Glycobiology, 2015, vol. 25, no. 9, 953–962 doi: 10.1093/glycob/cwv039 Advance Access Publication Date: 2 June 2015 Original Article

Analytical Glycobiology

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E Downloaded from http://glycob.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 31, 2015

Ippei Watanabe2,3, Tomoya Hikita2, Haruka Mizuno2, Risa Sekita2, Akira Minami2, Ami Ishii2, Yuka Minamisawa3, Kiyoshi Suzuki3, Hiroshi Maeda3, Kazuya I P J Hidari4, and Takashi Suzuki1,2 2

Department of Biochemistry, School of Pharmaceutical Science, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan, 3Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato-shi, Tokyo 207-0021, Japan, and 4Department of Food and Nutrition, Junior College Division, University of Aizu, 1-1 Aza-Kadota Yahata, Ikki-machi, Aizuwakamatsu-shi, Fukushima 965-8570, Japan 1

To whom correspondence should be addressed: Tel: +81-54-264-5725; Fax: +81-54-264-5725; e-mail: [email protected]

Received 23 April 2015; Revised 27 May 2015; Accepted 28 May 2015

Abstract Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6SDS) that possessed ∼60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and Liduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE. Key words: binding affinity, chondroitin sulfate E, glycosaminoglycan, monoclonal antibody, surface plasmon resonance

Introduction Chondroitin sulfate (CS) is a sulfated polysaccharide that consists of repetitive disaccharide units containing D-glucuronic acid (GlcA) and N-acetyl galactosamine (GalNAc) with a β1-3 linkage. CS is classified into four major type isomers based on structural diversity because sulfonates can be bound to various positions of the disaccharide units, i.e. chondroitin 4-sulfate (CSA), chondroitin 6-sulfate (CSC), chondroitin 2,6-sulfate (CSD) and chondroitin 4,6-sulfate (CSE). Dermatan sulfate (DS) (also known as CSB) is an epimer of CSA that contains L-iduronic acid (IdoA) instead of GlcA. CS/DS

chains are usually attached to extracellular matrix proteins to form proteoglycans in animal tissues. The structural heterogeneity of CS is apparently associated with the specific biological activities and functions (Lauder et al. 2001; Gama et al. 2006; Troeberg et al. 2014). CSE has been shown in many studies to interact with various bioactive proteins including fibroblast growth factors, midkine, pleiotrophin and heparin (Hep)-binding epidermal growth factor-like growth factor. The interactions of CSE with these proteins contribute to mitogenic and neurotropic activities (Rauvala et al. 2000; Zou et al. 2003; Tully et al. 2004). CSE is involved in anti-inflammation events through

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I Watanabe et al. (KS). In addition, we demonstrated that the antibodies are applicable to histochemical analysis.

Results Generation of monoclonal antibodies against CSE Fraction-1 prepared from squid cartilage is composed of the predominantly E-unit ([GlcAβ1-3GalNAc (4S, 6S)], 61.4%). However, it also contains A-units ([GlcAβ1-3GalNAc (4S)], 23.8%), C-units ([GlcAβ13GalNAc (6S)], 5.5%) and 0S-units ([GlcAβ1-3GalNAc], 9.3%) (Table I). We hypothesized that the structural heterogeneity of CS might induce antibodies that are broadly cross-reactive to CS isomers. By enhancing the E-unit content of CSE, it might be possible to develop highly specific antibodies to CSE. In order to obtain CSE with a high E-unit content, we prepared CSE by anion-exchange column chromatography and hyaluronidase treatment. We obtained a CSE fraction in which the E-unit content was increased up to 70%, termed Fraction-4 (Table I). We prepared a phosphatidylethanolamine (PE) conjugate of Fraction-4 and immunized mice with the conjugate (CSE-PE). By using CSE-PE immobilized microtiter plates for screening, the two hybridomas were obtained as anti-CSE antibodyproducing clones, E-12C and E-18H. The isotypes of these antibodies were IgM with kappa light chain (Supplementary data, Figure S1).

