhesion molecules; oligo/poly-Neu5Ac,Neu5Gc, hetero-oligomer/polymer composed of ␣238-linked Neu5Ac and Neu5Gc.

Oligo-Neu5Gc Unit in Mammalian Glycoproteins
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 ␣238-linked oligo/poly-Sia in mammalian glycoproteins and the immunospecificity of the presently available anti-␣238-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.