Identification and adipocyte differentiation-dependent expression of the unique disialic acid residue in an adipose tissue-specific glycoprotein, adipo Q.

Recently, we have shown that alpha 2,8-linked disialic acid (diSia) residue occurs in glycoproteins more frequently than ever recognized (Sato, C., Fukuoka, H., Ohta, K., Matsuda, T., Koshino, R., Kobayashi K., Troy, F. A., II, and Kitajima, K. (2000) J. Biol. Chem. 275, 15422--15431). In the course of identification of the diSia-containing glycoproteins in mammals, the 30-kDa glycoprotein was found in bovine serum. The 30-kDa glycoprotein was shown to be the bovine adipo Q, an adipocyte-specific protein, based on the partial amino acid sequences and the immuno-cross-reactivity with the recombinant mouse adipo Q. The bovine adipo Q was shown to have no N-linked but O-linked glycan(s) containing the diSia epitope, Neu5Ac alpha 2-->8Neu5Ac alpha 2-->3Gal. Furthermore, the diSia epitope was also found in the mouse adipo Q in serum as well as in the 3T3-L1 cells that are fully differentiated into adipocytes. Notably, among the known alpha 2,8-sialyltransferases, only the alpha 2,8-sialyltransferase III mRNA was detected in the 3T3-L1 cells at any stages of differentiation, and the recombinant alpha 2,8-sialyltransferase III could sialylate the purified bovine adipo Q. Thus, this study clearly provides the new findings that adipo Q is the diSia-containing glycoprotein and a physiological substrate of alpha 2,8-sialyltransferase III, whose substrates have not been identified so far.

␣2,8-Linked di/oligosialic acid (di/oligoSia) 1 chains containing 2-3 Sia residues are common structural units of gangliosides and are shown to be involved in various biological functions such as cell adhesion, cell differentiation, and signal transduction (1,2). Until recently, little attention has been paid to the existence of such short sialyl oligomers in glycopro-teins, whereas the polysialic acid (polySia) chains with more than 8 Sia residues have been well studied in neural cell adhesion molecule for the regulatory functions in the cell-cell interaction during development and differentiation (3,4) as well as in fish polysialoglycoprotein for the involvement in egg activation at fertilization (5). Recently, using the newly developed sensitive techniques (6 -8), we have shown that the di/ oligoSia containing up to 7 Sia residues occurs in glycoproteins more frequently than heretofore recognized and forms a new class of sialyl groups in glycoproteins (6, 8 -10). This finding raised several questions as to which proteins contain the di/ oligoSia groups, which enzymes are involved in the synthesis, and what is the biological significance of the modification in the glycoproteins. We have thus sought to identify the di/oligoSiacontaining serum glycoproteins and the involved enzymes in the synthesis of the di/oligoSia residue of the glycoproteins. In serum glycoproteins, it has been shown that bovine fetuin and ␣ 2 -macroglobulin (originally identified as a contaminant in the commercially available fetuin sample) contain the di/oligoSia structure (9).
In our further efforts to identify the di/oligoSia-containing glycoproteins in serum, in this study we clearly show that an adipose tissue-specific glycoprotein (adipo Q) contains the Neu5Ac␣238Neu5Ac␣233Gal structure in bovine and murine sera as well as in mouse 3T3-L1 cells that are differentiated into the adipocytes. Adipo Q is one of the serum glycoproteins (0.05% of total serum proteins) and is considered to play important roles in energy homeostasis (11,12). Interestingly, adipo Q has been reported not to be a glycoprotein because of the negative sensitivity to the digestion with endo-N-acetylglucosaminidase H during the de novo synthesis of adipo Q (11). So far carbohydrate modification of adipo Q has never been described in animals. This study also indicates that the ␣238 linkage of the diSia epitope in adipo Q is synthesized by an ␣2,8-sialyltransferase III (ST8Sia III) (13) but not ST8Sia II (STX) (14,15) nor ST8Sia IV (PST) (16) in the 3T3-L1-derived adipocytes. To our knowledge, this is the first identification of physiological substrate of ST8Sia III.

EXPERIMENTAL PROCEDURES
Materials-Clostridium perfringens sialidase, and endoproteinase Lys-C were purchased from Sigma. Arthrobacter ureafaciens sialidase was purchased from Nacalai Co. (Kyoto, Japan). Bicinchoninic acid protein assay kit was purchased from Pierce. 1,2-Diamino-3,4-methylenedioxybenzene was purchased from Dojindo (Kumamoto, Japan). Peptide:N-glycanase F was purchased from TAKARA (Kyoto Japan). DEAE-Sephadex A-25, Sephacryl S-300, Zn 2ϩ -chelating Sepharose, Sephadex G-25, Sephadex G-50, and protein-G-Sepharose resins, enhanced chemiluminescence (ECL) reagents, and CMP-[ 14 C]Neu5Ac (10.7 GBq/mmol) were purchased from Amersham Pharmacia Biotech. DEAE-Toyopearl 650M resins were purchased from Tosoh (Tokyo, * This research was supported in part by grants-in-aid for International Scientific Research, Joint Research, for Scientific Research on Priority Areas for Scientific Research (C) 10680581 (to K. K.) and the Japan Society for the Promotion of Science Research Fellows from the Ministry of Education, Science, Sports, and Culture (to C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF269230.
Cell Culture and Induction of Differentiation-Culture and induction of adipocyte differentiation of murine fibroblastic 3T3-L1 preadipocyte were carried out as described (12). Briefly, cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% FBS under 5% CO 2 at 37°C. For the confluent cells (day 0), differentiation was induced by administration of dexamethasone (0.25 M), isobutylmethylxthantine (200 M), and insulin (5 g/ml) for 2 days (day 2). The cells were then incubated with Dulbecco's modified Eagle's medium containing 10% FBS and 5 g/ml of insulin. Under these conditions, more than 90% of the cells acquire adipocyte morphology 8 days after the induction of differentiation (day 8). Culture medium was changed at a 2-day intervals, and adipocyte differentiation was observed under the microscope.
Purification of the Bovine 30-kDa Glycoprotein-FBS (500 ml) was mixed with polyethylene glycol (final concentration, 10%) and centrifuged at 10,000 ϫ g for 15 min. The pellet was dissolved in 0.1 M sodium phosphate (pH 6.5) and 0.8 M NaCl and applied to a Zn 2ϩ -chelating Sepharose column (1.8 ϫ 17 cm, equilibrated with 0.1 M sodium phosphate (pH 6.5), 0.8 M NaCl). The column was washed with 0.1 M sodium phosphate (pH 6.5) containing 0.8 M NaCl, 0.02 M sodium phosphate (pH 7.4) and 0.15 M NaCl and then eluted with 0.02 M sodium phosphate (pH 7.4), 0.15 M NaCl, and 0.02 M EDTA. The eluent was concentrated to 5 ml with ultrafiltration (YM-30, Amicon, Beverly, MA), and then applied to Sephacryl S-300 chromatography (1.2 ϫ 90 cm, 0.1 M NaCl, 10 mM Tris-HCl (pH 8.0)). The elution profile was monitored by absorbance at 280 nm and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Brilliant Blue staining. The fractions containing the 30-kDa glycoprotein (30 kDa-gp) were pooled, dialyzed against 10 mM Tris-HCl (pH 8.0), and then applied to DEAE-Toyopearl 650M (2.1 ϫ 2.3 cm, equilibrated with Tris-HCl (pH 8.0)) chromatography column with a linear gradient of 0 -0.6 M NaCl in the same buffer. The 30 kDa-gp-containing fractions were pooled and subjected to the SDS-PAGE/ Coomassie Brilliant Blue staining. The gel containing the 30 kDa-gp was cut and electroeluted to collect the protein.
Determination of Amino Acid Sequences-For determination of amino acid sequences, intact adipo Q and its proteolytic peptide frag-ments were blotted on a ProSorb TM sample preparation cartridge and analyzed on a protein sequencer model 610A (Applied Biosystems, Foster City, CA) as described (21). Homology searches were carried out at the National Center for Biotechnology Information by using the BLAST 2.0 program. For multiple alignments, DNASIS-Mac v3.0 (HI-TACHI, Tokyo, Japan) was used.
