Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor

From MDPI via Jisc Publications RouterHistory: accepted 2021-06-19, pub-electronic 2021-06-29Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): 978724Funder: Medical Research Council; Grant(s): MR/L007525/1The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems

High sensitivity (zeptomole) detection of BODIPY-labelled heparan sulfate (HS) disaccharides by ion-paired RP-HPLC and LIF detection enables analysis of HS from mosquito midguts

The fine structure of heparan sulfate (HS), the glycosaminoglycan polysaccharide component of cell surface and extracellular matrix HS proteoglycans, coordinates the complex cell signalling processes that control homeostasis and drive development in multicellular animals. In addition, HS is involved in the infection of mammals by viruses, bacteria and parasites. The current detection limit for fluorescently labelled HS disaccharides (low femtomole; 10-15 mol), has effectively hampered investigations of HS composition in small, functionally-relevant populations of cells and tissues that may illuminate the structural requirements for infection and other biochemical processes. Here, an ultra-high sensitivity method is described that utilises a combination of reverse-phase HPLC, with tetraoctylammonium bromide (TOAB) as the ion-pairing reagent and laser-induced fluorescence detection of BODIPY-FL-labelled disaccharides. The method provides an unparalleled increase in the sensitivity of detection by ∼six orders of magnitude, enabling detection in the zeptomolar range (∼10-21 moles; Anopheles gambiae mosquitoes that was achieved without approaching the limit of detection

Characterisation of luciferase-expressing <i>L</i>. <i>mexicana</i> transgenic lines.

<p>(A) Promastigote growth curve of parental <i>L</i>. <i>mexicana</i> M379 (open square) and transgenic cell lines expressing PRE9 (open triangle) and NanoLuc-PEST (filled square) monitored over a five day time course. Mean values are shown (n = 3) ± SD. The Y-axis was transformed by log₁₀. (B), (C) Cell density dilution series on the promastigote and axenic amastigote forms, respectively. Both X- and Y-axes were transformed by log₁₀ prior to regression analysis. Mean values are shown (n = 3) ± SD. (D), (E) Cycloheximide assay on the promastigote and axenic amastigote forms, respectively, was monitored over an eight hour time course. The X-axis was transformed by log₁₀ prior to regression analysis. Mean values are shown (n = 3) ± SD.</p

Galactokinase-like protein from Leishmania donovani: Biochemical and structural characterization of a recombinant protein.

Leishmaniasis is a spectrum of conditions caused by infection with the protozoan Leishmania spp. parasites. Leishmaniasis is endemic in 98 countries around the world, and resistance to current anti-leishmanial drugs is rising. Our work has identified and characterised a previously unstudied galactokinase-like protein (GalK) in Leishmania donovani, which catalyses the MgATP-dependent phosphorylation of the C-1 hydroxyl group of d-galactose to galactose-1-phosphate. Here, we report the production of the catalytically active recombinant protein in E. coli, determination of its substrate specificity and kinetic constants, as well as analysis of its molecular envelope using in solution X-ray scattering. Our results reveal kinetic parameters in range with other galactokinases with an average apparent Km value of 76 μM for galactose, V and apparent K values with 4.46376 × 10  M/s and 0.021 s , respectively. Substantial substrate promiscuity was observed, with galactose being the preferred substrate, followed by mannose, fructose and GalNAc. LdGalK has a highly flexible protein structure suggestive of multiple conformational states in solution, which may be the key to its substrate promiscuity. Our data presents novel insights into the galactose salvaging pathway in Leishmania and positions this protein as a potential target for the development of pharmaceuticals seeking to interfere with parasite substrate metabolism. [Abstract copyright: Copyright © 2024. Published by Elsevier B.V.

Activity of the six most potent MMV compounds against axenic and intramacrophage amastigotes.

<p>Previous EC₅₀ data for compounds against <i>Leishmania spp</i>. are shown in italics, where available.</p

Comparison of bioluminescence- and microscopy-based intramacrophage infection assays following treatment with a supralethal dose of Amphotericin B.

<p>(A) The bioluminescence-based assay was used to assess infection of PMA-differentiated THP-1 macrophages by stationary phase <i>L</i>. <i>mexicana</i> NanoLuc-PEST promastigotes. Infected cells were exposed to 0.8 μM Amphotericin B, or left untreated, for 72 hours. Mean values are shown (n = 4) ± SD. The Y-axis was transformed by log₁₀, and the data was analysed by paired, two-tailed T-test (<i>p</i><0.001). (B) The standard microscopy-based counting assay was used to assess infection of PMA-differentiated THP-1 macrophages by stationary phase <i>L</i>. <i>mexicana</i> NanoLuc-PEST promastigotes Infected cells were exposed to 0.8 μM Amphotericin B, or left untreated, for 72 hours. Infection indices were calculated as described in the methods. Mean values are shown (n = 4) ± SD. The data was analysed by paired, two-tailed T-test on the infection index data (<i>p</i> = 0.0017).</p

EC₅₀ values and assay parameters following treatment with Amphotericin B and Miltefosine against wild-type and NanoLuc-PEST expressing <i>L</i>. <i>mexicana</i> (see S5 Fig for full dataset).

