Accepted Manuscript Title: Inhibition of Leishmania amazonensis arginase by fucogalactan isolated from Agrocybe aegerita mushroom Authors: Renan Akio Motoshima, Tainara da F. Rosa, Léia da C. Mendes, Estefânia V. Silva, Sthefany R.F. Viana, Bruno Sérgio do Amaral, Dulce H.F. de Souza, Luciano M. Lião, Maria de Lourdes Corradi da Silva, Lorena R.F. de Sousa, Elaine R. Carbonero PII: DOI: Reference: S0144-8617(18)31019-1 https://doi.org/10.1016/j.carbpol.2018.08.109 CARP 14006 To appear in: Received date: Revised date: Accepted date: 5-6-2018 24-8-2018 25-8-2018 Please cite this article as: Motoshima RA, da F. Rosa T, da C. Mendes L, Silva EV, Viana SRF, do Amaral BS, de Souza DHF, Lião LM, de Lourdes Corradi da Silva M, de Sousa LRF, Carbonero ER, Inhibition of Leishmania amazonensis arginase by fucogalactan isolated from Agrocybe aegerita mushroom, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.08.109 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Inhibition of Leishmania amazonensis arginase by fucogalactan isolated from PT Agrocybe aegerita mushroom Renan Akio Motoshimaa, Tainara da F. Rosaa, Léia da C. Mendesa, Estefânia V. Silvaa,b, RI Sthefany R. F. Vianac, Bruno Sérgio do Amarald, Dulce H. F. de Souzad, Luciano M. Liãob, Unidade Acadêmica Especial de Química, Universidade Federal de Goiás, Regional Catalão, 75704-020 Catalão, N a U SC Maria de Lourdes Corradi da Silvae, Lorena R. F. de Sousaa, Elaine R. Carboneroa ,* b Laboratório de Ressonância Magnética Nuclear, Instituto de Química, Universidade Federal de Goiás, Campus M Samambaia, 74001-970 Goiânia, GO, Brazil c A GO, Brazil. Departamento de Engenharia Rural, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista “Júlio d Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, Km 235, 13565-905 PT São Carlos, SP, Brazil e ED de Mesquita Filho”, 18610-307 Botucatu, SP, Brazil Departamento de Química e Bioquímica, Faculdade de Ciências e Tecnologia, Universidade Estadual Paulista A CC E “Júlio de Mesquita Filho”, 19060-900 Presidente Prudente, SP, Brazil * Corresponding author at: E-mail addresses: elainecarbonero@gmail.com; elaine_carbonero@ufg.br (E.R. Carbonero) 2 Highlights A fucogalactan (FG-Aa) was purified from the medicinal mushroom Agrocybe aegerita.  The fucogalactan was highly branched by non-reducing ends of -Fucp.  FG-Aa was evaluated against arginase (ARG) from Leishmania amazonensis.  The mechanism of arginase inhibition promoted by the fucogalactan was competitive. A CC E PT ED M A N U SC RI PT  3 Abstract The inhibition of arginase from Leishmania spp. is considered a promising approach to PT the leishmaniasis treatment. In this study, the potential of a fucogalactan isolated from the RI medicinal mushroom Agrocybe aegerita was evaluated against arginase (ARG) from Leishmania amazonensis. The polysaccharide was obtained via aqueous extraction, and purified by freeze SC thawing and precipitation with Fehling solution. Its chemical structure was established by U monosaccharide composition, methylation analysis, partial acid hydrolysis, and NMR N spectroscopy. The data indicated that it is a fucogalactan (FG-Aa; Mw = 13.8 kDa), having a FG-Aa showed significant inhibitory activity on ARG with IC50 potency of 5.82 ± 0.57 M L-Fucp. A (16)-linked α-D-Galp main-chain partially substituted in O-2 by non-reducing end-units of α- µM. The mechanism of ARG inhibition by the heterogalactan was the competitive type, with ED Ki of 1.54 ± 0.15 µM. This is the first report of an inhibitory activity of arginase from L. amazonensis by biopolymers, which encourages us to investigate further polysaccharides as a CC E PT new class of ARG inhibitors. Keywords: Agrocybe aegerita; Fucogalactan; Chemical structure; Leishmania amazonensis; A Arginase; competitive inhibitor. 