Cloning of the Bovine Adipo Q cDNA-Total RNA was prepared from bovine fat tissue (0.3 g) using Trizol (Life Technologies, Inc.) according to the manufacturer's instructions. The oligo-dT-primed cDNA (1 g) from the total RNA was used as a template for the polymerase chain reaction (PCR) cloning. The primers, based on the mouse adipo Q nucleotide sequence (GenBank TM accession number U49915) used were 5Ј-GTTGGATGGCAGGCATCC-3Ј (nucleotides 122-140) and 5Ј-CCTG-GAGCCAGACTTGGTC-3Ј (nucleotides 634 -652). 3Ј-and 5Ј-rapid amplification of cDNA ends were carried out as described previously (22). Nucleotide sequences of the cloned cDNAs were determined by the dideoxy chain termination method (23) on a DNA sequencer model 373 (Applied Biosystems).
Production of the Glutathione S-Transferase-Adipo Q Fusion Protein-A DNA fragment coding for a truncated form of the mouse adipo Q, lacking the N-terminal 17 amino acids of the open reading frame, was amplified by PCR using the following primers containing EcoRI or XhoI site: 5Ј-GTGAATTCGAAGATGACGTTACTACAAC-3Ј and 5Ј-GAGTAGCTCGAGTCAGTTGGTATCATGGAAGA-3Ј. The amplified fragment (693 base pairs) was subcloned into pGEX 4T-1 (Amersham Pharmacia Biotech) through EcoRI and XhoI sites (pGEX-20). This plasmid encodes the recombinant mouse adipo Q consisting of glutathione S-transferase followed by the truncated form of the mouse adipo Q. The recombinant mouse adipo Q was expressed and prepared according to the manufacturer's instructions.
Preparation of Antisera against the Bovine 30 kDa-gp and the Recombinant Mouse Adipo Q-The bovine 30 kDa-gp (30 g/mouse) or the recombinant mouse adipo Q (30 g/rat) together with Freund's complete adjuvant were intraperitoneally injected into the Balb/c mice and Wistar rats (female, 8 weeks old), respectively. The animals were boosted twice with each protein (10 g/mouse or 30 g/rat) mixed with Freund's incomplete adjuvant every 2 weeks. Blood was collected 1 week after the last boost, and the serum was prepared.
Immunoprecipitation-Mouse sera that had been pretreated with protein G-Sepharose, 3T3-L1 cell lysates, and the culture medium at day 8 were subjected to immunoprecipitation using the anti-recombinant mouse adipo Q and S2-566 antibodies that had been incubated with protein G-Sepharose as described previously (24). In the case of S2-566, protein G was pre-incubated with rat anti-mouse IgM antibodies.
Reverse Transcriptase-PCR-The following degenerate oligonucleotide primers for mouse proteins were used: ST8Sia I (GenBank TM accession  , and the template cDNA. Twenty to 40 cycles were carried out on a thermal cycler; each cycle involved incubation at 94°C for 30 s, 51°C for 1 min, and 72°C for 2 min. The PCR amplification was found to be proportional to the initial amount of the cDNAs with GAPDH and the number of cycles (20 -25 cycles) of PCR (data not shown). Aliquots of the PCR products were separated on a 1.0% agarose gel and blotted onto Hybond-N ϩ membranes (Amersham Pharmacia Biotech). The membranes were then probed with the indicated digoxigenin-labeled cRNAs (ST8Sia I, II, III, IV, V, adipo Q, GAPDH; nucleotides, 48 -1014, 46 -1107, 7-1101, 72-1061, 176 -1140, 52-744, and -5-1013, respectively) that had been cloned from the mouse adult brain mRNAs for ST8Sia I-V and GAPDH and from the bovine fat tissue mRNA for adipo Q and sequenced.
Preparation of Soluble ST8Sia III Fused with Protein A-Full-length of ST8Sia III and pcDSA-STX were generously donated by Dr. Shuichi Tsuji (RIKEN, Japan). pcDSA-ST8Sia III encoding ST8Sia III lacking the first 39 amino acids fused with the IgM signal sequence, and protein A was constructed as described (13) using 5Ј-ACGAATTCACCACTC-CCAAGTACGCC-3Ј and 5Ј-GCCTCGAGTTCCATTTGGAGTTCTTA-3Ј. COS-7 cells (1.0 ϫ 10 6 ) were transfected with pcDSA-ST8Sia III (10 g) by the Lipofectin reagents (Life Technologies, Inc.) and further cultured for 48 h in Dulbecco's modified Eagle's medium supplemented with 10% FBS that had been pre-absorbed by IgG-Sepharose. The culture medium was collected, and the protein A-fused mouse ST8Sia III expressed in the medium was adsorbed to IgG-Sepharose (15 l of the resin/10 ml of culture medium) at 4 C for 16 h. The resin was collected by centrifugation, washed three times with cold PBS, suspended in an equal volume of 50% glycerol in serum free-Dulbecco's modified Eagle's medium, and used as the soluble enzyme.

Identification of the Bovine Serum 30 kDa-gp as Adipo
Q-Recently, we have suggested that several glycoproteins in bovine serum contain di/oligoSia residues based on the fluorometric C 7 /C 9 analysis, which is the highly sensitive, chem-ical method of detecting the ␣2,8-linked di/oligoSia residues (6). Among these glycoproteins, fetuin and ␣ 2 -macroglobulin were first identified (9). In the course of the purification of ␣ 2 -macroglobulin, it was suggested that 30 kDa-gp, which was co-purified with ␣ 2 -macroglobulin on Zn 2ϩ -chelating column chromatography (9), also had the diSia residues. As shown in Fig. 1, the 30 kDa-gp was first purified from FBS. After polyethylene glycol precipitation of FBS, the precipitate was successively applied to a Zn 2ϩ -chelating column chromatography (Fig. 1a), Sephacryl S-300 gel filtration, and DEAE-Toyopearl 650M chromatography (Fig. 1b). DEAE-Toyopearl 650M chromatography resulted in two pooled fractions, A and B, as shown in Fig. 1b. The 30 kDa-gp was eluted in fraction B, whereas ␣ 2 -macroglobulin was in fraction A (Fig. 1b). The 30 kDa-gp was further purified to homogeneity by subjecting the fraction B to the SDS-PAGE/electroelution (Fig. 1c). The C 7 /C 9 analysis showed that the purified 30 kDa-gp had the diSia residues (see Table I). The yield of the 30 kDa-gp was 33 mg from 100 ml of FBS.
For identifying the 30 kDa-gp, the amino acid sequences of the N-terminal part and the Lys-C proteolytic peptide fragments were determined. The amino acid sequence from the N terminus was EDNMEDPPL, and those of three internal peptide fragments that were obtained by the Lys-C digestion were ADNVNDST, GSVL, and GDQVWL. The homology search shows that exactly the same sequences as these three peptide fragments are found in the mouse adipo Q (12) and the human homologue named apm1 (26) (Fig. 2), although no identity with known proteins is found for the N-terminal sequence. It is thus suspected that the 30 kDa-gp is the bovine adipo Q. Since no sequence information for the bovine adipo Q was available, the bovine adipo Q was cloned by the PCR-based methods using the bovine adipose tissue RNA. The deduced amino acid sequence is shown in Fig. 2.
The sequence has 92 and 82% identities with the mouse and the human adipo Q, respectively, indicating that this cDNA clone codes for the bovine adipo Q. The bovine adipo Q consists of 240 amino acids with a secretory signal sequence at the N-terminal part (amino acids 1-17) and two potential N-glycosylation sites (Asn-45 and Asn-225). A collagenous region (amino acids 45-111) and a globular domain (amino acids 112- 240) are also present as is the case with the mouse adipo Q (11,12). The N-terminal 9-amino acid sequence (EDNMED-PPL) of the 30 kDa-gp is identical to the deduced amino acids 18 -26 in the cloned bovine adipo Q, indicating that the intact bovine serum adipo Q starts with Glu-18 (Fig. 2). This is consistent with the presumption that amino acids 1-17 are a signal peptide as in the mouse adipo Q (11,12). The three peptide fragments of the 30 kDa-gp are also assignable in the bovine adipo Q (Fig. 2). Taken all together, it is concluded that the 30 kDa-gp is the bovine adipo Q. Now that the 30 kDa-gp is identified to be the bovine adipo Q, we call the 30 kDa-gp the bovine adipo Q.
