Mark A. Skidmore

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions IMPLICATIONS FOR BINDING SPECIFICITY

Background: Heparan sulfate (HS) regulates the transport and signaling activities of fibroblast growth factors (FGF). Results: The molecular determinants of the interactions of FGFs and heparin were identified. Conclusion: There are clear molecular specificities determining the interactions of FGFs with the polysaccharide. Significance: The expansion of the FGFs in metazoan evolution parallels the diversification of the specificity of their interactions with heparin. The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit KD values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m−1 s−1 and FGF-9, 130,000 m−1 s−1). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

Glycosaminoglycan origin and structure revealed by multivariate analysis of NMR and CD spectra

Principal component analysis (PCA) is a method of simplifying complex datasets to generate a lower number of parameters, while retaining the essential differences and allowing objective comparison of large numbers of datasets. Glycosaminoglycans (GAGs) are a class of linear sulfated carbohydrates with diverse sequences and consequent complex conformation and structure. Here, PCA is applied to three problems in GAG research: (i) distinguishing origins of heparin preparations, (ii) structural analysis of heparin derivatives, and (iii) classification of chondroitin sulfates (CS). The results revealed the following. (i) PCA of heparin (13)C NMR spectra allowed their origins to be distinguished and structural differences were identified. (ii) Analysis of the information-rich (1)H and (13)C NMR spectra of a series of systematically modified heparin derivatives uncovered underlying properties. These included the presence of interactions between residues, providing evidence that a degree of degeneracy exists in linkage geometry and that a different degree of variability exists for the two types of glycosidic linkage. The relative sensitivity of each position (C or H nucleus) in the disaccharide repeating unit to changes in O-, N-sulfation and N-acetylation was also revealed. (iii) Analysis of the (1)H NMR and CD spectra of a series of CS samples from different origins allowed their structural classification and highlighted the power of employing complementary spectroscopic methods in concert with PCA.

Rapid Purification and High Sensitivity Analysis of Heparan Sulfate from Cells and Tissues TOWARD GLYCOMICS PROFILING

Studies on glycosaminoglycans and proteoglycans (PGs) have been hampered by difficulties in isolation and analysis by traditional methods that are laborious and lack sensitivity and throughput. Here we demonstrate a simple method for rapid isolation of proteoglycans (RIP) employing phenol/guanidine/chloroform reagent to purify heparan sulfate (HS) PGs quantitatively from various tissues and cells. We further show that this generic purification methodology, when applied in concert with a BODIPYTM fluorescent label, permits structural analyses on RIP-purified HS at ∼1,000-fold higher sensitivity than standard UV detection methods and ∼10–100-fold higher sensitivity than previous fluorescence detection methods. The utility of RIP-BODIPY methodology was demonstrated by rapid profiling of HS structural composition from small tissue samples, multiple mouse organs, and as little as a few thousand cultured cells. It was also used to generate novel insights into in vivo structural changes in HS from Sulf1 knock-out mice for the first time that differed significantly from previous observations limited to tissue culture experiments. RIP was also applied to purify HS for bioassay testing, exemplified by cell assays of fibroblast growth factor signaling activation; this generated data from 2-O-sulfotransferase knock-out mice and revealed an unexpected deficiency in fibroblast growth factor activation by HS from heterozygous mice. These data demonstrate that RIP will underpin emerging efforts to develop glycomics profiling strategies for HS and other glycosaminoglycans to explore their structure-function relationships in complex biological systems.

Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection

One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end–labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.

The conformation and structure of GAGs: recent progress and perspectives

The glycosaminoglycan (GAG) family of linear sulphated polysaccharides are involved in most regulatory processes in the extracellular matrix of higher organisms. The relationship between GAG substitution pattern and activity, however, remains unclear and experimental evidence suggests that subtle conformational factors play an important role. The difficulty of modelling these complex charged molecules shifts the burden of investigation towards experimental techniques. Recent advances in complementary physical-chemical, particularly spectroscopy-based approaches are reviewed, together with methods for analysing the resulting complex data. The prospects for combining some of these approaches and fitting them into the wider context of interactions, are also discussed.

The Activities of Heparan Sulfate and its Analogue Heparin are Dictated by Biosynthesis, Sequence, and Conformation

The glycosaminoglycan heparan sulfate (HS), is expressed on the surface of virtually all mammalian cells and is implicated in many crucial biological activities. The activities of HS and its close structural analogue heparin are mediated through interactions with proteins. However, the relationship between structure and activity is not simple, because the structure and conformation of HS and heparin are complex. This review surveys some of the relevant findings in HS/heparin chemistry, biochemistry, and biology.

Protein-GAG interactions: new surface-based techniques, spectroscopies and nanotechnology probes

New approaches, rooted in the physical sciences, have been developed to gain a more fundamental understanding of protein-GAG (glycosaminoglycan) interactions. DPI (dual polarization interferometry) is an optical technique, which measures real-time changes in the mass of molecules bound at a surface and the geometry of the bound molecules. QCM-D (quartz crystal microbalance-dissipation), an acoustic technique, measures the mass and the viscoelastic properties of adsorbates. The FTIR (Fourier-transform IR) amide bands I, II and III, resulting from the peptide bond, provide insight into protein secondary structure. Synchrotron radiation CD goes to much shorter wavelengths than laboratory CD, allowing access to chromophores that provide insights into the conformation of the GAG chain and of beta-strand structures of proteins. To tackle the diversity of GAG structure, we are developing noble metal nanoparticle probes, which can be detected at the level of single particles and so enable single molecule biochemistry and analytical chemistry. These new approaches are enabling new insights into structure-function relationships in GAGs and together they will resolve many of the outstanding problems in this field.

Disruption of Rosetting in Plasmodium falciparum 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 (IC 50 = 1.97 x 10 (-2) and 3.05 x 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.

Site-specific interactions of copper(II) ions with heparin revealed with complementary (SRCD, NMR, FTIR and EPR) spectroscopic techniques.

The interactions between Cu(II) ions and heparin were investigated using several complementary spectroscopic techniques. NMR indicated an initial binding phase involving specific coordination to four points in the structure that recur in slightly different environments throughout the heparin chain; the carboxylic acid group and the ring oxygen of iduronate-2-O-sulfate, the glycosidic oxygen between this residue and the adjacent (towards the reducing end) glucosamine and the 6-O-sulfate group. In contrast, the later binding phase showed little structural specificity. One- and two-dimensional correlated FTIR revealed that complex out of phase (asynchronous) conformational changes also occurred during the titration of Cu(II) ions into heparin, involving the CO and N-H stretches. EPR demonstrated that the environments of the Cu(II) ions in the initial binding phase were tetragonal (with slightly varied geometry), while the later non-specific phases exhibited conventional coordination. Visible spectroscopy confirmed a shift of the absorbance maximum. Titration of Cu(II) ions into a solution of heparin indicated (both by analysis of FTIR and EPR spectra) that the initial binding phase was complete by 15-20 Cu(II) ions per chain; thereafter the ions bound in the non-specific mode. Hetero-correlation spectroscopy (FTIR-CD) improved resolution and assisted assignment of the broad CD features from the FTIR spectra and indicated both in-phase and more complex out of phase (synchronous and asynchronous, respectively) changes in interactions within the heparin molecule during the titration of Cu(II) ions.

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.