Substrate specificity of antibodies E-12C and E-18H with GAGs and their chemical derivaives We used CSA, 6S-CSA, CSC, deS-CSC, CSD, CSE (Fraction-4), 6deS-CSE, DS, 6S-DS, HA, Hep, HS, HPN and KS to evaluate the substrate specificity of the antibodies. The average MW and disaccharide compositions of CS and CS derivatives are summarized in Table II. The reactivities of antibodies E-12C and E-18H to purified GAGs derived from animals and desulfated GAGs are shown in Figure 1. Both antibodies exclusively reacted with CSE (Fraction-4), but no reactivity to other GAGs was observed. The results indicated that both antibodies show no cross-reactivity. To investigate the participation of the negative charge density for the reactivity of the antibody, reactivities of CSE and Hep to the antibodies were compared. The sulfur content of CSE is the highest among CS isomers (9–10%). Although the sulfur content of Hep was 1.4-fold higher than that of CSE, neither of the antibodies reacted with Hep. These results suggested that the interaction of the antibodies with a substrate is not merely dependent on the negative charge density. Since 6deS-CSE prepared from Fraction-4 resulted in loss of reactivity to the antibodies, the E-unit was indispensable to the reactivity. We also evaluated the specificity of these antibodies by using artificial CSE and CSH, designated as 6S-CSA-p and 6S-DS, respectively. Disaccharide composition analysis showed that 6S-CSA-p contained 59.4% of E-units and that 6S-DS contained 66.0% of H-units (Supplementary data, Table SI). The reactivities of

Table I. MWs and disaccharide compositions of five isolated CSE fractions Fraction No.

1 2 3 4 5

ΔDi- (%)

MW (kDa)

68.0 56.7 24.1 14.6 8.7

Sulfur content (%)

0S

4S

6S

2S

(4,6)S

(2,4)S

(2,6)S

(2,4,6)S

9.3 8.0 8.1 6.4 9.3

23.8 25.2 23.1 17.5 19.5

5.5 5.8 5.7 5.7 4.5

0.0 0.0 0.0 0.0 0.0

61.4 61.0 63.0 70.4 66.7

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

The prepared fractions indicate Fractions-1, 2 3, 4 and 5, respectively (Supplementary data, Scheme S1).

9.3 9.4 9.5 10.0 9.6

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interaction with the adhesion molecules L- and P-selectins and various cytokines (Kawashima et al. 2002). CSE also shows an antiviral effect on dengue virus infection (Kato et al. 2010). To address the biological and pathological significance of CSE, most previous studies focused on the expression level of the gene encoding a responsible enzyme in CSE biosynthesis, GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST; Yamaguchi et al. 2007). GalNAc4S-6ST mRNA is expressed in neuronal cells of the mouse postnatal cerebellum, bone marrow-derived mast cells derived from mice and early mouse embryos (Salgueiro et al. 2006; Ishii and Maeda 2008; Ohtake et al. 2008; Ohtake-Niimi et al. 2010). In patients with astrocytic tumors, the expression level of GalNAc4S-6ST mRNA was shown to be significantly associated with poor prognosis (Kobayashi et al. 2013). These findings suggested that CSE might play important roles in biological and pathological phenomena in living bodies. However, its functions have not been clarified in detail because expression profiles of CSE in living bodies are not completely clear. Therefore, a method for highly specific analysis of CSE might be helpful for elucidation of CSE functions. A method using antibodies has been used widely for microanalysis of biological materials. Several monoclonal antibodies that recognize CS isomers have been generated. CSE-reactive monoclonal antibodies, MO-225, GD3G7 and IO3D9, have been reported. The antibody MO-225, which was selected by a group of hybridoma clones prepared against the CS proteoglycan of chick embryo limb buds, binds to CSD strongly and to CSC and CSE to a lesser extent (Yamagata et al. 1987). The antibody GD3G7, which was selected using phage display technology directed against rat embryo glycosaminoglycans (GAGs), binds to not only CSE but also CSA and CSH, which contains abundant H-units [IdoAβ1-3GalNAc (4S, 6S)] (ten Dam et al. 2007). The antibody IO3D9, which was selected by phage display technology using immobilized CSC, binds to CSE strongly and to CSA or CSC weakly (Smetsers et al. 2004). Thus, no antibodies that specifically recognize CSE have been established so far. Approximately 60% of disaccharide in CSE derived from squid cartilage is [GlcAβ1-3GalNAc (4S, 6S)], termed the E-unit. However, CSE also contains considerable amounts of disaccharides characterizing other CS isomers. To develop monoclonal antibodies that specifically react with the E-unit, we prepared a CSE fraction in which the content of the E-unit was over 70% as an immunogen. In the present study, we established anti-CSE monoclonal antibodies, termed E-12C and E-18H, by immunizing mice with the aforementioned CSE fraction. To investigate the binding specificity of the antibodies, indirect enzyme-linked immunosorbent assay (ELISA) was performed using immobilized GAG microtiter plates, such as CSA, 6S (6-O-sulfated)-CSA, CSC, deS (desulfated)-CSC, CSD, CSE, 6deS (6-O-desulfated)-CSE, DS, 6S-DS, hyaluronic acid (HA), Hep, heparan sulfate (HS), heparosan (HPN) and keratin sulfate

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Table II. MWs and disaccharide compositions of CS and CS derivatives used in indirect ELISA GAG