Immunoreactivity of Anti-bovine Adipo Q and Anti-recombinant Mouse Adipo Q Antibodies-For immunochemical characterization of the bovine adipo Q, cross-reactivity of polyclonal antibodies raised against the bovine adipo Q (30 kDa-gp) and the recombinant mouse adipo Q was tested. Three antisera raised against the bovine 30 kDa-gp in three mice designated MA1, MA2, and MA3 were tested for the reactivity with the recombinant mouse adipo Q, a 51-kDa glutathione S-transferase fusion protein of mouse adipo Q expressed in Escherichia coli. MA1 and MA3 were reactive with the recombinant mouse adipo Q, whereas MA2 was not. Fig. 3a shows the results for MA3. This cross-reactivity with the recombinant mouse adipo Q (Fig. 3a, lane 2) is consistent with the conclusion that the bovine serum 30 kDa-gp is the bovine adipo Q. Furthermore, two antisera against the recombinant mouse adipo Q, designated RA1 and RA2, were generated. RA2 had the cross-reactivity with the bovine adipo Q (Fig. 3b, lane 1), whereas RA1 did not (data not shown). The cross-reactivity of RA2 shows that the bovine adipo Q shares the common epitopes with the mouse adipo Q, which is also consistent with the above conclusion.
The bovine adipo Q was eluted at the pass-through fraction on Sephacryl S-300 chromatography (data not shown), indicating that the bovine adipo Q is larger than 150 kDa and is present as homooligomers of the 30-kDa subunit, as were in the cases with other animal adipo Q (11,27). The bovine adipo Q gave a 30-kDa band exclusively and a minor 60-kDa band on SDS-PAGE under the reducing conditions, whereas it gave a prominent 60-kDa band and minor 90-and 120-kDa bands under the nonreducing conditions (Fig. 3a, lane 3), as was reported for the mouse adipo Q (11).
The Bovine Serum Adipo Q Contains the diSia Structure-The bovine adipo Q was tested for the presence of the diSia by Western blot analysis using monoclonal antibody S2-566, which specifically recognizes Neu5Ac␣238Neu5Ac␣233Gal sequence (8). As shown in Fig. 4a, S2-566 was reactive with the bovine adipo Q but not with the exosialidase-treated adipo Q. These results indicate that the bovine adipo Q has the Neu5Ac␣238Neu5Ac␣233Gal sequence. Consistently, the fluorometric C 7 /C 9 analysis showed the presence of the internal Sia residues (Table I). The Sia composition analysis (Table I) showed that the bovine adipo Q contains on average 1.4 Sia residues in a molecule. It is estimated that the Sia residues are present as Sia␣23glycan and Sia␣238Sia␣23glycan in the ratio 1:4, because the molar proportion of the internal to terminal Sia residue was 0.8:1 (Table I). The immunostain of the bovine adipo Q with S2-566 remained unchanged after the peptide:N-glycanase F treatment (Fig. 4b). This suggests that the diSia epitope is linked to O-linked glycan chain(s).
The Mouse Adipo Q Contains the diSia Structure-To examine if modification of adipo Q with the diSia epitope is confined to the bovine adipo Q, we also characterized the mouse serum adipo Q. The mouse adipo Q was immunoprecipitated from mouse sera using the anti-recombinant mouse adipo Q antibody RA1 and analyzed by Western blotting using S2-566. As shown in Fig. 5a, mouse adipo Q was precipitated from mouse sera (upper panel), and the immunopu-

FIG. 2. Amino acid sequence of the bovine 30 kDa-gp and the comparison with the mouse and human adipo Q.
The sequence of the 30 kDa-gp has been deposited in GenBank TM as accession number AF269230. The sequences of the mouse and human adipo Q are referenced to the accession numbers U49915 and D45371, respectively. The putative signal peptide sequence is amino acids 1-17. Two potential N-glycosylation sites are italicized. Amino acid sequences for the N-terminal region and the three Lys-C proteolytic peptide fragments that were determined by the sequencing are underlined.
Adipo Q Secreted from Adipocyte-differentiated 3T3-L1 Cells Is Modified by the diSia Structure-It has been shown that the mouse adipo Q is biosynthesized in and secreted from mouse 3T3-L1 cells on the treatment with differentiation-inducing reagents (12). The 3T3-L1 cells were harvested at 0, 2, and 8 days after the differentiation induction, and the cell lysates and the culture supernatant and the cell lysates were subjected to immunoprecipitation by the anti-recombinant mouse adipo Q antibody RA1 or anti-diSia antibody S2-566. These precipitates were analyzed by SDS-PAGE/ Western blotting using RA1 (Fig. 5b). As shown in Fig. 5b  (upper panel), adipo Q was clearly detected in the immunoprecipitate by RA1 from the cell lysates and the culture supernatant at day 8 of differentiation, whereas faint stains and no stains of adipo Q were observed at days 2 and 0, respectively. Since RA1 does not recognize the bovine adipo Q, the stained bands are not derived from the bovine adipo Q that might be contaminated from the culture medium containing FBS. These results are consistent with the results of the expression of adipo Q mRNA (see Fig. 7b). Immunoprecipitated adipo Q both from the cell lysates and the culture medium was immunostained with S2-566 (Fig. 5b, lower panel). Eight days after differentiation, the cell lysates were subjected to immunoprecipitation by S2-566 followed by Western blot analysis using RA1, and the S2-566 was shown to immunoprecipitate the 30 kDa-gp that was recognized by RA1 (data not shown). It is thus suggested the mouse adipo Q synthesized in and secreted from adipocyte-differentiated 3T3-L1 cells contains the diSia epitope.
