Selective Detection of Protein Secondary Structural Changes in Solution Protein−Polysaccharide Complexes Using Vibrational Circular Dichroism (VCD)

A major challenge to understanding the fundamental structural basis of interactions between macromolecules in solution is how to measure their separate contributions. Particularly challenging is the interaction between proteins and polysaccharides. The polysaccharide component is often both very large (10 kDa−MDa) and immobile, or it undergoes anisotropic motion in solution, causing line broadening in NMR. Furthermore, they often exhibit signals in the very spectral regions normally employed for protein secondary structural analysis (FTIR and CD), and these signals cannot simply be subtracted because they occupy variable positions. The selective detection of protein secondary structural changes in aqueous complexes of proteins and polysaccharides, particularly the biologically important glycosaminoglycan class, is demonstrated here, exploiting a property of vibrational circular dichroism (VCD) that allows signals from proteins to be selectively detected. We show that polysaccharides, in contrast to proteins, which show well-documented and characteristic VCD signals for distinct secondary structural types, exhibit no VCD signals in the amide I‘ region despite containing N-acetyl groups. This is because the chromophores in the polysaccharides (in the CO bonds of N-acetyl and carboxylic acids groups) lack the regular geometric relationship to each other that characterizes stretches of defined protein secondary structure. We have exploited this hitherto unreported feature of VCD to enhance the contrast between proteins and bound polysaccharides in protein−polysaccharide complexes in solution. This enables the direct observation of protein secondary structural changes in protein−polysaccharide complexes in solution and will advance understanding of the structural basis of these interactions

How To Find a Needle (or Anything Else) in a Haystack: Two-Dimensional Correlation Spectroscopy-Filtering with Iterative Random Sampling Applied to Pharmaceutical Heparin

Risks of contamination of the major clinical anticoagulant heparin can arise from deliberate adulteration with unnatural or natural polysaccharides, including heparin from other animal sources, other natural products, or artifacts of manufacture, and these can escape detection by conventional means. Currently, there is no generally applicable, objective test recommended by regulators that can detect these in pharmaceutical heparin, and this continues to leave heparin exposed to contamination risks. Two-dimensional correlation spectroscopic-filtering with iterative random sampling (2D-COS-firs) is reported. It employs a difference covariance matrix with iterative random sampling, and is capable of revealing contamination in pharmaceutical heparin to a high level of sensitivity irrespective of the nature of those features. The technique is suitable to any situation in which a comparison of a single entity to a family of heterogeneous entities, particularly natural products and biosimilars, needs to be made, and will find application in pharmaceutical monitoring, manufacturing quality control, materials science, biotechnology, and metabolomic investigations

Differentiation of Generic Enoxaparins Marketed in the United States by Employing NMR and Multivariate Analysis

The U.S. Food and Drug Administration defines criteria for the equivalence of Enoxaparin with Lovenox, comprising the equivalence of physiochemical properties, heparin source material and mode of depolymerization, disaccharide building blocks, fragment mapping and sequence of oligosaccharide species, biological and biochemical assays, and <i>in vivo</i> pharmacodynamic profile. Chemometric analysis of the NMR spectra, utilizing both ¹H and ¹H–¹³C HSQC NMR experiments, of Lovenox and Enoxaparin, the latter being the generic version of the former, revealed that Lovenox and the four Enoxaparin compounds produced by Sandoz (Enoxaparin and Fibrinox), Winthrop, and Amphastar exhibit dissimilarities in terms of their composition. All of the collected samples had expiry dates between 2012 and 2015. These studies, in addition to chromatographic analysis, highlighted signatures that differentiated the branded material from the generic products

Disruption of Rosetting in <i>Plasmodium falciparum</i> Malaria with Chemically Modified Heparin and Low Molecular Weight Derivatives Possessing Reduced Anticoagulant and Other Serine Protease Inhibition Activities