1. Introduction 4 Leishmaniasis is a parasitic infection that remains nowadays, affecting millions of people per year around the world (WHO, 2018). The chemotherapeutics currently used against leishmaniasis have several side effects and resistance issues (Rojo et al., 2015). In regarding to the need for new approaches for human leishmaniasis treatments, polysaccharides are macromolecules with a great structural diversity that have been shown leishmanicidal and PT antitumor activities related to immunomodulatory effects (Adriazola et al., 2014; Amaral et al., RI 2015; Kangussu-Marcolino et al., 2015; Moretão, Zampronio, Gorin, Iacomini, & Oliveira, 2004; Valadares et al., 2011, 2012). SC The polysaccharides are considered relevant responsible agents for biological response modification related to medicinal usage of mushrooms (Meng, Liang, & Luo, 2016; Rathore, U Prasad, & Sharma, 2017). Agrocybe aegerita, commonly known as “black poplar”, “Pioppino” N or “Yanagi-matsutake” mushroom, is used as a traditional Chinese herbal medicine and A recognized for its potential health benefit. Several biological activities, such as antioxidant (Lo M & Cheung, 2005; Petrović et al., 2015), anti-inflammatory (Diyabalanage, Mulabagal, Mills, ED DeWitt, & Nair, 2008), antimicrobial (Petrović et al., 2014) and antitumoral properties (Diyabalanage et al., 2008; Liang et al., 2011; Lin, Ching, Lam, & Cheung, 2017; Yang et al., PT 2009) have been described for this species, which were attributed to its secondary compounds (phenolic compounds, indole derivatives, among others), polysaccharides, and lectins. However, CC E the polysaccharides related to biological activities from A. aegerita have not been chemically characterized up to now. A In attempt to improve antileishmanial efficacy for drug design, new enzymes have been explored as molecular targets for therapeutic intervention, such as arginase (ARG) from Leishmania amazonensis (da Silva, Zampieri, Muxel, Beverley, & Floeter-Winter, 2012; Robertson, 2005). ARG is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L- ornithine and urea carrying out reactions for essential metabolites for Leishmania spp. 5 development. TH2 cytokine activation increases ARG expression leading to the establishment of Leishmania infection, thus arginase appears as a promising target against leishmaniasis (BlañaFouce et al., 2012; Colotti & Ilari, 2011; da Silva et al., 2008; da Silva, M.F.L., 2012). Inhibitors of arginase have been searched in order to identify new antileishmanial leads (da Silva, Maquiaveli, & Magalhães, 2012; de Sousa et al., 2014a, b; Maquiaveli et al., 2016). PT Among the natural products pointed as ARG inhibitors, glycoside compounds from plants have RI shown potency in decreasing arginase catalytic activity (da Silva, E.R., 2012; de Sousa et al., 2014a; Maquiaveli et al., 2016). In silico studies have showed interaction between SC glucopyranose and the active site of the enzyme (Maquiaveli et al., 2016). In a search for new arginase inhibitors, the heteropolysaccharide (FG-Aa) isolated from U A. aegerita was structurally characterized and investigated by ARG enzymatic assay. FG-Aa N was identified as potent ARG inhibitor and its mechanism of action was determined. A Furthermore, the identified polysaccharide was revealed as a new class of natural products as ED 2. Experimental M ARG inhibitors. PT 2.1. Biological material Fresh Agrocybe aegerita (3.0 kg) was donated by Yuki Cogumelos Company (Owner: CC E José Francisco Ramos Fernandes Viana), located in Araçoiaba da Serra, State of São Paulo, Brazil, in September 2015. The fungus was grown on culture substrate constituted of eucalyptus A sawdust (2%), wheat bran (8%), corn bran (5%), soybean bran (4%), and calcitic limestone (1%). After freeze-dried, the fruiting bodies of A. aegerita were reduced to 9.7% of the original weight, resulting in ~300 g of dry matter. 