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

Heparin Derivatives as Inhibitors of BACE-1, the Alzheimer's β-Secretase, with Reduced Activity against Factor Xa and Other Proteases

Heparan sulfate (HS) regulates processing of the amyloid precursor protein by the Alzheimer's β-secretase (BACE-1). An HS analogue, porcine intestinal mucosal heparin, was systematically modified at the principal positions of O-sulfation and N-sulfation/acetylation and tested for BACE-1 inhibitory and anti factor Xa activities. The derivative with the highest anti-BACE-1 to anti-Xa activity ratio contained N-acetyl and 2-O- and 6-O-sulfates and also exhibited attenuated activities against cathepsin-D and renin, two other structurally related aspartyl proteases

Supplemental Figure and Table Legends from Enhanced Tumorigenic Potential of Colorectal Cancer Cells by Extracellular Sulfatases

Supplemental Figure and Table Legends</p

Supplementary Tables 1 - 2, Figures 1 - 2 from Enhanced Tumorigenic Potential of Colorectal Cancer Cells by Extracellular Sulfatases

Supplementary 1: Table 1 - Sequences of shRNA used in the study. Supplementary 2: Table 2 - Sequences of primers used in the study. Supplementary 3: mRNA level of SULFs after shRNA knockdown. Supplementary 4: mRNA expression of HS biosynthetic enzymes</p

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

Differential Scanning Fluorimetry Measurement of Protein Stability Changes upon Binding to Glycosaminoglycans: A Screening Test for Binding Specificity

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein−GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument

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

Software Tool for the Structural Determination of Glycosaminoglycans by Mass Spectrometry

Structural elucidation of glycosaminoglycans (GAGs) is one of the major challenges in biochemical analysis. This is mainly because of the diversity of GAG sulfation and N-acetylation patterns and variations in uronate isomers. ESI-MS and recently MALDI-MS methodologies are important strategies for investigating the molecular structure of GAGs. However, the interpretation of MS data produced by these strategies must take into account a large number of variables (including the number of monosaccharide residues, acetylations, sulfate groups, multiple charges, and exchanges between different cations). We have developed a bioinformatics tool to assist this complex interpretation task. The software is based on GlycoWorkbench, a tool for semiautomatic interpretation of glycan MS data. The tool generates the sugar backbones in all their variants (GAG family, composition, acetylation positions, and number of sulfates) and automatically matches them with the selected MS peaks. The backbones corresponding to a given peak are validated against the selected MS/MS peaks by generating all possible fragmentations. Native chondroitin sulfate and heparin oligosaccharides as well as chemically modified heparin oligomers have been successfully analyzed by MALDI- and ESI-MS and MS/MS, and the results of the semiautomated annotation of these mass spectra are presented here

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

Human (α2→6) and Avian (α2→3) Sialylated Receptors of Influenza A Virus Show Distinct Conformations and Dynamics in Solution

Differential interactions between influenza A virus protein hemagglutinin (HA) and α2→3 (avian) or α2→6 (human) sialylated glycan receptors play an important role in governing host specificity and adaptation of the virus. Previous analysis of HA–glycan interactions with trisaccharides showed that, in addition to the terminal sialic acid linkage, the conformation and topology of the glycans, while they are bound to HA, are key factors in regulating these interactions. Here, the solution conformation and dynamics of two representative avian and human glycan pentasaccharide receptors [LSTa, Neu5Ac-α(2→3)-Gal-β(1→3)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc; LSTc, (Neu5Ac-α(2→6)-Gal-β(1→4)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc] have been explored using nuclear magnetic resonance and molecular dynamics simulation. Analyses demonstrate that, in solution, human and avian receptors sample distinct conformations, topologies, and dynamics. These unique features of avian and human receptors in solution could represent distinct molecular characteristics for recognition by HA, thereby providing the HA–glycan interaction specificity in influenza