Chemical and Immunochemical Detection of diSia-containing Glycoproteins in 3T3-L1 Cells before and after Differentiation-3T3-L1 cells were harvested, homogenized, and subjected to SDS-PAGE followed by blotting on the PVDF membrane. The membrane was cut into 11 equal pieces according to the descending molecular mass region and analyzed for diSia by the fluorometric C 7 /C 9 method. The results of the fluorometric C 7 /C 9 analysis are shown as molar proportions of internal to total Sia residues (C 9 /C T ) and as apparent ϽDPϾ values in Fig. 6a. After differentiation, the C 9 /C T value increased especially at 20 -45 and Ͼ120 kDa after differentiation. This suggests that the relative amount of diSia-to-monosialyl structure increases in glycoproteins after differentiation. The results of Western blotting are shown in Fig. 6b. The anti-diSia antibody (1E6) visualized several glycoproteins of both differentiated and undifferentiated 3T3-L1 cells, although more components were detected for the differentiated cells than the undifferentiated cells (Fig. 6b, 1E6). S2-566 also visualized several components for the differentiated 3T3-L1 cells (Fig. 6b, S2-566). The intensity and number of the S2-566-reactive bands increased at the 20 -45-kDa region after differentiation, consistent with the increase in the C 9 /C T value in the same region (Fig. 6b,  diSia). No component was detected by anti-polySia antibody (735) or antioligo ϩ polySia antibody (OL28) (Fig. 6b, polySia  and oligoSia), suggesting that the oligo/polySia chain with more than four Sia residues is not present in glycoproteins of 3T3-L1 cells, as estimated by the immunospecificity of these antibodies. All these results indicate that 3T3-L1 cells have the ability to synthesize the diSia residues in various glycoproteins of the cells, including adipo Q (Fig. 5b), and the amounts of diSia epitope was higher in the fully adipocytedifferentiated cells than in the undifferentiated cells.
Expression of ␣2,8-Sialyltransferases in 3T3-L1 Cells during Differentiation-To gain an insight into the biosynthesis of the diSia structure of adipo Q in 3T3-L1 cells, the expression of mRNA for the known ␣2,8-sialyltransferases, including ST8Sia I and V, which only utilize glycolipids as sub-  5. Immunodetection of the diSia epitope in the mouse adipo Q immunopurified from sera and the 3T3-L1 cells. a, the mouse adipo Q immunoprecipitated (IP) from sera using rat anti-recombinant mouse adipo Q antibodies (RA1) was immunostained with anti-mouse adipo Q (RA1) and S2-566 after it was treated (ϩ) or untreated (Ϫ) with sialidases (0.5 milliunit/ml) as described under "Experimental Procedures." b, the cell lysates of the 3T3-L1 cells at 0-, 2-, and 8-day post-differentiation and the culture supernatant of the cells at day 8 (8M) were subjected to the immunoprecipitation using antimouse adipo Q, RA1, followed by immunoblotting with anti-mouse adipo Q (RA1) and S2-566. strates, ST8Sia II (STX) and ST8Sia IV (PST), which utilize glycoproteins, and ST8Sia III, which utilizes both glycoproteins and glycolipids, was examined by the reverse transcriptase-PCR method. Only the ST8Sia III mRNA was detected at any stages of differentiation (Fig. 7a, days 0, 2, and  8), whereas four other mRNAs were not detected even after 40 cycles of amplifications. As controls, we used mouse adult brain, where the low expression of the ST8Sia II and IV mRNAs and the high expression of ST8Sia I, III, and V mRNAs are reported (28), and these results were reproduced as shown in Fig. 7a. Semi-quantitative reverse transcriptase-PCR analysis suggested that expression of the adipo Q mRNA was prominent at day 8 (Fig. 7b), as reported previously (11,12). This expression profile of the adipo Q mRNA is coincidental with protein expression of adipo Q as shown in Fig. 5b. On the other hand, the expression level of ST8Sia III mRNA of days 2 and 8 was 1.3 times higher than that of day 0 (Fig.  7, b and c). These results suggest that ST8Sia III, but not other known sialyltransferases, is involved in the synthesis of the diSia epitope of the mouse adipo Q.