2.2. Extraction and purification procedures for polysaccharides from A. aegerita 6 The dried fruiting bodies of Agrocybe aegerita (~300 g), were pulverized and extracted with water at ~10 ºC for 8 h (x 5, 1 L). The extract was filtered and after centrifugation at 9000 rpm at 20 ºC for 15 min a clear solution was obtained. The polysaccharides were precipitated by addition of excess EtOH (3:1; v/v) to the concentrated supernatant, and then recovered by centrifugation at 9000 rpm at 15 ºC for 10 min. The crude polysaccharide fraction was dissolved PT in H2O, dialyzed against distilled water for 48 h to remove low-molecular-weight carbohydrates, and freeze-dried, giving rise to fraction CW-Aa. This fraction was then dissolved in distilled RI water and the solution submitted to freezing followed by mild thawing at 4 oC (Gorin & SC Iacomini, 1984), giving cold water-soluble (SCW-Aa) and insoluble fractions (ICW-Aa), which were separated by centrifugation (9000 rpm at 10 ºC for 10 min). SCW-Aa fraction was treated U with Fehling’s solution (Jones & Stoodley, 1965), and precipitated Cu++ complex (FPCW-Aa) N was removed by centrifugation at 9000 rpm for 10 min, at 15 C. The precipitate was neutralized M A with HOAc, dialyzed against tap water (48 h), deionized with mixed ion exchange resins, and then freeze-dried. Fehling treatment was repeated one more cycle on fraction FPCW-Aa, giving A CC E PT ED the further purified fraction FP2CW-Aa that was denominate FG-Aa. 7 Dried fruiting bodies of Agrocybe aegerita (300 g) H2O at ~10 ºC for 8 h (x 5) Residue I Aqueous extract Ethanol supernatant Ethanol precipitate (CW-Aa, 13.2 g) Insoluble fraction (ICW-Aa, 1.4 g) RI - Freeze-thawing - Centrifugation PT EtOH (3:1; v/v) Soluble fraction (SCW-Aa, 11.5 g) U SC -Treatment with Fehling solution; - Centrifugation and dialysis; - Deionized with ion-exchange resins; Centrifugation and dialysis Fehling precipitate (FPCW-Aa, 1.8 g) N Fehling supernatant (FSCW-Aa, 3.1 g) ED M A -Treatment with Fehling solution; - Centrifugation and dialysis; - Deionized with ion-exchange resins; Centrifugation and dialysis Fehling precipitate II (FP2CW-Aa, 924 mg) HETEROGALACTAN (FG-Aa) CC E A. aegerita PT Fig. 1. Scheme of extraction and purification of the heteropolysaccharide from 2.3. Monosaccharide composition Monosaccharide components of the polysaccharides were identified and their ratios were A determined following hydrolysis with 1 M TFA for 8 h at 100 C, and conversion to alditol acetates (GC-MS) by successive NaBH4 reduction, and acetylation with Ac2O-pyridine (1:1, v/v) for 12 h at room temperature (Wolfrom & Thompson, 1963a, b). The resulting alditol acetates were analyzed by gas chromatography-mass spectrometry (GC-MS) and identified by their typical retention times and electron impact profiles. GC-MS analysis was performed with an 8 Agilent 7820A gas chromatograph interfaced to an Agilent 5975E quadrupole mass spectrometer, fitted with split/splitless capillary inlet system, an Agilent G4513A autosampler, and a capillary HP5-MS column (30 m x 0.25 mm i.d.). Injections of 1 uL were made in the splitless mode at injection temperature of 250 °C and detector at 280 ºC. The column oven temperature was initially hold at 75 °C for 1 min, programmed at 35 ºC.min-1 to 100 