Severe malaria has been, in part, associated with the ability of parasite infected red blood cells to aggregate together with uninfected erythrocytes to form rosettes via the parasite protein PfEMP-1. In this study, inhibitors of rosetting by the Plasmodium falciparum strain R-29, based on chemically modified heparin polysaccharides (IC50 = 1.97 × 10−2 and 3.05 × 10−3 mg·mL−1) and their depolymerized, low molecular weight derivatives were identified with reduced anticoagulant and protease (renin, pepsin, and cathepsin-D) activities. Low molecular weight derivatives of the two most effective inhibitors were shown to have distinct minimum size and strain-specific structural requirements for rosette disruption. These also formed distinct complexes in solution when bound to platelet-factor IV

Unravelling Structural Information from Complex Mixtures Utilizing Correlation Spectroscopy Applied to HSQC Spectra

The first use of <i>statistical</i> correlation spectroscopy to extract chemical information from 2D-HSQC spectra, termed HSQC correlation spectroscopy (<b>HSQCcos</b>), is reported. <b>HSQCcos</b> is illustrated using heparin, a heterogeneous polysaccharide, whose diverse composition causes signals in HSQC spectra to disperse. <b>HSQCcos</b> has been used to probe the chain modifications that cause this effect and reveals hitherto unreported structural details. An interesting finding was that the signal for position 2 of trisulfated glucosamine [N-, 3-O-, and 6-O-sulfated] (<b>A</b>*) is bifurcated, owing to the presence of <b>A</b>* residues in both the “normal” antithrombin binding site and also at the nonreducing end of the molecule, which is reported in intact heparin for the first time. The method was also applied to investigating the environment around other rare sequences/disaccharides, suggesting that the disaccharide; 2-O-sulfated iduronic acid linked to 6-O-sulfated N-glucosamine, which contains a free amine at position 2, is adjacent to the heparin linkage region. <b>HSQCcos</b> can extract chemically related signals from information-rich spectra obtained from complex mixtures such as heparin

Atomic Details of the Interactions of Glycosaminoglycans with Amyloid‑β Fibrils

The amyloid plaques associated with Alzheimer’s disease (AD) comprise fibrillar amyloid-β (Aβ) peptides as well as non-protein factors including glycos­amino­glycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aβ self-assembly and can impede fibril clearance; thus, they may be accessory molecules in AD. Here we report the first high-resolution details of GAG–Aβ fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aβ fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly 13C-labeled octa­saccharide heparin analogue enabled magic-angle spinning solid-state NMR of the GAG bound to 3Q fibrils, and measurements of dynamics revealed a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular 13C–15N dipolar dephasing is consistent with close (<5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aβ40 fibrils and a major non-protein component of AD plaques, and they reveal that GAG–amyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture

Using NMR to Dissect the Chemical Space and <i>O</i>‑Sulfation Effects within the <i>O</i>- and <i>S</i>‑Glycoside Analogues of Heparan Sulfate

Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of “heparanome” and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin–spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described

Cations Modulate Polysaccharide Structure To Determine FGF−FGFR Signaling: A Comparison of Signaling and Inhibitory Polysaccharide Interactions with FGF-1 in Solution

For heparan sulfate (HS) to bind and regulate the activity of proteins, the polysaccharide must present an appropriate sequence and adopt a suitable conformation. The conformations of heparin derivatives, as models of HS, are altered via a change in the associated cations, and this can drastically modify their FGF signaling activities. Here, we report that changing the cations associated with an N-acetyl-enriched heparin polysaccharide, from sodium to copper(II), converted it from supporting signaling through the fibroblast growth factor receptor (FGF-1−FGFR1c) tyrosine kinase signaling system to being inhibitory in a cell-based BaF3 assay. Nuclear magnetic resonance and synchrotron radiation circular dichroism (SRCD) spectroscopy demonstrated that the polysaccharide conformation differed in the presence of sodium or copper(II) cations. Electron paramagnetic resonance confirmed the environment of the copper(II) ion on the N-acetyl-enriched polysaccharide was distinct from that previously observed with intact heparin, which supported signaling. Secondary structures in solution complexes of polysaccharides with FGF-1 (which either supported signaling through FGFR1c or were inhibitory) were determined by SRCD. This allowed direct comparison of the two FGF-1−polysaccharide complexes in solution, containing identical molecular components and differing only in their cation content. Subtle structural differences were revealed, including a reduction in the level of disordered structure in the inhibitory complex