CSA DS CSC CSD CSE de6S-CSE deS-CSC

ΔDi- (%)

MW (kDa)

16.8 16.4 42.1 31.7 14.6 12.7 7.5

Sulfur content (%)

0S

4S

6S

2S

(4,6)S

(2,4)S

(2,6)S

(2,4,6)S

2.2 5.9 1.9 2.5 6.4 18.0 91.2

97.8 75.5 17.8 30.5 17.5 82.0 0.0

0.0 8.4 68.2 39.6 5.7 0.0 8.7

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 1.1 1.3 1.4 70.4 0.0 0.0

0.0 8.0 0.0 0.0 0.0 0.0 0.0

0.0 1.0 10.8 26.2 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0

6.8 7.0 7.4 8.1 10.0 5.7 0.6

the antibodies to 6S-CSA-p and 6S-DS are shown in Figure 2. The antibodies E-12C and E-18H reacted with 6S-CSA-p similar to CSE (Fraction-4). According to Habuchi et al. (1977), CSE contains small amounts of glucose branches linked to C-6 of the GalNAc moieties. On the other hand, it seems that 6S-CSA does not contain such a branching structure. This result indicated that glucose-branching moieties were not involved in the interaction of the antibodies with CSE. In contrast, 6S-DS was not recognized by the antibodies. 6S-DS contains the H-unit as the major disaccharide isomeric of the E-unit. The antibodies could strictly distinguish the structural difference between GlcA and IdoA in the polysaccharides.

Affinity analysis of antibodies E-18H and E-12C with CSE and 6S-CSAs To investigate the effects of molecular sizes of CSE on interaction with these antibodies, five CSE fractions were prepared as shown in Supplementary data, Scheme S1 ( properties of them shown in Table I). The influence of MW of CSE on the affinity of the antibodies was analyzed by surface plasmon resonance. The antibodies E-12C and E-18H

significantly bound to each CSE fraction. The responses of the antibodies on sensorgrams increased in a dose-dependent manner of the antibodies (Supplementary data, Figure S2). Each antibody did not bind to the 6deS-CSE-immobilized surface (data not shown). The KD values were calculated by fitting a 1: 1 Langmur-binding model and are shown in Table III. The KD value of each fraction to the antibodies was less than 10−9 M. All CSE fractions had high affinity interaction with the antibodies. In addition, the intensity of the affinity increased depending on the MW of CSE. Next, we prepared four kinds of artificial CSE containing different amounts of the E-unit to evaluate affinities of the antibodies with them. The E-unit contents of them were 14.1, 32.7, 45.9 and 59.4% and they were termed 6S-CSA-l, m, h and p, respectively (Supplementary data, Table SI). The antibodies E-12C and E-18H bound to 6S-CSA-m, h and p. The responses of the antibodies on sensorgrams increased in a dosedependent manner of the antibodies (Supplementary data, Figure S3). The kinetic parameters for the interaction of each 6S-CSA with these antibodies are summarized in Table IV. As the E-unit content increased, the KD values decreased. In other words, the intensity of affinity was dependent on the E-unit content. However, sensorgrams

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Fig. 1. Reactivities of antibodies E-12C and E-18H with GAGs. The reactivity was examined by indirect ELISA. Biotinylated GAGs were immobilized at 50 ng/well on 96-well streptavidin-conjugated microtiter plates. Antibodies were added to the wells at 125 ng/well. (A) Reactivity of E-12C with GAGs. (B) Reactivity of E-18H with GAGs. The values represent the mean ± standard deviations (n = 3).

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Immunohistochemical detection of CSE

Table III. Kinetic parameters for the interaction of antibodies E-12C and E-18H with immobilized CSE Antibody

CSE fraction no.

MW (kDa)

ΔDi-(4,6)S (%)

ka (M−1s−1)

kd (s−1)

KD (M)

E-12C

1 2 3 4 5 1 2 3 4 5

68.0 56.7 24.1 14.6 8.7 68.0 56.7 24.1 14.6 8.7

61.4 61.0 63.0 70.4 66.7 61.4 61.0 63.0 70.4 66.7

(3.5 ± 1.7) × 107 (3.0 ± 0.1) × 107 (3.8 ± 1.1) × 106 (5.7 ± 0.2) × 106 (3.7 ± 0.6) × 106 (2.9 ± 1.7) × 107 (3.4 ± 0.7) × 107 (6.3 ± 1.2) × 106 (7.3 ± 0.5) × 106 (4.3 ± 0.1) × 106