ºC (5 min), PT then 45 ºC.min-1 to 150 ºC, hold for 5 min, 55 ºC.min-1 to 200 ºC (15 min), and 65 ºC.min-1 to 240 ºC (2 min.) for quantitative analysis of the alditol acetates. Helium was the carrier gas at a RI flow rate of 1 mL.min-1. Electron impact (EI) analysis was performed with the ionization energy 2.4. Methylation analysis of polysaccharides U SC set at 70 eV. N Per-O-methylation of the purified fraction (FP2CW-Aa; 12 mg) was carried out using A NaOH-Me2SO-MeI (Ciucanu & Kerek, 1984). The per-O-methylated derivatives (1 mg) were M hydrolyzed with 1 M TFA (250 μL) for 10 h at 100 C, followed by evaporation to dryness. The ED resulting mixture of O-methylaldoses was reduced with NaBD4 and acetylated with Ac2Opyridine (1:1, v/v) for 12 h at room temperature (Wolfrom & Thompson, 1963a, b) to give a PT mixture of partially O-methylated alditol acetates, which was analyzed by GC-MS as above cited (item 2.3). For quantitative analysis of the partially O-methylated alditol acetates (PMAAs) was CC E used a DB-225 capillary column (30 m x 0.25 mm i.d.) held at 50 °C during injection and later programmed to 220 °C (constant temperature) at 40 °C min-1. PMAAs were identified from m/z A of their positive ions, by comparison with standards (Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005). The relative percentage of the resulting PMAAs was calculated by determination of the each peak area using the Agilent ChemStation software. 2.5. Determination of polysaccharide homogeneity and molecular weight 9 The homogeneity of FP2CW-Aa was determined by high performance steric exclusion chromatography (HPSEC) coupled to a refractive index (RI) detector model RID 10A. The chromatography system consisted of an HPLC pump (Model Shimadzu-10AD), a manual injection valve (Shimadzu) fitted with a 200-µL loop and an Ultrahydrogel column (7.8 x 300 mm) system (Waters) with exclusion limit of 7x106, 4x105, 8x104 and 5x103 Da arranged in PT series. The mobile phase was 0.1 M NaNO3 with sodium azide (0.03%), and a flow rate 0.6 RI mL/min. Data analysis was performed using LC solution software (Shimadzu Corporation). To determine the average molecular weight of FP2CW-Aa the standard curve of dextran with U SC molecular weights of 670, 410, 266, 150, 72.2, 60.0, 40.2, 22.8, and 9.4 kDa was made. 2.6. Partial acid hydrolysis of heterogalactan N FP2CW-Aa (88 mg) was partially hydrolyzed with 0.2 M TFA (2 mL) for 3 h at 100 ºC. A After neutralization with NaOH, the material was dialyzed (2 kDa cut-off membrane) against M distilled water. The retained fraction was lyophilized (HPFP2-Aa, 59 mg) and analyzed by 13C ED NMR. PT 2.7. Nuclear magnetic resonance (NMR) spectroscopy NMR spectra (1H, 13C, HSQC-DEPT, HSQC-TOCSY and HSQC-NOESY) were CC E obtained using a 500 MHz Bruker Avance spectrometer incorporating Fourier transform. Analyses were performed at 50 or 70 C on samples dissolved in D2O or Me2SO-d6. Chemical A shifts are expressed in  relative to the internal standard tetramethylsilane (TMS) ( = 0.0 for 13C and 1H) or Me2SO-d6 (=39.70 and 2.50 for 13C and 1H signals, respectively). 2.8. Arginase activity measurements 10 The expression and purification of recombinant ARG of L. amazonensis was performed as previously described (de Sousa et al., 2014b), as well detailed conditions of assay are the same previously established by de Sousa et al. (2014a, b). The samples and negative control were briefly diluted in MilliQ water. The polysaccharide FG-Aa (10 µM) was incubated with arginase solution (CHES buffer solution at PT pH 9.6; Sigma-Aldrich) for 10 min at 37 °C. Then, the substrate L-arginine (Sigma-Aldrich) was RI added to the