Preparation and Characterization of Glycosaminoglycan Chemokine Coreceptors

Kitic, Nikola; Gschwandtner, Martha; Derler, Rupert; Gerlza, Tanja; Kungl, Andreas J.

doi:10.1016/bs.mie.2015.09.018

Cited by 3 publications

(7 citation statements)

References 29 publications

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“…HS was isolated using a previously published protocol [ 49 ]. The lung tissues were stored at −80 °C.…”

Section: Methodsmentioning

confidence: 99%

“…An amount of 50 µg of enzymatically depolymerized HS sample was separated by strong anion exchange chromatography on a Shimadzu UFLC- HPLC system connected to a UV-VIS detector (20AD, Kyoto, Japan), as described earlier [ 49 ]. All preparations except for the tumor lobe were prepared thrice, and SAX was measured once for each preparation using a ProPac PA1 analytical LC column (Thermo Fisher, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The binding affinity of HS to recombinant CCL2 and CXCL8, was measured by isothermal fluorescence titration, as already described [ 49 , 80 ]. Fluorescence quenching due to protein GAG interaction was measured under increasing GAG concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…HS was isolated using a previously published protocol [49]. The lung tissues were stored at −80 • C. For HS isolation, the tissue was thawed overnight at 4 • C and cut to pieces of approximately 1 cm 3 .…”

Section: Isolation Of Heparan Sulfatementioning

confidence: 99%

“…In this study, we isolated HS postmortem from human lungs of five donors using a previously published protocol [ 49 ] and elucidated their structure—overall chain length and disaccharide composition—as well as protein-binding behavior. For this purpose, we applied size exclusion chromatography (SEC-HPLC), enzymatic depolymerization, strong anion exchange chromatography (SAX-HPLC), and isothermal fluorescence titration (IFT).…”

Section: Introductionmentioning

confidence: 99%

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Isolation and Characterization of Heparan Sulfate from Human Lung Tissues

et al. 2021

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Glycosaminoglycans are a class of linear, highly negatively charged, O-linked polysaccharides that are involved in many (patho)physiological processes. In vitro experimental investigations of such processes typically involve porcine-derived heparan sulfate (HS). Structural information about human, particularly organ-specific heparan sulfate, and how it compares with HS from other organisms, is very limited. In this study, heparan sulfate was isolated from human lung tissues derived from five donors and was characterized for their overall size distribution and disaccharide composition. The expression profiles of proteoglycans and HS-modifying enzymes was quantified in order to identify the major core proteins for HS. In addition, the binding affinities of human HS to two chemokines—CXCL8 and CCL2—were investigated, which represent important inflammatory mediators in lung pathologies. Our data revealed that syndecans are the predominant proteoglycan class in human lungs and that the disaccharide composition varies among individuals according to sex, age, and health stage (one of the donor lungs was accidentally discovered to contain a solid tumor). The compositional difference of the five human lung HS preparations affected chemokine binding affinities to various degrees, indicating selective immune cell responses depending on the relative chemokine–glycan affinities. This represents important new insights that could be translated into novel therapeutic concepts for individually treating lung immunological disorders via HS targets.

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“…HS was isolated using a previously published protocol [ 49 ]. The lung tissues were stored at −80 °C.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Isolation Of Heparan Sulfatementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Isolation and Characterization of Heparan Sulfate from Human Lung Tissues

et al. 2021

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Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides

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Derler

Gesslbauer

et al. 2019

Biochimica et Biophysica Acta (BBA) - General Subjects

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Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

et al. 2021

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As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600–680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50–500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

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Preparation and Characterization of Glycosaminoglycan Chemokine Coreceptors

Cited by 3 publications

References 29 publications

Isolation and Characterization of Heparan Sulfate from Human Lung Tissues

Isolation and Characterization of Heparan Sulfate from Human Lung Tissues

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides

Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

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