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

Abstract

22 23 *Corresponding authors: m.a.skidmore@keele.ac.uk. Tel: +44 (0)1782 733945 24 marissa.maciej-hulme@radboudumc.nl Tel: +31 (0)243610553 25 26 Abstract 29 The fine structure of heparan sulfate (HS), the glycosaminoglycan polysaccharide component of cell 30 surface and extracellular matrix HS proteoglycans, coordinates the complex cell signalling processes 31 that control homeostasis and drive development in multicellular animals. In addition, HS is involved in 32 the infection of mammals by viruses, bacteria and parasites. The current detection limit for 33 fluorescently labelled HS disaccharides that is in the low femtomole range (10 -15 mol), has effectively 34 hampered investigations of HS composition from small, functionally-relevant populations of cells and 35 tissues. Here, an ultra-high sensitivity method is described that utilises a combination of reverse-36 phase HPLC, with tetraoctylammonium bromide (TOAB) as the ion-pairing reagent and laser-induced 37 fluorescence detection of BODIPY-FI-labelled disaccharides. The method provides an unparalleled 38 increase in the sensitivity of detection by ~ six orders of magnitude, to the zeptomolar range (~10 -21 39 moles), enabling detection of <1000 labelled molecules. This facilitates determination of HS 40 disaccharide compositional analysis from minute biological samples, as demonstrated by analysis of 41 HS isolated from the midguts of Anopheles gambiae mosquitoes that was achieved without 42 approaching the limit of detection. 43 44 49 in multicellular animals. HS, which is displayed at the mammalian cell surface, is also known to 50 interact with viruses (e.g. HIV 1 and Zika virus 2,3 ) and other cells, including pathogenic 51 microorganisms (e.g. Toxoplasma gondii 4,5 , Plasmodium falciparum 5,6 , and Leishmania parasites 7-9 ) 52 and is often involved in the process of infection. In addition, diffusible HS oligosaccharide fragments 53 released by heparanase activity are thought to exert influence further afield 10 . 54 The biosynthesis of HS occurs in the endoplasmic reticulum and Golgi, where the nascent 55 chain is modified during de novo synthesis on the protein core. Specific enzymes either transfer 56 sulfate groups (N-deacetylase/sulfotransferases, 6-O-, 2-O-, and 3-O-sulphotransferases) to 57 glucosamine or uronate residues, or epimerise (C5-epimerase) β-D-glucuronate to α-L-iduronate units 58 in the chain. Together, these enzymes produce distinct sulfation patterns both at the disaccharide 59 level and in the completed polysaccharide. For HS, the modification enzymes act in an incomplete and 60 interdependent fashion to form domains, consisting of regions of high sulfation flanked by intermediate 61 sulfation 11 . Following synthesis, the removal of 6-O-sulfate groups from the HS polysaccharide by the 62 sulfatases (Sulf 1 and 2), may also occur 12,13 , potentially creating further diversity in the HS chain 14 . 63Owing to the relatively poor detection sensitivity inherent to carbohydrates compares to other 64 biomolecules, heterogeneous HS ch...

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

“…This enabled the real-time, ratiometric quantification of sulfate hydrolysis in solution, by comparing the change in the retention time of the separated sulfated substrate (6S-GlcNAc-BODIPY) and desulfated product (GlcNAc-BODIPY) of the enzyme assay. The fluorescent label 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-propionic acid, hydrazide (BODIPY-FL hydrazide) was selected (see below), despite a relatively high initial cost, owing to its high extinction coefficient (80 000 M −1 cm −1 ) and its stability over a variety of pH values (pH 3–12) that is critical for analysis in buffer-sensitive assays [ 35 ]. The labelled substrate was detected in a 20 nl volume in-line via LED-induced fluorescence, resulting in a time-dependent leading sulfated peak and a smaller secondary peak that represented the initial non-sulfated substrate ( Figure 2A ).…”

Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

“…The upper layer (aqueous phase) was dialyzed against 5 × 5 L baths of Milli-Q H2O using SnakeSkin dialysis membranes (MWCO 3500 Da, Thermo Fisher Scientific) and dried using a Savant SC210A Speed-Vac concentrator (Thermo Fisher Scientific). Isolation of HS glx from extracted glycocalyx was performed as described previously ( Guimond et al, 2009 ; Maciej-Hulme et al, 2020 ). In brief, dialyzed and concentrated glycocalyx extracts were digested with 125 mU of Chondroitinase ABC (Sigma-Aldrich) in 25 mM Tris, 2 mM Mg(Ac) 2 pH 8 for 18 h before fractionation by anion exchange chromatography using DEAE-sepharose CL-6B beads (Sigma-Aldrich) equilibrated in PBS.…”

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

“…This enabled the real-time, ratiometric quantification of sulfate hydrolysis in solution, by comparing the change in retention time of the separated sulfated substrate (6S-GlcNAc-BODIPY) and de-sulfated product (GlcNAc-BODIPY) of the enzyme assay. The fluorescent label 4,4-difluoro-5,7-dimethyl-4bora-3a,4a-diaza-s-indacene-3-propionic Acid, hydrazide (BODIPY-FL hydrazide) was selected (see below), despite a relatively high initial cost, owing to its high extinction coefficient (80,000 M -1 cm -1 ) and its stability over a variety of pH values (pH 3 -12) that is critical for analysis in buffer-sensitive assays [34]. The labelled substrate was detected in a 20 nl volume in-line via LED-induced fluorescence, resulting in a time-dependent leading sulfated peak and a smaller secondary peak that represented the initial non-sulfated substrate (Figure 2A).…”