reaction (50 mM of CHES buffer and 50 mM of L-arginine at pH 9.6), incubating similarly for 10 min at 37 °C. Thereafter, the second reaction takes place (Urea kit - Bioclin, SC Brazil) and urease catalysis allowed to determine the indirect ARG activity by measuring the absorbance of indophenol blue at 600 nm using a Varian Cary UV/Visible spectrophotometer. U Indophenol was generated by reaction of ammonia and Berthelot´s reagent, from which, 10 µL N of arginase reaction mixture were added to 500 µL of a solution (100 mM phosphate buffer, pH A 6.8, 300 mM salicylate, 5.0 mM sodium nitroprusside, and 10000 IU urease) and incubated for M 10 min at 37 °C and 500 µL of a second solution (10 mM NaOCl and 1.5 M NaOH) was added ED and incubated similarly. Additionally, uncoupled assay was performed to ensure ARG inhibitory activity. PT IC50 of FG-Aa was determined by rate measurements with inhibitor concentrations ranging from 0.7 to 355 µM. The enzymatic assay was performed in duplicate and titration of CC E inhibitor was reproduced three times in independent experiments. Data fitting for IC50 were processed using 4 parameters logistic equation. A The kinetics experiments were performed by increasing substrate concentrations (6.25- 100 mM) with 1.4, 3.5 and 7.0 µM of inhibitor. A control was used without the addition of inhibitor. The type of inhibition was determined analyzing kinetics data by Lineweaver-Burk, Dixon and Cornish-Bowden plots. The Ki was calculated by using the Dixon equations (Cortés, Cascante, Cárdenas, & Cornish-Bowden, 2001; de Sousa et al., 2015; Dixon, 1953): 11 v0/vi = 1 + ([I]/ Kiapp), and Ki = Kiapp/(1 + [L -Arg]/Km), with [L-Arg] = 50.0 mM and Km = 18.3 ± 1.4 mM The experimental data were analyzed with the program GraFit® (Erithacus Software Ltd.: Horley, Surrey, UK, 2006). PT 3. Results and Discussion RI The crude polysaccharide fraction (CW-Aa, 13.2 g) was isolated from the freeze-dried fruiting bodies (300 g) of the mushroom Agrocybe aegerita, via cold water extraction (Fig. 1). It SC showed to be composed of galactose (Gal, 48.9%) as its main component, in addition to fucose (Fuc, 25.0%), glucose (Glc, 26.0%), and traces of mannose (Man), according to GC-MS results U of its derived alditol acetates. Fractionation of CW-Aa by freeze/thawing process gave water- N soluble (SCW-Aa, 11.5 g) and insoluble (ICW-Aa, 1.4 g) polysaccharidic fractions, which were A separated by centrifugation. M Fraction SCW-Aa, composed of Fuc (16.9 %), Man (5.7%), Gal (52.8%), and Glc ED (24.6%), was treated with Fehling solution twice, sequentially, giving rise to a precipitate (FP2CW-Aa, 924 mg), which was homogeneous (Mw/Mn = 1.18) on HPSEC (Fig. 2), and had Mw PT 13.8 kDa. FP2CW-Aa contained mainly fucose (29.0%), and galactose (65.8%) as monosaccharide A CC E components, suggesting the presence of a fucogalactan (named as FG-Aa). 12 Fig. 2. The standard curve of dextran (A) and HPSEC chromatogram (B) of FP2CW-Aa fraction. The glycosidic linkages pattern of FG-Aa was determined by methylation procedure. Analysis by GC–MS of the partially O-methylated alditol acetates showed a highly branched structure, containing non-reducing end units of Fucp (2,3,4-Me3-Fuc; 31.3%), 6-O-substituted PT (2,3,4-Me3-Gal; 33.0%) and 2,6-di-O-substituted (3,4-Me2-Gal; 33.7%) of Galp units, together with minor amounts of non-reducing end units of Galp (2,3,4,6-Me4-Gal, 2.0%). RI The anomeric region of the 1H NMR spectrum of the polysaccharide FG-Aa (FP2CW-Aa SC fraction) contained three signals of H-1 at  5.09, 5.04, and 4.99 (Fig. 3), which were assigned as residue A, residue B, and residue C, respectively, and accordingly in the anomeric region of the C-NMR, three carbon resonances appeared at  104.10, 100.87, and 101.02 (Fig. 4). The U 13 5.05 A 4.99 5.00 4.95 ppm 1.0 5.10 1.0 5.15 1.0 CC E PT ED M 5.04 5.09 N relative areas of peak A, B, and C in the 1H-NMR spectrum were 1:1:1 (Fig. 3). Fig. 3. Anomeric region of 1H NMR spectrum of fucogalactan (FG-Aa) from A. aegerita, analyzed in A D2O at 50 °C (chemical shifts are expressed in δ ppm). 