I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Ana S.
Luis
,
Arnaud
Basle
,
Dominic P.
Byrne
,
Gareth S. A.
Wright
,
James A.
London
,
Chunsheng
Jin
,
Niclas G.
Karlsson
,
Gunnar C.
Hansson
,
Patrick A.
Eyers
,
Mirjam
Czjzek
,
Tristan
Barbeyron
,
Edwin A.
Yates
,
Eric C.
Martens
,
Alan
Cartmell
Diamond Proposal Number(s):
[21970, 18598]
Abstract: Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.
|
Jun 2022
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Ana S.
Luis
,
Chunsheng
Jin
,
Gabriel
Vasconcelos Pereira
,
Robert W. P.
Glowacki
,
Sadie R.
Gugel
,
Shaleni
Singh
,
Dominic P.
Byrne
,
Nicholas A.
Pudlo
,
James A.
London
,
Arnaud
Basle
,
Mark
Reihill
,
Stefan
Oscarson
,
Patrick A.
Eyers
,
Mirjam
Czjzek
,
Gurvan
Michel
,
Tristan
Barbeyron
,
Edwin A.
Yates
,
Gunnar C.
Hansson
,
Niclas G.
Karlsson
,
Alan
Cartmell
,
Eric C.
Martens
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease.
|
Oct 2021
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the ‘sulfation problem’ is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
|
Jan 2020
|
|
|
|
Abstract: 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 in vivo 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.
|
Aug 2015
|
|
B23-Circular Dichroism
|
Carrie A.
Duckworth
,
Scott E.
Guimond
,
Paulina
Sindrewicz
,
Ashley J.
Hughes
,
Neil S.
French
,
Lu-Yun
Lian
,
Edwin A.
Yates
,
D. Mark
Pritchard
,
Jonathan M.
Rhodes
,
Jeremy E.
Turnbull
,
Lu-Gang
Yu
Diamond Proposal Number(s):
[9218]
Open Access
Abstract: Concentrations of circulating galectin-3, a metastasis promoter, are greatly
increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated
heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin
derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated
cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these
modified heparin derivatives and their ultra-low molecular weight sub-fractions
had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their
ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives
(with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections
of the derivatives) abolished the circulating galectin-3-mediated increase in lung
metastasis of human melanoma and colon cancer cells in nude mice. Structural
analysis using nuclear magnetic resonance and synchrotron radiation circular
dichroism spectroscopies showed that the modified heparin derivatives bind to
the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified,
non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding
inhibitors with substantial potential as anti-metastasis/cancer drugs.
|
Jun 2015
|
|
|
|
Abstract: Phage display antibodies are widely used to follow heparan sulfate (HS) expression in tissues and cells. We demonstrate by ELISA, that cations alter phage display antibody binding profiles to HS and this is mediated by changes in polysaccharide conformation, demonstrated by circular dichroism spectroscopy. Native HS structures, expressed on the cell surfaces of neuroblastoma and fibroblast cells, also exhibited altered antibody binding profiles following exposure to low mM concentrations of these cations. Phage display antibodies recognise conformationally-defined HS epitopes, rather than sequence alone, as has been assumed, and resemble proteins in being sensitive to changes in both charge distribution and conformation following binding of cations to HS polysaccharides.
|
Apr 2015
|
|
|
|
Abstract: Influenza remains a serious health threat, with resistance to frontline drugs becoming more common, and new treatments urgently sought. One strategy for the inhibition of the attachment of influenza to host cells is to employ chemically modified heparins, capable of effectively competing with the multivalent interactions involved. In an assay of H5N1 influenza viral invasion comprising a H5 pseudotyped HIV system, selective removal of the sulfate groups from heparin (IC50 ~22 × 10−9 g mL−1) allowed the retention of inhibitory activity in the products (IC50 ~4 × 10−9 g mL−1) while significantly reducing their anticoagulant activities. Chemically modified anionic polysaccharides offer a potential source of effective inhibitors of viral attachment, which are suitable for further optimisation.
|
Jan 2015
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[9218, 5843]
Abstract: Presented herein is a novel method for the detection of tryptophan-ligand interactions utilising magnetic circular dichroism. Two examples are presented, one a small molecule and another, a large polysaccharide, heparan sulfate. These data are also the first reported example of a Trp:glycosaminoglycan interaction shown by magnetic circular dichroism.
|
Dec 2014
|
|
B23-Circular Dichroism
|
Noemi
Veraldi
,
Ashley J.