ST8Sia III Can Utilize Adipo Q as a Substrate-To make sure if ST8Sia III can utilize adipo Q as a substrate, adipo Q purified from FBS and CMP-[ 14 C]Neu5Ac were incubated with the recombinant ST8Sia III, and the incorporated radioactivity on the substrate was measured. Based on the internal to terminal Sia ratio for the bovine adipo Q (Table I), at least 20% of the Sia residues in adipo Q are present as the mono-Sia residues. Therefore, the purified adipo Q is expected to be a substrate for ST8Sia that requires the pre-existing mono-Sia residue. As shown in Fig. 8, radioactivity in the bovine adipo Q fraction increased in an incubation time-dependent manner. Thus, it is demonstrated that adipo Q is sialylated by the ST8Sia III. DISCUSSION Recently, it has been demonstrated that di/oligoSia-containing glycoproteins occur in nature more frequently than ever recognized, as analytical methods for detecting the di/ oligoSia structures have been improved (8). In this study, we identified adipo Q from bovine and mouse sera as a new member of the diSia-containing glycoproteins using the chemical and immunochemical methods. Bovine adipo Q has been cloned and found to consist of 240 amino acids with a secretory signal sequence (amino acid 1-17) followed by collagenous (amino acids 45-111) and globular domains (amino acids 112-240) like the mouse (12) and human adipo Q (26). Reverse transcriptase-PCR for ST8Sia I to V, adipo Q, and GAPDH mRNAs in mouse 3T3-L1 cells and mouse adult brain. a, reverse transcriptase-PCR and Southern blotting was carried out using cDNAs from the 3T3-L1 cells at 0-, 2-, and 8-day postdifferentiation as templates and the primers as described under "Experimental Procedures." Aliquots of the PCR products amplified for 40 cycles were analyzed for the expression of the mRNAs for ST8Sia I to V, adipo Q, and GAPDH by 1.0% agarose gel electrophoresis and/or Southern blotting. As positive controls, mouse adult brain was used for the detection of these mRNAs. b, semi-quantitative analysis of the amounts of mRNAs, which code for ST8Sia III, adipo Q, and GAPDH. 20-and 25-cycle-amplified PCR products using primers for ST8Sia III, adipo Q, and GAPDH were analyzed using 1.0% agarose gel electrophoresis. c, relative amounts of mRNAs for adipo Q and ST8Sia III to that of GAPDH (internal control) at days 0, 2, and 8. The PCR products were analyzed at 20 cycles for adipo Q and 25 cycles for ST8Sia III using densitograph. The amounts of cDNAs coding for ST8Sia III and adipo Q at day 0 are set equal to 1.0. The bovine adipo Q gives a band of 30 kDa on SDS-PAGE under the reducing conditions, whereas it gives bands at 60, 90, and 120 kDa under the nonreducing conditions (Fig. 3a,  lane 3), and it is eluted at Ͼ150 kDa on Sephacryl S-300 chromatography (data not shown). These results suggest that the bovine adipo Q forms oligomers of the 30-kDa subunit. All of these features for the bovine adipo Q are common with those of the mouse adipo Q (11,12,27). It has been believed that the mouse adipo Q is not a glycoprotein, because endo-N-acetylglucosaminidase H treatment has no effect on the molecular mass of the molecule at any stages of the biosynthesis (11). For the bovine adipo Q, it is suggested that the N-linked glycan chain does not exist. Of two potential Nglycosylation sites (Asn-45 and Asn-225), at least Asn-255 is not glycosylated because Asn was detected by the amino acid sequence analysis (Fig. 2). Furthermore, the adipo Q was not susceptible to peptide:N-glycanase F treatment (Fig. 4b).