90 80 70 60 50 40 30 20 ppm SC 100 RI PT 18.42 80.53 74.60 72.54 72.47 72.42 72.35 71.98 71.65 71.36 71.28 71.16 70.03 69.85 69.48 104.10 101.02 100.87 13 Fig. 4. 13C NMR spectrum of fucogalactan (FG-Aa) from A. aegerita, analyzed in D2O at 70 °C. U All units showed an -configuration by high-frequency H-1 ( 5.09, 5.04, and 4.99) and N low-frequency C-1 signals ( 104.10, 100.87, and 101.02) (Fig. 3-5) (Agrawal, 1992). A Further NMR experiments, HSQC-DEPT (Fig. 5) and HSQC-TOCSY (Fig. 6), were M performed in order to elucidate the structure of this heteropolysaccharide (FG-Aa). The ED connectivities observed in the HSQC-TOCSY spectrum allowed to assign all carbons and protons of the each unit (Table 1S). Once the protons had been identified, the chemical shifts of PT their corresponding carbons were confirmed by HSQC-DEPT analysis (Fig. 5; Table 1). 60 CC E A5 B3 65 B6a C6a C6b B 6b 70 C5 B 5 B 4 C4 A 3 A2 C3 C2 A4 75 80 A B2 85 90 ppm A6 18 B1 C1 95 100 20 1.5 1.4 1.3 1.2 ppm 105 A1 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 ppm 14 Fig. 5. HSQC-DEPT spectrum of fucogalactan (FG-Aa) from A. aegerita, with amplified insert of the C-6 region of Fucp, analyzed in D2O at 50 °C. A= non reducing ends -Fucp; B = 2,6-di-O-substituted Galp units; C = 6-O-substituted -Galp units. ppm AC6 Residue A 30 50 PT 40 AC5 70 A AC4 AC3 C2 AH5 80 AH6 SC 60 90 100 3.5 3.0 2.5 A 70 H4 BH5 BH3 B BH1 65 BC3 BC6 BC4 M BC5 75 80 BC2 ED 85 1.5 ppm N Residue B 60 2.0 BH6/b 4.0 BH2 4.5 BH6/a 5.0 U AC1 ppm 90 95 PT 100 BC1 105 5.0 4.8 4.4 A 75 4.0 CH5 65 70 4.2 Residue C CH1 60 4.6 3.8 3.6 ppm CH6/b 5.2 CH6/a CC E 5.4 ppm CC6 CC5 CC3 CC2 CC4 80 85 90 95 100 CC1 105 5.4 5.2 5.0 4.8 4.6 4.4 RI AH4 AH3 AH2 AH1 4.2 CH4 CH2 CH3 4.0 3.8 3.6 ppm 15 Fig. 6. HSQC-TOCSY spectrum of fucogalactan (FG-Aa) from A. aegerita, analyzed in D2O at 50 °C. A= non reducing ends -Fucp; B = 2,6-di-O- substituted -Galp units; C = 6-O- substituted -Galp units. On the basis of NMR analyses, the identities of the monosaccharide residues A, B, and C PT were established (Tables 1 and 1S). Residue A was assigned as -L-fucopyranosyl unit. This was strongly supported by the presence of characteristics 1H ( 1.25) and 13C ( 18.42) signals RI for a CH3 group, besides typical carbon chemical shifts (C-1 to C-6) corresponding to the SC standard values of methyl glycosides (Agrawal, 1992). The downfield shifts of the C-2 and C-6 at δ 80.53 and 70.03, respectively, indicated that residue B was a 2,6-di-O-substituted α-D- U galactopyranose unit. In residue C, the downfield shift of the C-6 at δ 69.48 (C-6) confirmed the A N presence of 6-O-substituted α-D-galactopyranose. M A partial acid hydrolysis eliminated almost all -Fucp units from FP2CW-Aa, as shown ED by 13C NMR spectrum (Fig. 7), which contained main signals characteristics of a linear (16)linked -galactopyranan (C-1, 98.82; C-2, 68.55; C-3, 69.59; C-4, 69.02; C-5, 68.96; C-6, 66.51) PT (Oliveira et al., 2018). An interresidue cross peak AH-1/BC-2 in the HSQC-NOESY experiment 69.59 69.02 68.96 68.53 66.51 A CC E (residue A). 98.82 further confirmed the substitution at O-2 of the residue B by non-reducing ends of -Fucp 105 100 95 90 85 80 75 70 65 ppm 16 Fig. 7. 