(5.5 ± 1.0) × 10−4 (1.0 ± 1.6) × 10−3 (8.2 ± 0.3) × 10−4 (2.0 ± 0.2) × 10−3 (1.4 ± 0.1) × 10−3 (3.8 ± 0.8) × 10−4 (8.5 ± 1.6) × 10−4 (7.6 ± 1.1) × 10−4 (1.8 ± 0.1) × 10−3 (1.2 ± 0.03) × 10−3

(1.7 ± 0.5) × 10−11 (3.4 ± 0.4) × 10−11 (2.3 ± 0.9) × 10−10 (3.6 ± 0.5) × 10−10 (3.7 ± 0.7) × 10−10 (1.5 ± 0.5) × 10−11 (2.5 ± 0.4) × 10−11 (1.2 ± 0.1) × 10−10 (2.5 ± 0.2) × 10−10 (2.9 ± 0.1) × 10−10

E-18H

CSE fractions with different MWs containing more than 60% of E-units, designated as Fractions-1, 2 3, 4 and 5 (Table I). The values represent the mean ± standard deviations (n = 3).

Table IV. Kinetic parameters for the interaction of antibodies E-12C and E-18H with immobilized 6S-CSA Antibody

6-O-sulfated CSA

MW (kDa)

ΔDi-(4,6)S (%)

ka (M−1s−1)

kd (s−1)

KD (M)

E-12C

L m h p l m h p

19.0 19.6 18.0 20.1 19.0 19.6 18.0 20.1

14.1 32.7 45.9 59.4 14.1 32.7 45.9 59.4

NB (8.0 ± 1.1) × 105 (4.0 ± 0.5) × 105 (1.0 ± 5.5) × 106 NB (9.6 ± 0.1) × 105 (7.1 ± 2.6) × 105 (1.4 ± 0.3) × 106

NB (8.4 ± 0.2) × 10−3 (1.4 ± 0.2) × 10−3 (7.4 ± 0.3) × 10−4 NB (9.0 ± 0.3) × 10−3 (1.2 ± 0.1) × 10−3 (6.0 ± 0.2) × 10−3

NB (1.0 ± 0.1) × 10−8 (3.5 ± 0.4) × 10−9 (7.8 ± 3.1) × 10−10 NB (9.4 ± 0.4) × 10−9 (1.8 ± 0.7) × 10−9 (4.3 ± 1.0) × 10−10

E-18H

6S-CSAs with different E-unit contents and almost the same MWs were designated as 6S-CSA-l, m, h and p. The values represent the mean ± standard deviations (n = 3). NB, not bound.

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Fig. 2. Reactivities of antibodies E-12C and E-18H with 6S-CSA and 6S-DS. Biotinylated GAGs were immobilized at 50 ng/well on 96-well streptavidinconjugated microtiter plates. 6S-CSA-p and 6S-DS contained 59.4% of E-units and 66.0% of H-units, respectively. Antibodies were added to the wells at 125 ng/well. (A) Reactivity of E-12C with GAGs. (B) Reactivity of E-18H with GAGs. The values represent the mean ± standard deviations (n = 3).

To investigate the capability of the antibodies E-12C and E-18H for immnohistochemical staining, we prepared cryosections from squid cartilage. Staining of the sections was performed using the antibody E-12C. As shown in Figure 3, the entire area of the section was positively stained by the antibody (Figure 3A), including the extracellular matrix of the cell (Figure 3D), but not by normal mouse IgM (Figure 3C). With chondroitinase ABC digestion (50 mU/coverslip, 37°C, 2 h) before the immunostaining, the staining property of the sections practically disappeared (Figure 3B and E). The staining property of the antibody E-18H was similar to that of the antibody E-12C (data not shown). GalNAc4S-6ST mRNA expression was observed in OVCAR3 cells (ten Dam et al. 2007). For staining of CSE molecules, OVCAR3 cells were incubated with the antibody E-12C or E-18H. The results using the antibody E-12C are shown in Figure 4. By visualizing with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgM, the cells were clearly stained with the antibody E-12C (Figure 4A). OVCAR3 cells were also stained with the antibody E-18H (data not shown). These staining properties disappeared with chondroitinase ABC digestion before immunostaining (Figure 4D). As for this dyeing, the placement of the cells accorded with 4',6-diamidino-2phenylindole (DAPI) dyeing. Furthermore, rat tissues were stained with the antibody E-18H. As shown in Figure 5, the choroid plexus in the rat brain slice showed intense fluorescence with the antibody

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(Figure 5A). This fluorescence was obviously attenuated by treatment with 12.5 mU/coverslip chondroitinase ABC at 37°C overnight (Figure 5D). The results indicated that antibodies are useful for immnohistochemical staining.

