Abstract
An important characteristic of malaria parasite Plasmodium falciparum-infected red blood cells (IRBCs) is their ability to adhere to host endothelial cells and accumulate in various organs. Sequestration of IRBCs in the placenta, associated with excess perinatal and maternal mortality, is mediated in part by adhesion of parasites to the glycosaminoglycan chondroitin sulfate A (CSA) present on syncytiotrophoblasts lining the placental blood spaces. To define key structural features for parasite interactions, we isolated from CSA oligosaccharide fractions and established by electrospray mass spectrometry and high performance liquid chromatography disaccharide composition analysis their differing chain length, sulfate content, and sulfation pattern. Testing these defined oligosaccharide fragments for their ability to inhibit IRBC adhesion to immobilized CSA revealed the importance of non-sulfated disaccharide units in combination with 4-O-sulfated disaccharides for interaction with IRBCs. Selective removal of 6-O-sulfates from oligo- and polysaccharides to increase the proportion of non-sulfated disaccharides enhanced activity, indicating that 6-O-sulfation interferes with the interaction of CSA with IRBCs. Dodecasaccharides with four or five 4-O-sulfated and two or one non-sulfated disaccharide units, respectively, comprise the minimum chain length for effective interaction with IRBCs. Comparison of the activities of CSA and CSB oligo- and polysaccharides with a similar sulfation pattern and content achieved from partial desulfation demonstrated that glucuronic acid rather than iduronic acid residues are important for IRBC binding.
Highlights
An important characteristic of malaria parasite Plasmodium falciparum-infected red blood cells (IRBCs) is their ability to adhere to host endothelial cells and accumulate in various organs
Selective removal of 6-O-sulfates from oligo- and polysaccharides to increase the proportion of non-sulfated disaccharides enhanced activity, indicating that 6-O-sulfation interferes with the interaction of chondroitin sulfate A (CSA) with IRBCs
This suggested that non-sulfated disaccharide units may be important elements of the structural motif required for parasite adhesion acid; GlcUA, glucuronic acid; IdoUA, iduronic acid; GalNAc, N-acetylgalactosamine; SAX, strong anion exchange; APS, aminopropyl silica; ES-MS, electrospray mass spectrometry; 0S, ⌬UA-GalNAc; 4S, ⌬UAGalNAc(4S); 6S, ⌬UA-GalNAc(6S); HexA, hexuronic acid; HPLC, high performance liquid chromatography
Summary
Preparation and Fractionation of CS Oligosaccharide Fragments— Chondroitin sulfate A (from bovine trachea, Sigma; average molecular mass: 45,500 kDa, based on low angle laser light scattering), B (from porcine intestinal mucosa, Sigma), and C (from shark cartilage, Sigma) were partially depolymerized by controlled digestion with chondroitin lyase ABC (EC 4.2.2.4, from Proteus vulgaris, Sigma) essentially as described previously [12, 13]. CSB oligosaccharide fragments were prepared but on a smaller scale (100 mg of polysaccharide and 0.5 units of chondroitin lyase ABC in 4 ml of solution). A linear gradient of NaH2PO4 (solvent A, 0.1 M; and solvent B, 1.0 M; 30 –50% B in 40 min) was used to elute SAX-HPLC fractions F6C-2 and F6C-3. De-6-O-sulfated CSA dodecasaccharide fraction (dF6) and the subfractions derived from it, dF6A to dF6G containing 0 to 6 sulfates, respectively, were obtained essentially as described above for CSA oligosaccharide fragments. A gradient of NaCl (solvents A and B as above), 0 – 80% B in 45 min was used for the elution of disaccharides at a flow rate of 1 ml/min with detection at UV 232 nm. Samples were randomized and coded, and cytoadherence assays were carried out blinded
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