However, the fluorometric C 7 /C 9 analysis and Western blot analysis using monoclonal antibody S2-566 indicate that the bovine and mouse serum adipo Q contain the diSia structure ( Figs. 4 and 5a). We have also shown that the bovine adipo Q contains carbohydrates (Man, Glc, GlcNAc, Gal, and Sia) by the gas-liquid chromatography and fluorometric HPLC analysis. 2 It is thus concluded that adipo Q has the diSia epitope most possibly on its O-linked glycan(s). Adipo Q is secreted by 3T3-L1 cells that are differentiated into adipocytes under insulin control (12). Here we show that the mouse adipo Q secreted from the adipocyte-differentiated 3T3-L1 cells also has the diSia epitope (Fig. 5b). The presence of the diSia epitope in adipo Q not only from serum but also from the adipocyte-differentiated 3T3-L1 cells suggests the ubiquitous modification of adipo Q with the diSia epitope.
Adipo Q is a serum protein (0.05% of total serum protein) secreted from adipose tissue (11,12). The mouse adipo Q is also designated as ACRP30 (adipocyte complement-related protein 30), and its human homologue is designated as apm-1 (adiponectin) (26) or GBP28 (27). Adipo Q is considered to play an important role in energy homeostasis because the adipo Q mRNA decreases in adipose tissue in both ob/ob mice and obese humans (12). The three-dimensional structure of its C-terminal globular domain has close structural homology to tumor necrosis factor-␣ (29), which is also secreted by adipocytes and implicated in insulin resistance and energy control (30). Recently, it has been shown that human adiponectin appears to dysregulate the growth of myelomonocytic progenitors and the function of macrophages (31) and accumulation of macrophages when endothelial barrier is injured (32). Now that adipo Q is shown to have the diSia epitope on the molecule, it is conceivable that this glycotope may be functionally involved in these physiological phenomena. Elucidation of biological functions of the diSia epitope of adipo Q is under way in our laboratory. It should be noted that a leptin-binding protein, designated OB-BP2, has the ability to bind the ␣2,8and ␣2,3-Sia residues of glycoproteins and glycolipids (33). OB-BP2 is structurally classified as a member of siglec (sialic acid binding immunogloblulinlike lectin) and is named siglec-5 (34). OB-BP2 has separate binding sites for the Sia residues and leptin on the molecule. Considering that the expression of adipo Q is related with obesity (11,12) like leptin, it would be interesting to see if the diSia-containing adipo Q binds leptin through OB-BP2 to regulate the leptin-mediated energy homeostasis.
We also show that several glycoproteins other than adipo Q also have the diSia epitope in adipocyte-undifferentiated and differentiated mouse 3T3-L1 cells by the fluorometric C 7 /C 9 analysis and the immunoreactivity with the anti-diSia antibodies (Fig. 6, a and b). It appears that the diSia-containing glycoproteins increase after differentiation (Fig. 6b). These results suggest that 3T3-L1 cells are useful for biosynthetic study of the diSia epitope of these glycoproteins. So far, no enzyme involved in the synthesis of diSia epitope in glycoproteins has ever been identified. The ST8Sia III is a candidate enzyme for disialylation because the enzyme has been shown to catalyze the synthesis of the diSia epitope in both glycoproteins and glycolipids in vitro (13), although the physiological substrates of this enzyme have remained unidentified. To identify the enzyme responsible for the synthesis of the diSia epitope, we sought it in 3T3-L1 cells that not only secrete the diSia-containing adipo Q but also express several diSia-containing glycoproteins (Fig. 6). Interestingly, of known five ␣2,8-sialyltransferases, only ST8Sia III was detected in the 3T3-L1 cells. This strongly suggests that ST8Sia III is involved in the synthesis of the diSia epitope on various glycoproteins including adipo Q in 3T3-L1 cells. The semiquantitative analysis of mRNAs for ST8Sia III and adipo Q in 3T3-L1 cells shows that ST8Sia III is expressed constitutively, although its expression is 1.3-fold higher after differentiation. On the other hand, adipo Q is expressed only after differentiation. Therefore, the synthesis of the diSia epitope on adipo Q in 3T3-L1 cells appears to be regulated by the expression level of ST8Sia III. We also identified mRNA for ST8Sia III in mouse fat tissues. 3 Furthermore, it is shown that adipo Q purified from bovine serum is sialylated by the mouse recombinant ST8Sia III. All these results indicate that adipo Q is a physiological substrate of ST8Sia III. To our knowledge, this is the first example of physiological substrates for ST8Sia III.