<p>EC₅₀ values and assay parameters following treatment with Amphotericin B and Miltefosine against wild-type and NanoLuc-PEST expressing <i>L</i>. <i>mexicana</i> (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006639#pntd.0006639.s005" target="_blank">S5 Fig</a> for full dataset).</p

Peroxisome proliferator-activated receptor ɣ agonist mediated inhibition of heparanase expression reduces proteinuria

Background: Proteinuria is associated with many glomerular diseases and a risk factor for the progression to renal failure. We previously showed that heparanase (HPSE) is essential for the development of proteinuria, whereas peroxisome proliferator-activated receptor ɣ (PPARɣ) agonists can ameliorate proteinuria. Since a recent study showed that PPARɣ regulates HPSE expression in liver cancer cells, we hypothesized that PPARɣ agonists exert their reno-protective effect by inhibiting glomerular HPSE expression. Methods: Regulation of HPSE by PPARɣ was assessed in the adriamycin nephropathy rat model, and cultured glomerular endothelial cells and podocytes. Analyses included immunofluorescence staining, real-time PCR, heparanase activity assay and transendothelial albumin passage assay. Direct binding of PPARɣ to the HPSE promoter was evaluated by the luciferase reporter assay and chromatin immunoprecipitation assay. Furthermore, HPSE activity was assessed in 38 type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARɣ agonist pioglitazone. Findings: Adriamycin-exposed rats developed proteinuria, an increased cortical HPSE and decreased heparan sulfate (HS) expression, which was ameliorated by treatment with pioglitazone. In line, the PPARɣ antagonist GW9662 increased cortical HPSE and decreased HS expression, accompanied with proteinuria in healthy rats, as previously shown. In vitro, GW9662 induced HPSE expression in both endothelial cells and podocytes, and increased transendothelial albumin passage in a HPSE-dependent manner. Pioglitazone normalized HPSE expression in adriamycin-injured human endothelial cells and mouse podocytes, and adriamycin-induced transendothelial albumin passage was reduced as well. Importantly, we demonstrated a regulatory effect of PPARɣ on HPSE promoter activity and direct PPARy binding to the HPSE promoter region. Plasma HPSE activity of T2DM patients treated with pioglitazone for 16/24 weeks was related to their hemoglobin A1c and showed a moderate, near significant correlation with plasma creatinine levels. Interpretation: PPARɣ-mediated regulation of HPSE expression appears an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice. Funding: This study was financially supported by the Dutch Kidney Foundation, by grants 15OI36, 13OKS023 and 15OP13. Consortium grant LSHM16058-SGF (GLYCOTREAT; a collaboration project financed by the PPP allowance made available by Top Sector Life Sciences & Health to the Dutch Kidney Foundation to stimulate public-private partnerships)

The role of heparan sulfate in host macrophage infection by Leishmania species

The leishmaniases are a group of neglected tropical diseases caused by parasites from the Leishmania genus. More than 20 Leishmania species are responsible for human disease, causing a broad spectrum of symptoms ranging from cutaneous lesions to a fatal visceral infection. There is no single safe and effective approach to treat these diseases and resistance to current anti-leishmanial drugs is emerging. New drug targets need to be identified and validated to generate novel treatments. Host heparan sulfates (HSs) are abundant, heterogeneous polysaccharides displayed on proteoglycans that bind various ligands, including cell surface proteins expressed on Leishmania promastigote and amastigote parasites. The fine chemical structure of HS is formed by a plethora of specific enzymes during biosynthesis, with various positions (N-, 2-O-, 6-O- and 3-O-) on the carbon sugar backbone modified with sulfate groups. Post-biosynthesis mechanisms can further modify the sulfation pattern or size of the polysaccharide, altering ligand affinity to moderate biological functions. Chemically modified heparins used to mimic the heterogeneous nature of HS influence the affinity of different Leishmania species, demonstrating the importance of specific HS chemical sequences in parasite interaction. However, the endogenous structures of host HSs that might interact with Leishmania parasites during host invasion have not been elucidated, nor has the role of HSs in host-parasite biology. Decoding the structure of HSs on target host cells will increase understanding of HS/parasite interactions in leishmaniasis, potentiating identification of new opportunities for the development of novel treatments