Discussion Monoclonal antibodies recognizing CSE domains including antibodies MO-225, GD3G7 and IO3D9 have been reported (Yamagata et al. 1987; Smetsers et al. 2004; ten Dam et al. 2007). However, in contrary to their expectations, these antibodies show cross-reactivity with CS isomers. For example, the antibody IO3D9 was selected by using immobilized CSC; however, it recognized CSE, CSA and CSC. We hypothesized that the cross-reactivity of antibodies was caused by the structural heterogeneity in the CS molecule. We attempted to obtain CSE fractions with larger amounts of the E-unit by digestion with hyaluronidase, which susceptibly reacts with low sulfation moieties of the CS molecule. We obtained a CSE fraction containing over 70% of E-units with molecular size decreased to ∼ 1/5 (Table I, Fraction-4). Using that fraction, we finally established two hybridomas that produce a CSE-specific monoclonal antibody, termed E-12C and E-18H. In most previous studies, the specificity of anti-CS antibodies was evaluated by ELISA immobilized CS isomers, e.g. Sugiura et al. (2012). We also used purified GAGs derived from animals and chemically synthesized GAGs for ELISA to evaluate the specificity of the antibodies E-12C and E-18H. As shown in Figure 1, the E-unit is

involved in the substrate recognition of the antibodies because selective 6deS-CSE showed no reactivity to the antibodies. We also evaluated the reactivity against four kinds of artificial CSE with different E-unit contents, which were prepared by selective 6S-CSA (Supplementary data, Table SI). The binding affinity of the antibodies to the artificial CSE was enhanced depending on the E-unit content. Several repetitive E-units may be required for epitope recognition of the antibodies. According to Habuchi et al. (1977), CSE contains small amounts of glucose branches linked to C-6 of the GalNAc moieties. CSA has never been reported to contain such a branching structure. Therefore, we used an artificial CSE prepared from CSA to compare the reactivities to native CSE (Supplementary data, Table SI, 6S-CSA-p). As can be seen in Figure 2, the antibodies showed almost equal reactivities to native CSE and artificial CSE. The results suggested that the glucose branches in the native CSE may not be necessary for antigen recognition of these antibodies. The antibody GD3G7 shows moderate reactivity to CSH of which the major disaccharide is [IdoAβ1-3GalNAc (4S, 6S)] (H-unit). To evaluate the reactivity of the antibodies with CSH, we prepared an artificial CSH from DS possessing [IdoAβ1-3GalNAc (4S)] as the major disaccharide (Supplementary data, Table SI, 6S-DS). Neither of the antibodies, E-12C and E-18H, were able to react with the artificial CSH, indicating that the antibodies strictly recognized the E-unit [GlcAβ1-3GalNAc (4S, 6S)]. In other words, the antibodies distinguished the structural difference between GlcA and IdoA in the GAG molecules.

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Fig. 3. Immunohistochemical staining of squid cartilage. (A) Cryosection of squid cartilage stained with the antibody E-12C. (B) Reactivity of the antibody E-12C was lost after chondroitinase ABC digestion. (C) No staining was observed with normal mouse IgM. (D) and (E) are magnified images of (A) and (B), respectively. Biotinylated anti-mouse IgM antibody was added as the secondary antibody. Bound antibodies were visualized using peroxidase-labeled streptavidin and 3,3′-diaminobenzidine. Scale bar = 500 µm (A–C) and 50 µm (D and E). This figure is available in black and white in print and in colour at Glycobiology online.

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In artificial CSEs with almost the same MWs and different E-unit contents, the binding affinity of the antibodies to them increased depending on the E-unit content as described above. Furthermore, we focused on CSEs with different MWs on binding affinity to the antibodies. As shown in Table III, the KD values depended on MW of the CSE molecules. The results suggested that the length of polysaccharide chains of CSE affects the binding affinity. To determine the expression of E-unit epitopes in squid cartilage and an ovarian carcinoma cell line, we performed immunostaining analysis using the antibodies E-12C and E-18H. Both samples were stained by the antibodies, whereas chondroitinase-pretreated samples were not. Moreover, we also demonstrated that the choroid plexus in the rat brain slice was stained with the antibody E-18H. These findings strongly suggest that these antibodies detect E-unit epitopes expressed on the tissues and cultured cells. By mass spectrometry analysis techniques, it has become possible to detect trace the amounts of CSs. However, a substantial amount of materials and laborious work are still required to determine the

detailed structures in CS chains. The antibodies directed towards the E-unit that we presented here will significantly contribute to the analysis of CS, especially for rapid and simple profiling, combined with current analytical methods, e.g. disaccharide composition analysis or GalNAc4S-6ST gene expression analysis. In conclusion, we have established two monoclonal antibodies, E-12C and E-18H, that specifically react with CSE. These antibodies are applicable to both ELISA and immunohistochemical analysis. They might be helpful for the elucidation of the biological and pathological significance of CSE.

