Glycosaminoglycans as naturally occurring combinatorial libraries: developing a mass spectrometry-based strategy for characterization of anti-thrombin interaction with low molecular weight heparin and heparin oligomers.
Heparin is a densely charged polysaccharide, which is best known for its anticoagulant activity, although it also modulates a plethora of other biological processes. Unlike biopolymers whose synthesis is strictly controlled by a unique genetic template, heparin molecules exhibit a remarkable degree of structural heterogeneity, which poses a serious challenge for studies of heparin-protein interactions. This analytical challenge is often dealt with by reducing the enormous structural repertoire of heparin to a model small molecule. In this paper, we describe a different approach inspired by the experimental methodologies from the arsenal of combinatorial chemistry. Interaction of anti-thrombin III (AT) with heparinoids is studied using a mixture of oligoheparin molecules of fixed degree of polymerization, but varying chemical composition (heparin hexasaccharides obtained by size exclusion chromatography of an enzymatic digest of porcine intestinal heparin with bacterial heparinase), as well as a heparin-derived pharmaceutical preparation Tinzaparin (heparin oligosaccharides up to a 22-mer). AT binders are identified based on the results of ESI MS measurements of complexes formed by protein-oligoheparin association. Additionally, differential depletion of free heparin oligomers in solution in the presence of AT is used to verify the binding preferences. ESI MS characterization of oligoheparin-AT interaction under partially denaturing conditions allowed the conformer specificity of the protein-polyanion binding to be monitored. A model emerging from these studies invokes the notion of a well-defined binding site on AT, to which a flexible partner (heparin) adapts to maximize favorable intermolecular electrostatic interactions. This study demonstrates the enormous potential of ESI MS as an analytical tool to study the interactions of highly heterogeneous glycosaminoglycans with their cognate proteins outside of the commonly accepted reductionist paradigm, which reduces the intrinsic complexity of heparin by using structurally defined homogeneous low molecular weight mimetics.
Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, Massachusetts 01003, USA.
Rinat R
RR
Abzalimov
Paul L
PL
Dubin
Igor A
IA
Kaltashov
D000990:Antithrombin III / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D020650:Combinatorial Chemistry Techniques; D019281:Dimerization; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D006025:Glycosaminoglycans; D006495:Heparin, Low-Molecular-Weight; D000990:Antithrombin III; D006493:Heparin