Hughes
,
Timothy R.
Rudd
,
Huw B.
Thomas
,
Steven W.
Edwards
,
Lynsay
Hadfield
,
Mark A.
Skidmore
,
Giuliano
Siligardi
,
Cesare
Cosentino
,
Janis K.
Shute
,
Annamaria
Naggi
,
Edwin A.
Yates
Diamond Proposal Number(s):
[8027]
Abstract: An attractive strategy for ameliorating symptoms arising from the multi-faceted processes of excessive and/or continual inflammation would be to identify compounds able to interfere with multiple effectors of inflammation. The well-tolerated pharmaceutical, heparin, is capable of acting through several proteins in the inflammatory cascade, but its use is prevented by strong anticoagulant activity. Derivatives of heparin involving the periodate cleavage of 2,3 vicinal diols in non-sulfated uronate residues (glycol- split) and replacement of N-sulphamido- with N-acetamido- groups in glucosamine residues, capable of inhibiting neutrophil elastase activity in vitro, while exhibiting attenuated anticoagulant properties, have been identified and characterised. These also interact with two other important modulators of the inflammatory response, IL-8 and TNF-alpha. It is therefore feasible in principle to modulate several activities, while minimising anticoagulant side effects, providing a platform from which improved anti- inflammatory agents might be developed.
|
Oct 2014
|
|
|
|
Stefano
Elli
,
Eleonora
Macchi
,
Timothy R.
Rudd
,
Rahul
Raman
,
Guillherme
Sassaki
,
Karthik
Viswanathan
,
Edwin
Yates
,
Zachary
Shriver
,
Annamaria
Naggi
,
Giangiacomo
Torri
,
Ram
Sasisekharan
,
Marco
Guerrini
Open Access
Abstract: The glycan receptor binding and specificity of influenza A viral hemagglutinin (HA) are critical for virus infection and transmission in humans. However, ambiguities in the interpretation of the receptor binding specificity of hemagglutinin from human- and avian-adapted viruses have prevented an understanding of its relationship with aerosol transmissibility, an exclusive property of human-adapted viruses. A previous conformational study, which we performed, indicated that human and avian receptors sample distinct conformations in solution. On the basis of detailed nuclear magnetic resonance (NMR) studies provided herein, we offer evidence of the distinct structural constraints imposed by hemagglutinin receptor binding sites on the glycan conformational space upon binding. The hemagglutinin from the SC18 virus, which has efficient aerosol transmissibility in humans (human-adapted), imposed the most stringent constraints on the conformational space of the human glycan receptor (LSTc), compared to single (NY18) or double (AV18) amino acid HA mutants, a property correlating to the ligand–HA binding strength. This relationship was also observed for the avian-adapted HA, where the high affinity binding partner, AV18, imposed the most stringent conformational constraints on the avian receptor, compared to those imposed by NY18. In particular, it is interesting to observe how different HAs when binding to human or avian glycosidic receptors impose significantly different conformational states, in terms of the states sampled by the glycosidic backbone and/or the entire molecule shape (linear or bent), when compared to the corresponding unbound glycans. Significantly, we delineate a “characteristic NMR signature” for the human adapted hemagglutinin (SC18) binding to human glycan receptors. Therefore, the conformational space constraints imposed by the hemagglutinin receptor binding site provide a characteristic signature that could be a useful tool for the surveillance of human adaptation of other (such as H7N9 and H5N1) deadly influenza viruses.
|
Jul 2014
|
|