Materials and methods Purified GAGs derived from animals CSA from a sturgeon notochord or whale cartilage, DS from a chicken cockscomb, CSC from shark cartilage, CSD from shark cartilage, CSE from cartilage in Japanese flying squid Todarodes pacificus, HA from

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Fig. 4. Immunohistochemical staining of ovarian carcinoma OVCAR3 cells. (A) OVCAR3 cells stained with the antibody E-12C. (D) Reactivity of the antibody E-12C was reduced after treatment with chondroitinase ABC. (B and E) Nucleic acid staining with DAPI. (C and F) Merged images. Reactivity of antibody E-12C was visualized using FITC-conjugated anti-IgM secondary antibody. Scale bar = 100 µm. This figure is available in black and white in print and in colour at Glycobiology online.

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a chicken cockscomb, HS from a bovine kidney, HPN from E. coli K5 strain and KS from a bovine cornea were obtained from Seikgaku Corp. (Tokyo, Japan). Hep from a bovine lung was purchased from Scientific Protein Laboratories LLC (WI, USA).

Chemically synthesized GAGs deS-CSC was prepared according to the method of Kantor and Schubert (1957). 6deS-CSE, 6S-CSA and 6S-DS were prepared according to the method of Nagasawa et al. (1979, 1986). 6S-CSA-l, m, h and p were prepared by adding the sulfur reagent 2, 4, 6 and 8 equivalent per disaccharide unit, respectively. Biotinylated-GAGs were prepared according to the method of Osmond et al. (2002).

Determination of average molecular weight The average molecular weight (MW) of GAG was estimated by gel exclusion column chromatography using an high-performance liquid chromatography (HPLC) system equipped with a serial combination

of TSK-gel PWXLG4000, PWXLG3000 and PWXLG2500 (Tosoh Corp.) under a constant flow rate at 0.6 mL/min of 0.2 M NaCl at 40°C (Rice et al. 1985). GAG solutions with concentrations of 2 mg/ml were injected into the columns and detected with the refractive index. MW was calculated by LC solution software (Tosoh Corp.) using the standard curve of CS of which absolute MW was already determined by a light scattering application.

Disaccharide composition analysis Unsaturated disaccharides from CS and DS were prepared by the conventional method (Saito et al. 1968; Suzuki et al. 1968). The disaccharides were separated by an ion-pairing reversed phase HPLC system equipped with a DOCOSIL SP 100 column (Senshu Scientific Corp.). A mixed solvent containing four different solutions, H2O, 0.2 M NaCl, 10 mM tetra-n-butylammonium hydrogen sulfate and 50% acetonitrile, was used for the column eluent. The mixed ratio of the solutions was controlled by an HPLC software program during

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Fig. 5. Immunohistochemical staining of rat choroid plexus. (A) The choroid plexus in the rat brain slice was stained with the antibody E-18H. (D) Reactivity of the antibody E-18H was reduced after treatment with chondroitinase ABC. (B and E) Nucleic acid staining with DAPI. (C and F) Merged images. Reactivity of antibody E-18H was visualized using the FITC-conjugated anti-IgM secondary antibody. Scale bar = 1 mm. This figure is available in black and white in print and in colour at Glycobiology online.

960 analysis. The eluted disaccharides were derivatized at their reducing ends with a fluorophore 2cyanoacetamide to achieve sensitivity. The labeled compounds were detected by in-line fluorescence (excitation at 346 nm and emission at 410 nm) (Toyoda et al. 2000). An unsaturated disaccharide kit containing ΔDi-0S, ΔDi-4S, ΔDi-6S, ΔDi-2S, ΔDi-(4,6)S, ΔDi-(2,6)S, ΔDi-(2,4)S and ΔDi-(2,4,6)S (Seikagaku Corp.) was used as a standard. Each sulfur content (%) was calculated by the following numerical formula: 32 × [molar concentration of sulfur per disaccharide unit]/[MW of disaccharide unit/1000] × [ peak area ratio of disaccharide unit (%)]. Total sulfur content (%) was calculated by integrating each sulfur content (%).

