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| Bar graphs (based on triplicate experiments with standard deviation) of normalized PrP binding preference to surface heparin by competing with different GAGs. One asterisk and two asterisks denote the statistical comparison between control and each sample (**p < 0.01; *p < 0.05). (A) Full length PrP (M23-230) concentration was 63 nM, concentrations of GAGs in solution were 1,000 nM. (B) Delta PrP (M23-230 59-90) concentration was 125 nM, concentrations of GAGs in solution were 1,000 nM. (C) N-terminal PrP (M23-109) concentration was 125 nM, concentrations of GAGs in solution were 1,000 nM. All measurements (A-C) were made using the same SPR chip immobilized with heparin (average molecular weight ∼15 kDa).
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Self-propagating form of the prion protein (PrP
Sc
) causes many neurodegenerative diseases, such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). Heparin is a highly sulfated linear glycosaminoglycan (GAG) and is composed of alternating D-glucosamine and L-iduronic acid or D-glucuronic acid sugar residues. The...
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Context 1
... screened inhibition capability of GAGs of different structures (Figure 1), including unfractionated heparin, HS, chondroitin sulfate type A (CS-A), CS-C, CS-D, CS-E, DS, disulfated DS (Dis-DS), and keratan sulfate (KS), against interactions between PrP constructs and immobilized heparin ( Figure 5). All GAGs tested were used at 1,000 nM. ...
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... GAGs tested were used at 1,000 nM. For full length PrP, only unfractionated heparin was capable of inhibiting PrP-heparin interactions by 80% while the rest of GAGs showed negligible inhibition ( Figure 5A). Unfractionated heparin inhibited M23-230 59-90 PrP and heparin interactions by ∼90% and varying degree of inhibition was observed by other GAGs ranging from 20 to 60% inhibition (Figure 5B). ...
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... full length PrP, only unfractionated heparin was capable of inhibiting PrP-heparin interactions by 80% while the rest of GAGs showed negligible inhibition ( Figure 5A). Unfractionated heparin inhibited M23-230 59-90 PrP and heparin interactions by ∼90% and varying degree of inhibition was observed by other GAGs ranging from 20 to 60% inhibition (Figure 5B). This reinforces the idea of weakened binding interaction to immobilized heparin due to lack of one putative heparin binding motif and potential change in 3-D structure as described above. ...
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... reinforces the idea of weakened binding interaction to immobilized heparin due to lack of one putative heparin binding motif and potential change in 3-D structure as described above. Lastly, inhibition ranging from 20 to 90% was demonstrated by various GAGs for inhibiting M23-109 PrP-heparin binding (Figure 5C), however, the preferred structure of GAG was different from those of full length or M23-230 59-90 PrP, suggesting a different mode of binding then was observed in competition assays utilizing varying chain length heparin oligosaccharides (Figure 4C). ...
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Neurodegenerative diseases are characterized by the deposition of specific protein aggregates, both intracellularly and/or extracellularly, depending on the type of disease. The extracellular occurrence of tridimensional structures formed by amyloidogenic proteins defines Alzheimer’s disease, in which plaques are composed of amyloid β-protein, whil...
Citations
... To understand the structure-activity relationships of the sulfation patterns in HS, Seeberger, 4,5 Linhardt, 6,7 Liu, 8,9 Boons, 10,11 Weigel, 12 DeAngelis, 13 Hung, 14, 15 Huang, [16][17][18] and others [19][20][21][22][23][24] have developed chemical and chemoenzymatic synthetic approaches to well-dened HS oligosaccharides that have signicantly advanced our understanding of HS biology. Nevertheless, the chemical synthesis of HS oligosaccharides oen requires exquisite synthetic route design and skillful carbohydrate chemistry manipulations, resulting in high production cost and scalability challenges. ...
Heparan sulfate (HS) glycosaminoglycans are widely expressed on the mammalian cell surfaces and extracellular matrices and play important roles in a variety of cell functions. Studies on the structure-activity relationships of HS have long been hampered by the challenges in obtaining chemically defined HS structures with unique sulfation patterns. Here, we report a new approach to HS glycomimetics based on iterative assembly of clickable disaccharide building blocks that mimic the disaccharide repeating units of native HS. Variably sulfated clickable disaccharides were facilely assembled into a library of mass spec-sequenceable HS-mimetic oligomers with defined sulfation patterns by solution-phase iterative syntheses. Microarray and surface plasmon resonance (SPR) binding assays corroborated molecular dynamics (MD) simulations and confirmed that these HS-mimetic oligomers bind protein fibroblast growth factor 2 (FGF2) in a sulfation-dependent manner consistent with that of the native HS. This work established a general approach to HS glycomimetics that can potentially serve as alternatives to native HS in both fundamental research and disease models.
