Discovery of a TNF-α Antagonist Using Chondroitin Sulfate Microarrays

Abstract

Conjugation of CS Oligosaccharides to 1,2-(Bisaminooxy)ethane for Microarray ProductionOzonolysis of the anomeric allyl group and linkage of CS compounds 1-4 1,2 to 1,2-(bisaminooxy)ethane 3 proceeded as follows: oligosaccharide (0.51 µmol) was dissolved in MeOH (500 µL) and cooled to -78 °C. O 3 was bubbled through the reaction until a blue color persisted (1 min). The reaction was then purged with N 2 until colorless, quenched with Ph 3 P beads (3 mg), and gradually warmed to rt over 12 h. It was filtered and the product concentrated to afford the desired aldehyde as a white solid. The aldehyde (0.51 µmol) was then reacted for 14 h at rt with 1,2-(bisaminooxy)ethane hydrochloride (1.4 mg, 15 µmol) that had been dissolved in H 2 O (100 µL) and pH adjusted to 5.0 with 1 M NaOH. The resulting oxime product was purified using a SepPak C18 column (500 mg, H 2 O) and Sephadex G-10 (CS-E The relative concentrations of the aminooxy oligosaccharides were calibrated to one another using the carbazole assay for uronic acid residues. 4 Briefly, the acid borate reagent (1.5 mL of 0.80 g sodium tetraborate, 16.6 mL H 2 O, and 83.3 mL H 2 SO 4 ) was added to 20-mL glass vials with Teflon caps. The Carbohydrate MicroarraysSolutions of the aminooxy oligosaccharides (in 300 mM NaH 2 PO 4 , pH 5.0, 10 µL/well in a 384-well plate) were arrayed on Hydrogel Aldehyde slides (NoAb Biodiscoveries) by using a Microgrid II arrayer (Biorobotics) to deliver sub-nanoliter volumes at rt and 50% humidity. Concentrations of carbohydrates ranged from 0 -500 µM. The resulting arrays were incubated in a 70% humidity chamber at rt for 12 h and then stored in a low humidity, dust-free dessicator. The pH and reaction time were Non-specific attachment of CS oligosaccharides lacking the aminooxy linker (e.g., compounds 1-4) was not observed. Prior to use, the arrays were outlined with a hydrophobic pen (Super Pap Pen, Research Products International) to create a boundary for the protein treatments and rinsed three times with H 2 O. The slides were then blocked by treatment with NaBH 4 (125 mg) in 140 mM NaCl, 2.7 mM KCl, 5.4 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 (phosphate buffered saline, PBS, 50 mL) at rt for 5 min with gentle rocking and washed five times for 3 min with PBS. For all incubations, the slides were placed in a covered pipette tip box. Human TNF-α (Peprotech), FGF-1 (R&D Systems; both reconstituted to 2 µM in 0.1% Triton X-100 in PBS), cell culture supernatant containing monoclonal anti-CS-A antibody, or cell culture supernatant containing monoclonal anti-CS-E antibody (both 1:1 in 0.1% Triton X-100 in PBS) were spotted onto the slides in 250 µL quantities, and incubated statically at rt for 2 h. The slides were then washed as previously described and incubated with the appropriate primary antibody [anti-TNF-α (Peprotech) or anti-FGF-1 (R&D Systems); 1:1000 in 0.1% Triton X-100 in PBS] for 2 h at rt with gentle rocking. Following the incubation, the slides were washed as previously described and treated in the dark at rt with a seconda...

Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

“…These data indicate that once formed, the TNF-α-TNFR1 complex cannot bind to CS-E tetrasaccharides on the array. Thus, CS-E, TNF-α, and TNFR1 do not appear to form a ternary complex, consistent with previous studies (21) and further validating the use of microarrays to rapidly probe the interactions of GAGs with multimeric protein complexes.…”

“…The binding of proteins to GAGs was examined using carbohydrate microarrays containing either synthetic tetrasaccharides of defined sulfation sequence (21,22) or naturally occurring polysaccharides representing various GAG classes (23). Microarrays of synthetic tetrasaccharides displaying the CS-A, CS-C, or CS-E sulfation motifs (SI Appendix, Fig.…”

“…As final validation of our computational and carbohydrate microarray approaches, we examined the interaction of CS with TNF-α, a proinflammatory cytokine involved in autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis (33,34). Previous studies in our laboratory have demonstrated that a tetrasaccharide displaying the CS-E sulfation motif binds to TNF-α and antagonizes its interaction with TNFR1, thereby inhibiting TNF-α-induced cell death (21). To test our microarray approach, we incubated TNF-α, TNFR1-Fc, or a 1∶1 mixture of TNF-α:TNFR1-Fc with CS tetrasaccharide microarrays.…”

A sulfated carbohydrate epitope inhibits axon regeneration after injury

“…An important implication of these results is that blocking CS-E interactions may prevent the inhibition caused by CSPGs and promote axon regeneration. To generate a CS-E blocking agent, we raised a monoclonal antibody against a pure synthetic CS-E tetrasaccharide (28). Although antibodies have been generated previously using CS polysaccharides as antigens (29,30), their specificity has been limited by the structural heterogeneity of natural polysaccharides.…”

Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells

“…We tried to investigate about the binding capacity of CS-E to RANKL, however, the strong and stable interactions were not seen (data not shown). Sulfated GAGs are capable of binding with many kinds of proteins, including cytokines, growth factors, and matrix proteins, for example, OPG, TRAP 22) , tumor necrosis factor-α 23) , macrophage antigen 1 (Mac-1) 24) , and fibronectin 25) . It is thought that different activity of CS structures on osteoclast differentiation results from possible multiple interactions of each CS structure with various growth factors or adhesion molecules.…”