Squid cartilage was treated by the method of Kawai et al. (1966) with some modification, and 24 g of CSE, referred to as Fraction-1, was obtained. To prepare CSE fractions with different MWs and amounts of the E-unit, Fraction-1 was digested with bovine testicular hyaluronidase under different conditions as shown in Supplementary data, Scheme S1. First, 4.0 g of Fraction-1 was dissolved in 50 mM phosphate buffered saline (PBS) ( pH 5.3) and digested with the hyaluronidase (1000 units for 1 g of CSE) at 37°C for 30 min. The CSE fraction (Fraction-2) was purified by a combination of dialysis, activated carbon treatment, filtration and ethanol precipitation (3.5 g). Next, 17.0 g of Fraction-1 was dissolved in distilled water and applied to an anion-exchanger column HPA-25 (7.1 × 45 cm). After washing with 5 volumes of distilled water, the column was eluted stepwise with increasing NaCl concentration (1.5 and 3.5 M). After concentration and dialysis, the elution fraction was recovered from each fraction by ethanol precipitation. The 1.5 M NaCl fraction (1.5 g) was digested with hyaluronidase (1000 units for 1 g of CSE) at 37°C for 24h (Fraction-3; 1.2 g). The 3.5 M NaCl fraction was equally divided into 4 g and treated with hyaluronidase under different conditions. To prepare Fraction-4, hyaluronidase treatment was performed at 48,000 and 16,000 U for 24 h and purified (3.0 g). After digested two times 51,000 U hyaluronidase for 24 h, 3.0 g of CSE was obtained as Fraction-5. The average MWs and disaccharide compositions of the five recovered fraction were analyzed (Table I). Fraction-4 containing the highest E-unit content among the fractions was used as an immunogen. For the preparation of CSE-PE, Fraction-4 was covalently coupled with dipalmitoyl PE via the reducing terminal of the polysaccharide according to the method of Sugiura et al. (1993).

Mice immunization, cell fusion and cloning of antibody-producing hybridomas Three 6-week-old male C3H/HeN mice were immunized six times by tail vein injection with CSE-PE pre-mixed with the cell wall of Salmonella minnesota. At 3 days after the sixth injection, B cells from the spleen of each mouse were prepared and fused with the mouse myeloma cell line PAI (NIBIO, Japan) at a ratio of 5:1 (lymphocytes to myeloma cells) using 50% (w/v) polyethylene glycol 4000. The fused cells in RPMI 1640 medium supplemented with 10% fetal bovine serum and hypoxanthine-aminopterin-thymidine mixture were seeded onto 96-well culture plates (3–5 × 105 cells/well) and cultured at 37°C under 5% CO2. After 2 weeks of incubation, antibody titer in the culture supernatants was evaluated by a solid-phase binding assay using CSE-PE immobilized on screening microtiter plates. Hybridomas that produced an antibody of interest were cloned by the limiting dilution method and cultured in a cloning medium, S-Clone (Eidia Corp.). In 2 weeks of cloning, the hybridomas that produced an

antibody were transferred to larger cell culture. After performing the same cloning procedure twice, hybridomas producing monoclonal antibodies of interest were established. The isotypes of monoclonal antibodies were determined by using a mouse monoclonal antibody isotyping kit (Sigma-Aldrich Corp.).

Indirect ELISA CSE-PE and biotinylated GAGs were immobilized at 50 ng/well onto 96-well plastic microtiter plates. The wells were blocked for 1 h at room temperature with a blocking buffer (1% human serum albumin in PBS) and rinsed with PBS. The culture supernatants were added to the wells and incubated for 1 h at room temperature. After washing with PBS, a horseradish peroxidase-conjugated secondary antibody was added to the blocking buffer and incubated for 1 h at room temperature. After washing with PBS, CSE-bound antibodies were detected by adding O-phenylenediamine solution to each well and incubating for 15–30 min at room temperature. After adding an equal volume of stopping solution (1 M HCl), the absorbance was measured at 492 nm against a reference filter at 630 nm.

Surface plasmon resonance analysis This experiment was performed on a Biacore 3000 (GE Healthcare). Biotinylated GAGs were immobilized on a CM5 sensor chip (GE Healthcare Life Sciences) according to the manufacturer’s instructions. The binding experiments were performed by using the GAGimmobilized sensor chips and various concentrations of the culture supernatants. Affinity parameters, the association (ka) and dissociation (kd) rate constants and equilibrium dissociation constant (KD), were determined with BIAevaluation software (Version 4.1.1) (GE Healthcare) using a 1:1 Langmur binding model. The KD value was calculated from the ratio (ka/kd).

Immunohistochemical staining of squid cartilage For immunostaining, cryosections of squid cartilage were fixed with neutral buffered formalin. After blocking with 0.1% casein in PBS for 1 h at room temperature, the sections were incubated with the culture supernatants for 1 h at 37°C and washed with PBS. Biotinylated anti-mouse IgM antibody (Nichirei Biosciences Inc.) was added, and then the section was incubated for 10 min at room temperature and washed with PBS. CSE-antigen on the tissue sections was visualized by adding peroxidase-labeled streptavidin and 3,3′-diaminobenzidine as a chromogen. As a negative control, mouse IgM or the tissue section treated with chondroitinase ABC was used. Cryosections were observed using a biological microscope with a 4 × or 40 × objective lens (Olympus).