... Initial results show that the Noggin heparin binding kinetics are heterogeneous which is commonly observed for heparin binding proteins ( Figure 1a). 7,20 We thus fit the data to a two to one heterogeneous ligand binding model (Table S1), which assumes variability in the heparin sulfation patterns with two independent binding sites for Noggin on the heparin ligand. The binding kinetics show that both binding events have similar association rate constants (k on ), but different dissociation rate constants (k off ), with one binding event having a noticeably slower dissociation than the other resulting in a biphasic dissociation. ...
Extracellular matrix proteins are most often defined by their direct function that involves receptor binding and subsequent downstream signaling. However, these proteins often contain structural binding regions that allow for the proper localization in the extracellular space which guides its correct function in a local and temporal manner. The regions that serve a structural function, although often associated with disease, tend to have a limited understanding. An example of this is the extracellular matrix protein Noggin; as part of the bone morphogenetic protein inhibitor family, Noggin serves a crucial regulatory function in mammalian developmental stages and later periods of life. Noggin's regular function, after its expression and extracellular release, is mediated by its retention in close proximity to the cellular surface by glycosaminoglycans, specifically heparin and heparan sulfate. Using a biophysical hybrid method approach, we present a close examination of the Noggin heparin binding interface, study its dynamic binding behaviors and observe supramolecular Noggin assemblies mediated by heparin ligands. This confirms previously suggested models of non‐covalent protein assemblies mediated through glycosaminoglycans that exist in the extracellular matrix. Further, structural analyses through molecular dynamics simulations allowed us to determine contribution energies for each protein residue involved in ligand binding and correlate this to disease associated mutation data. Our combination of various biophysical and computational methods that characterize the heparin binding interface on Noggin and its protein dynamics expands on the functional understanding of Noggin and can readily be applied to other protein systems.
... As mentioned above, this difference may be due to sex, but more importantly, it is the proteins' function. ITC and SPR are suitable methods for characterizing the protein-ligand or protein-protein interactions via thermodynamic and kinetic changes occurring during binding [50][51][52]. Based on the SPR results, GOBP2 is better than GOBP1 regarding the number of binding proteins and the binding ability. From this result, we can hypothesize that when GOBP2 binds to a variety of other proteins, a complex is formed, and it dramatically broadens the binding range of the GOBP2 protein, especially with a solid binding ability with respect to CSPs. ...
The GOBP2 protein has a unique function in the yellow peach moth (Conogethes punctiferalis (Guenée)). Several general odorant-binding proteins (GOBPs) have been identified in various lepidopteran species, but the functional difference between GOBP1 and GOBP2 in recognition of host plant odorants is still unknown. The functions of GOBP1 and GOBP2 in the yellow peach moth were evaluated in this study by using the CRISPR-Cas9 system. The results revealed the importance of GOBP2 in the olfaction mechanism in the yellow peach moth. The perception of the GOBP1-knockout larvae toward feeding decreased but did not reach a significant level while knocking out the GOBP2 and GOBP1/2 genes resulted in huge differences. On the other hand, electroantennograms (EAGs) and wind tunnel tests showed that the sensitivity of GOBP2 knockout adults to odorants decreased more than that of GOBP1 knockout individuals. The results of STRING database text mining grabbed our attention in protein-protein interaction studies. In this research, we first proved the existence of physical interactions between GOBPs and chemosensory proteins (CSPs) through the surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) methods. Interestingly, GOBP1 and GOBP2 could not interact with each other, but they could interact with CSPs. The interaction results indicated that GOBP2 could physically interact with CSP15, CSP5, and OBP17, whereas GOBP1 could bind only with CSP5 and CSP10, and its association constant (ka) was also more substantial than that of GOBP1. These results strongly suggest the importance of the function of GOBP2 in the perception of host plant odorants by the yellow peach moth.
