Abstract
Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding analyses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(beta 1----3)N-acetylgalactosamine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase digestion results in progressive reduction in its inhibitory activity. The results suggest that the epitope has a requirement for a long stretch of a disaccharide-repeating structure for a better fit to the antibody.
Highlights
Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains
The glucuronic acid residues, on the other hand, can be sulfated [10] at either position 2 or position 3 [11].It appears that most if not allchondroitin sulfate proteoglycans carry copolymers of these sulfated disaccharide units inwhich the percentages of the disaccharides vary with the source and age of the tissue
We have prepared a number of monoclonal antibodies against two distinct chondroitin sulfate proteoglycans, PG-H [16] and PG-M [17], isolated from 12-day-old chick embryo epiphysial cartilage and stage 22-23 chick embryo limb buds, respectively
Summary
Proteoglycans-The following proteoglycans were prepared by previously described methods: PG-Mand [35S]sulfate-labeledPG-M from stage 22-23 chick embryo limb buds [17]; PG-H ( X ) , [35S]. NaCl and applied to a Diaion HPA-10 (anion exchange resin) column linkages to nonsulfated glucuronosyl residues [29].Briefly, a solution (2.6 X 55 cm) that had been equilibrated with 1M NaCl. The column of 100 mgof shark fin chondroitin sulfate Fraction 111 in 2 ml of was washed with 600 ml of 1 M NaCl and eluted stepwise with 0.025 M Tris buffer, pH 7.2, containing 0.4 mg of calcium acetate, 2.5. After chondroitin sulfate was precipitated with ethanol, washed, and dried centrifugation at 900 X g for 5 min, the clear supernatant was as above:yield,1.9 g
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