Disaccharides

SKOV3 exosomes were labeled with carboxyfluoresceine diacetate succinimidyl-ester (CFSE) (Invitrogen) as previously described [12 (link)]. Briefly, exosomes (20 μg) collected after a 100,000 × g ultracentrifugation were incubated with 7.5 μM CFSE for 30 min at 37°C in a final volume of 200 μl PBS containing 0.5% BSA. Labeled exosomes (Exos-CFSE) were 65-fold diluted with DMEM supplemented with 10% vesicles-free fetal calf serum and pelleted by ultracentrifugation for 16 h at 10,0000 × g, 12°C. Exos-CFSE were resuspended in DMEM and incubated with SKOV3 cells at 37 or 4°C.
When indicated Exos-CFSE or cells were treated for 30 min with 100 μg/ml proteinase K, or for 2 h with 15 mU neuraminidase from V. cholerae or from A. urefaciens (Roche), before uptake. SKOV3 cells were also incubated, 30 min prior to and during uptake, with the inhibitors 10 μg/ml chlorpromazine, 5 μg/ml cytochalasin D, 50 μM 5-ethyl-N-isopropyl amiloride (EIPA) or 2% methyl-beta-cyclodextrin, or with 150 mM of the monosaccharides D-glucose, D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine, and the disaccharide β-lactose (Sigma).
Uptake assays were always performed in the presence of the compounds and analyzed after 2 or 4 h by immunofluorescence microscopy or flow cytometry.

A neoglycoprotein (XXXG-BSA) was prepared by coupling a heptasaccharide containing 3 xylosyl and 4 glucosyl residues (XXXG, Megazyme, Bray, Ireland) to BSA by reductive amination [42 (link)]. XXXG (30 mg) was dissolved in 1.0 ml of 0.2 M sodium borate buffer pH 9.0. This was followed by the addition of 20 mg BSA and then 30 mg of sodium cyanoborohydride. The mixture was maintained in a water bath at 50°C with occasional mixing. After 24 h the pH was adjusted to pH 4.0 by the addition of 45 μl of 80% (v/v) acetic acid. The solution was then dialysed extensively against distilled water with several changes over 4 days.
Rat immunization, hybridoma preparation and cloning procedures were performed as described previously [34 (link)]. Two male Wistar rats were injected with 100 μg XXXG-BSA in complete Freund's adjuvant administered subcutaneously on day 0, with the same amount administered with incomplete Freund's adjuvant on days 33 and 71. On day 145, a selected rat was given a prefusion boost of 100 μg XXXG-BSA in 1 ml PBS by intraperitoneal injection. The spleen was isolated three days later for isolation of lymphocytes and fusion with rat myeloma cell line IR983F [43 ]. Antibodies were selected by ELISA using tamarind xyloglucan as antigen. Subsequent characterization was by means of a glycan microarray of cell wall polymers [28 (link)] and competitive inhibition ELISAs using the xyloglucan XXXG heptasaccharide from tamarind xyloglucan and a series of related xyloglucan oligosaccharides. A mixture of the XXLG and XLXG octasaccharide isomers and the XLLG nonasaccharide were derived from tamarind xyloglucan as described [44 (link)] and purified by HPLC using Tosoh TSK Gel Amide column (21.5 × 300 mm) eluted with 65% aqueous acetonitrile. Cellotetraose GGGG was prepared by acetolysis of cellulose [45 ] and separated from the mixture of deacetylated oligosaccharides by HPLC as above. The sample of pea xyloglucan was a gift from Marie-Christine Ralet (INRA, Nantes, France). ELISAs were carried out as described previously [6 (link)] and in all cases immobilised antigens were coated at 50 μg/ml. Mannan, tamarind xyloglucan polymers, isoprimeverose and xylose disaccharide were obtained from Megazyme, Bray, Ireland. The selected antibody, an IgG2c, was designated LM15.

Biotinylated heparin was synthesized by adapting a previously reported procedure (Thakar et al., 2014 (link)). First, a solution containing heparin (4 mM, Sigma-Aldrich), 10 mM acetate buffer (made from glacial acetic acid (Carl Roth, Karlsruhe, Germany) and sodium acetate (Sigma-Aldrich) at pH 4.5) and aniline (100 mM, Sigma-Aldrich) was prepared. Biotin-PEG₃-oxyamine (3.4 mM, Conju-Probe, San Diego, United States) was added to the heparin solution and allowed to react for 48 h at 37°C. The final product was dialyzed against water for 48 h by using a dialysis membrane with a 3.5 kDa cutoff. The final solution was then lyophilized and stored at −20°C. For further use, the conjugates were diluted to the desired concentrations in buffer. The obtained biotinylated heparin was characterized by biotin-streptavidin binding assays using QCM-D. The average mass of the heparin isolate, when anchored to the surface, was estimated at 9 kDa (∼18 disaccharide units) by QCM-D analysis (Srimasorn et al., 2022 (link)).

Sugar chips immobilized with heparin or synthetic sulfated disaccharides derived from HS, CS, and DS were purchased from SUDx-Biotec (Kagoshima, Japan) as previously described [25 (link)]. Briefly, synthetic disaccharides having a lipoyl group were used as ligands. Lipoic acid is reduced to dihydrolipoic acid, resulting in two SH groups in the molecule. SH groups of sugar derivatives were readily adsorbed on the surface of gold-coated tips. The sugar chip was set on a prism with refractive oil (nD = 1.518, Cargille Laboratories, Cedar Grove, NJ, U.S.A.) in an SPR apparatus SPR670M (Moritex, Saitama, Japan). SPR measurements were conducted at room temperature, according to the manufacturer's instructions, using various concentrations of purified cochlin solutions expressed by the S2 cells. The cochlin-containing solution was applied and washed with running buffer. The difference in the response between the equilibrium before running and after washing was recorded as the binding signal.