... Low molecular weight heparin interacts with murine PrP, leading to a transient oligomerization/aggregation process . The octapeptide region has also been shown to be important for interaction at neutral pH Kim et al., 2020), while a second binding site in the C-terminal region of the protein under acidic conditions has also been suggested . In addition, low molecular weight heparin acts as an inhibitor of PrP fibrillation, indicating its therapeutic potential (Vieira et al., , 2014. ...
Prion diseases have been described in humans and other mammals, including sheep, goats, cattle, and deer. Since mice, hamsters, and cats are susceptible to prion infection, they are often used to study the mechanisms of prion infection and conversion. Mammals, such as horses and dogs, however, do not naturally contract the disease and are resistant to infection, while others, like rabbits, have exhibited low susceptibility. Infection involves the conversion of the cellular prion protein (PrPC) to the scrapie form (PrPSc), and several cofactors have already been identified as important adjuvants in this process, such as glycosaminoglycans (GAGs), lipids, and nucleic acids. The molecular mechanisms that determine transmissibility between species remain unclear, as well as the barriers to transmission. In this study, we examine the interaction of recombinant rabbit PrPC (RaPrP) with different biological cofactors such as GAGs (heparin and dermatan sulfate), phosphatidic acid, and DNA oligonucleotides (A1 and D67) to evaluate the importance of these cofactors in modulating the aggregation of rabbit PrP and explain the animal’s different degrees of resistance to infection. We used spectroscopic and chromatographic approaches to evaluate the interaction with cofactors and their effect on RaPrP aggregation, which we compared with murine PrP (MuPrP). Our data show that all cofactors induce RaPrP aggregation and exhibit pH dependence. However, RaPrP aggregated to a lesser extent than MuPrP in the presence of any of the cofactors tested. The binding affinity with cofactors does not correlate with these low levels of aggregation, suggesting that the latter are related to the stability of PrP at acidic pH. The absence of the N-terminus affected the interaction with cofactors, influencing the efficiency of aggregation. These findings demonstrate that the interaction with polyanionic cofactors is related to rabbit PrP being less susceptible to aggregation in vitro and that the N-terminal domain is important to the efficiency of conversion, increasing the interaction with cofactors. The decreased effect of cofactors in rabbit PrP likely explains its lower propensity to prion conversion.
In recent years, the scientific community has been trying to tackle different diseases by using unifying and holistic approaches based on the concept that it is possible to target apparently very different diseases under a comprehensive general scheme. In other words, various different diseases have been grouped together under the label of "conformational diseases", because the triggering cause for each malady is the misfolding of a specific protein, whose dyshomeostasis and accumulation cause all the other downhill biomolecular events characteristic of each different disease. In a parallel manner, analytical techniques have developed to investigate protein misfolding and accumulation, so as to give a valid technical support to the investigation of conformational diseases. In this scenario, surface plasmon resonance (SPR) has widely contributed to study many different aspects correlated to conformational diseases, offering the advantages of real time investigations, use of small amounts of biological materials and possibility to mimic the cellular environments without recurring to the use of fluorescent tags. In this review, after a brief introduction about conformational diseases and the SPR technique, a thorough description of the various uses of SPR to investigate the biomolecular mechanisms involved in these diseases is given in order to provide the reader with an exhaustive list as well as a critical perspective of the use of SPR for such topic. The case of Alzheimer's disease is discussed at a deeper level. We hope that this work will make the reader aware of all the possible SPR experimental approaches, which can be used to develop new possible therapeutic strategies to tackle conformational diseases.
Aggregation of amyloid beta into amyloid plaques in the brain is a hallmark characteristic of Alzheimer's disease. Therapeutics aimed at preventing or retarding amyloid formation often rely on detailed characterization of the underlying mechanism and kinetics of protein aggregation. Surface plasmon resonance (SPR) spectroscopy is a robust technique used to determine binding affinity and kinetics of biomolecular interactions. This approach has been used to characterize the mechanism of aggregation of amyloid beta but there are multiple pitfalls that need to be addressed when working with this and other amyloidogenic proteins. The choice of method for analyte preparation and ligand immobilization to a sensor chip can lead to different theoretical and practical implications in terms of the mathematical modelling of binding data, different mechanisms of binding and the presence of different interacting species. This review examines preparation methods for SPR characterisation of the aggregation of amyloid beta and their influence on the findings derived from such studies.