Immunohistochemical staining of the carcinoma cell line The human ovarian cancer cell line OVCAR3 was obtained from the ATCC. OVCAR3 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin G and 100 μg/mL streptomycin. OVCAR3 cells on collagencoated coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with Tris-buffer saline containing 0.1% Tween 20, the samples were blocked with Blocking One solution (Nacalai), followed by incubation with the culture supernatants in the same solution overnight at 4°C. The resulting coverslips were incubated with FITC-conjugated anti-mouse IgM as a visualizing antibody (Sigma-Aldrich Corp.) for 1 h, and coverslips were mounted on glass slides using the ProLong Gold antifade reagent with DAPI

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Iisolation of CSE and preparation of CSE-PE as an immunogen for mice immunization

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Isolation and characterization of monoclonal antibodies specific for CSE (Invitrogen). Images were taken by using an IX71 fluorescence microscope (Olympus).

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buffered saline; PE, L-α-phosphatidylethanolamine; 2S, 2-O-sulfated; 4S, 4-O-sulfated; 6S, 6-O-sulfated.

Immunohistochemical staining of rat choroid plexus

Supplementary data Supplementary data for this article is available online at http://glycob. oxfordjournals.org/.

Funding This work was supported by grants-in-aid for Scientific Research on Priority Areas (24570168 to KIPJH) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported by a Cooperative Research Grant of the Institute of Tropical Medicine, Nagasaki University, 2015 (KIPJH, University of Aizu).

Acknowledgements The authors are grateful to Masataka Arihara (Seikgaku Corp.) for helpful discussion. We also thank Yuniko Shibata (Seikgaku Corp.) for skillful technical support about isolation of CSE.

Conflict of interest None declared.

Abbreviations CS, chondroitin sulfate; CSA, chondroitin sulfate A; CSC, chondroitin sulfate C; CSD, chondroitin sulfate D; CSE, chondroitin sulfate E; CSH, chondroitin sulfate H; DAPI, 4’,6-diamidino-2-phenylindole; deS-CSC, desulfated chondroitin sulfate C; 6deS-CSE, 6-O-desulfated chondroitin sulfate E; DS, dermatan sulfate; ΔDi-0S, ΔHexA1-3GalNAc; ΔDi-4S, ΔHexA1-3GalNAc(4S); ΔDi-6S, ΔHexA1-3GalNAc(6S); ΔDi-2S, ΔHexA(2S)1-3GalNAc; ΔDi-(4,6)S, ΔHexA13GalNAc(4S,6S); ΔDi-(2,4)S, ΔHexA(2S)1-3GalNAc(4S); ΔDi-(2,6)S, ΔHexA (2S)1-3GalNAc(6S); ΔDi-(2,4,6)S, ΔHexA(2S)1-3GalNAc(4S,6S); ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; GAG, glycosaminoglycan; GalNAc, N-acetyl-D-galactosamine; GalNAc4S-6ST, GalNAc 4-sulfate 6-O-sulfotransferase; GlcA, D-glucuronic acid; HA, hyaluronic acid; HAT, hypoxanthine-aminopterin-thymidine mixture; Hep, heparin; ΔHexA, 4,5-unsaturated hexuronic acid; HPLC, high-performance liquid chromatography; HPN, heparosan; HS, heparan sulfate; IdoA, L-iduronic acid; KS, keratin sulfate; MS, mass spectrometry; MW, molecular weight; PBS, phosphate

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Immunohistochemical staining of rat brain slices was described by Minami et al. (2011). Briefly, the rats coronal brain slices (3 mm in thickness) were embedded in Tissue-Tek OCT compound (Sakura Fine Technical, Tokyo, Japan). After freezing, the brain slices were cut into 10-µm-thick sections at −20°C using a Cryotome CM3050 S (Leica Biosystems, Germany). The sections were fixed with 4% paraformaldehyde for 15 min and blocked in PBS ( pH 7.4) containing 2% goat serum for 30 min. As a negative control, the sections were treated with chondroitinase ABC. The sections were incubated with the antibody E-18H at 4°C overnight and then incubated with FITCconjugated goat anti-mouse IgM (Sigma-Aldrich Corp.) at 27°C for 1 h. Counterstaining was performed with 1 µg/mL DAPI. After mounting with an anti-fade reagent (Invitrogen), images were acquired using a fluorescence microscope ( filter sets: ex/em, BP460–495/ BA510-550 for FITC; BP330-385/BA420 for DAPI). The background level of fluorescence was determined by staining the sections and using only the secondary antibody. Staining was performed at least twice using different rats, and the reproducibility was confirmed.

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Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galac...
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