Marcelo A. Lima

Heparan sulfate and heparin interactions with proteins

Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed.

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

The amyloid plaques associated with Alzheimers 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 (13)C-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 (13)C-(15)N 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.

High-sensitivity visualisation of contaminants in heparin samples by spectral filtering of 1H NMR spectra

A novel application of two-dimensional correlation analysis has been employed to filter (1)H NMR heparin spectra distinguishing acceptable natural variation and the presence of foreign species. Analysis of contaminated heparin samples, compared to a dataset of accepted heparin samples using two-dimensional correlation spectroscopic analysis of their 1-dimensional (1)H NMR spectra, allowed the spectral features of contaminants to be recovered with high sensitivity, without having to resort to more complicated NMR experiments. Contaminants, which exhibited features distinct from those of heparin and those with features normally hidden within the spectral mass of heparin could be distinguished readily. A heparin sample which had been pre-mixed with a known contaminant, oversulfated chondroitin sulfate (OSCS), was tested against the heparin reference library. It was possible to recover the (1)H NMR spectrum of the OSCS component through difference 2D-COS power spectrum analysis of as little as 0.25% (w/w) with ease, and of 2% (w/w) for more challenging contaminants, whose NMR signals fell under those of heparin. The approach shows great promise for the quality control of heparin and provides the basis for greatly improved regulatory control for the analysis of heparin, as well as other intrinsically heterogeneous and varied products.

A non-hemorrhagic hybrid heparin/heparan sulfate with anticoagulant potential

The structural characterization and the anticoagulant potential of a novel heparin/heparan sulfate-like compound from the heads of Litopenaeus vannamei shrimp are described. While it is distinct from either heparin or heparan sulfate, enzymatic depolymerization and nuclear magnetic resonance spectroscopy analyses revealed that this molecule does share some structural features with heparin, such as the high degree of N- and 6-O-sulfation and minor N-acetylation, and with heparan sulfate, in the glucuronic acid content. Its ability to stabilize human antithrombin explains its significant anticoagulant activity in aPTT and Factor-Xa inhibition assays. Interestingly, in contrast to mammalian heparin, the shrimp compound displayed negligible hemorrhagic effect. Together, these findings have particular interest since they reveal a novel molecule with significant anti-Xa activity coupled with low bleeding effects which make the shrimp heparin/HS-like compound a potential alternative for mammalian heparin.

Antithrombin stabilisation by sulfated carbohydrates correlates with anticoagulant activity

Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning fluorimetry, correlated with the anticoagulant activity of heparin and heparin-related saccharides. Similar conformational changes were induced in native AT by a variety of active and inactive heparin-related sulfated carbohydrates, measured in solution using synchrotron radiation circular dichroism, and their anticoagulant activities. Measurement of native AT stabilisation provides a convenient assay for prospective anticoagulants and represents an additional parameter by which to compare biosimilar heparins.

A heparin-like compound isolated from a marine crab rich in glucuronic acid 2-O-sulfate presents low anticoagulant activity

A natural heparin-like compound isolated from the crab Goniopsis cruentata was structurally characterized and its anticoagulant and hemorrhagic activities were determined. Enzymatic and nuclear magnetic resonance analysis revealed that its structure is rich in disulfated disaccharides, possessing significant amounts of 2-O-sulfated-β-D-glucuronic acid units. Furthermore, low amounts of trisulfated disaccharide units containing 2-O-sulfated-α-L-iduronic acid were detected, when compared to mammalian heparin. In addition, this heparin-like structure showed negligible in vitro anticoagulant activity and low bleeding potency, facts that make it a suitable candidate for the development of structure-driven, heparin based therapeutic agents with fewer undesirable effects.

A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities.

A heparin-like glycosaminoglycan from shrimp containing high levels of 3-O-sulfated D-glucosamine groups in an unusual trisaccharide sequence.

The detailed characterization of a novel heparin-like glycosaminoglycan purified from the viscera (heads) of the shrimp Litopenaeus vannamei is reported. Structural analysis performed by mono- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed it to be rich in both glucuronic acid and N,6-sulfated glucosamine residues. The key peculiarities were its high 3-O-sulfated glucosamine content compared to mammalian heparins; a residue which is usually associated with the antithrombin (AT) binding site, and the location of these residues within 2-O-sulfated iduronate and glucuronate-containing sequences (I2S-A(∗)-G), a situation not found in mammalian heparin. It also exhibited higher molecular weight (∼36kDa) than conventional heparin (∼16kDa) but, negligible anticoagulant activity (∼5IU/mg compared to heparin ∼190IU/mg) and stabilization of AT, which has been linked directly to anticoagulation activity. A high affinity fraction, eluting at a similar salt concentration (0.75-1.5M NaCl) from an antithrombin affinity column, to the high affinity fraction of heparin, also showed only weak thermal stabilization of AT (+∼2°C). These structural peculiarities may help elucidate more clearly the relationship between structure and function of sulfated polysaccharides, and provide useful model compounds with which to better understand interactions of biological significance.

New Applications of Heparin and Other Glycosaminoglycans

Heparin, the widely used pharmaceutical anticoagulant, has been in clinical use for well over half a century. Its introduction reduced clotting risks substantially and subsequent developments, including the introduction of low-molecular-weight heparin, made possible many major surgical interventions that today make heparin an indispensable drug. There has been a recent burgeoning of interest in heparin and related glycosaminoglycan (GAG) polysaccharides, such as chondroitin sulfates, heparan sulfate, and hyaluronate, as potential agents in various applications. This ability arises mainly from the ability of GAGs to interact with, and alter the activity of, a wide range of proteins. Here, we review new developments (since 2010) in the application of heparin and related GAGs across diverse fields ranging from thrombosis and neurodegenerative disorders to microbiology and biotechnology.

Lepstospira interrogans shotgun phage display identified LigB as a heparin-binding protein

LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue.