METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycoaminoglycans (GAGs); heparin, heparan sulfate, dermatan and chondroitin sulfate are involved in important physiological and pathological processes including regulation of enzymes and signaling molecules in response to cellular damage (wounding, infection, tumorigenesis), bacterial infection, etc. via direct interaction with intra- and inter-cellular proteins. Over the past decade an increasing number of proteins have been discovered to interact with GAGs. In order to identify these proteins as well as understand these interactions and structural motifs related to GAG-protein interaction we developed an improved LC-MS based binding assay method. In this work, an LC-MS method using an amide-based normal phase chromatography column was used to identify heparin structures that mediate antithrombin III binding. Heparin was digested with heparin lyase-I under conditions optimized to achieve partial digestion. Oligosaccharides were then fractionated using preparative size exclusion chromatography (SEC). Sugars with degree of polymerization (dp) 6, 8, 10 were respectively mixed with ATIII. The unbound sugars were separated from the complex using high performance SEC. The complex was applied to a reverse phase cartridge and the bound oligosaccharides eluted with 2M ammonium acetate. The eluted fractions were then characterized by LC-MS using a 250 ¿m x 15 cm TSK-gel Amide-80 column packed in house. The column was equilibrated in mobile phase at 1 ¿L/min with 95%A:5%B (A=95%ACN, B=10%ACN, 50 mM ammonium formate pH 4.4. Oligosaccharides were eluted with a gradient of 5% to 75% B over 60 min directly into a QSTAR Pulsar-I mass spectrometer operating in the negative ion mode. Capillary normal phase LC/MS yielded sensitive and reproducible data allowing facile characterization of the bound or unbound heparin oligosaccharides. Unambiguous resolution of 30 pmol of heparin dp6 could be achieved using the current LC/MS system. The following structures were detected: [¿HexA, HexA, GlcN, SO3, Ac] [1,2,3,6,1], [1,2,3,7,1], [1,2,3,7,0], [1,2,3,8,0], [1,2,3,9,0]. The elution time of the dp6 fraction compounds is based on polar interactions of the sulfated group with the stationary phase. The complete resolution of those compounds at non-overlapping retention times demonstrate without any ambiguity that each structure [1,2,3,X,Y] is not generated by a sulfate loss through in-source CID of the [1,2,3,X+1,Y] structure. Furthermore, 3 glycoforms of composition [1,2,3,6,1] were detected. The same type of results was observed for the dp8 and dp10 fractions. The same mixture of heparin dp6 was subjected to the previously described ATIII binding protocol. Among all the structures detected in the pre-bound dp6 mixture, only two binders were detected after elution from the protein, namely [1,2,3,8,0] and [1,2,3,9,0]. In addition to the ability of these two species to overcome the non-equilibrium conditions of the SEC column and three rounds of washes, their binding specificity was further confirmed with two independent experiments. In the first experiment, the ATIII was rendered biologically inactive by heat inactivation and subsequently mixed with heparin dp6. In the second experiment, the active protein was mixed with the unspecific oligosaccharide Chondroitin sulfate B. In both experiments, no oligosaccharides were detected after the workup, confirming the ability of the optimized protocol to eliminate non-specific binding. The work will be expanded to the dp8 and dp10 fractions.