13C NMR spectrum of partially degraded fucogalactan (FG-Aa) from A. aegerita, analyzed in Me2SO-d6 at 70 °C, chemical shifts are expressed in ppm. According to the molar ratio of the residues, determined by 1H NMR and methylation PT analysis, and partial acid hydrolysis results, it could be concluded that the fucogalactan from A. aegerita (FG-Aa) consist of a (1→6)-linked α-D-galactopyranosyl main-chain, substituted at RI O-2 by non-reducing end units of -L-Fucp, on the average of one to every second residues of SC the backbone (Fig. 8), which has been supported by previous studies about similar heteropolysaccharides (Li et al., 2016; Ruthes, Rattmann, Carbonero, Gorin, & Iacomini, 2012; HO O N U Ye et al., 2008). O H C 3 HO A B HO O H3C OH HO OH O C OH CH O 3 n ED HO A O M O Fig. 8. Proposed structure for fucogalactan from A. aegerita (FG-Aa) PT Bioactive fucogalactans have been found from several macrofungi (Li et al., 2016; CC E Ruthes et al., 2012; Ye et al., 2008). However, despite the general structure in common, FG-Aa had higher fucose content among the fucogalactans already described. In order to investigate the biological action of the fucogalactan now isolated, it was A evaluated for probable arginase inhibitory effect. The characterized heteropolysaccharide isolated showed 60.5% of inhibition of ARG catalytic activity, when evaluated at 10 µM, with IC50 potency of 5.82 ± 0.57 µM. Previously, glycoflavones and a phenylethanoid glycoside (verbascoside) were found as inhibitors of ARG with potency ranging from 2.0 to 12.2 µM (da Silva, Maquiaveli, & Magalhães, 2012; de Sousa et al., 2014a; Maquiaveli et al., 2016). 17 Verbascoside is a competitive inhibitor of ARG and among the interactions showed by docking in the active site; H-bonds were established between glucopyranose and different ARG side chains (Maquiaveli et al., 2016). By the present kinetics experiments carried out with FGAa and ARG, a competitive behavior was observed (Fig. 9) supporting the findings that monosaccharide units interact in the active site. PT The plot of velocity as a function of substrate (Michaelis±Menten equation) (Fig. 9 A) RI showed that Vmax values were kept constant at all inhibitor concentrations, otherwise apparent Km values increased with increasing inhibitor concentration referring to a competitive inhibition SC type. The experimental data was processed using other approaches as well, Lineweaver-Burk, Dixon and Cornish-Bowden plots (Fig. 9 B, C, and D, respectively). In combination, the plots U showed the characteristics of the ARG inhibition manner by FG-Aa, which compete with the N substrate for the pool of free enzyme molecules with Ki of 1.54 ± 0.15 µM. 0.06 20 0 20 40 60 80 L-arginine (mM) C PT 1.8 1.6 1.4 1.2 1 CC E [L-Arg]/Velocity (mM/µmol.min-1) 2 0.02 0 0.8 -0.02 [L-Arg] = 25 mM [L-Arg] = 12.5 mM [L-Arg] = 6.25 mM 0.6 0.4 0 2 4 0.08 [L-Arg] = 100 mM [L-Arg] = 60 mM 0.04 [L-Arg] = 50 mM [L-Arg] = 25 mM [L-Arg] = 15 mM 0.02 0 0 -2 0.02 0.04 0.06 1/L-arginine (mM)-1 D 0.2 -4 0 0.06 [L-Arg] = 100 mM [L-Arg] = 60 mM [I] = 3.5 µM [I] = 7.0 µM 100 1/Velocity (µmo/min)-1 0 [I] = 1.4 µM 0.04 M [I] = 7.0 µM 40 Control A [I] = 3.5 µM 60 B 1/V (µmol/min)-1 [I] = 1.4 µM ED Velocity (µmol/min) Control A 80 6 8 -8 -6 -4 -2 0 2 4 6 8 A inhibitor (µM) Fig. 9. Competitive inhibition of FG-Aa (Ki = 1.54 ± 0.15 µM). (A) Direct Inhibitor (µM) plot of velocity as a function of substrate; (B) Lineweaver-Burk plot; (C) Cornish-Bowden plot; and (D) Dixon plot. Data were expressed as the mean of independent assays. This is the first report of an inhibitory activity of arginase from L. amazonensis by a biopolymer with high molecular weight. This result encourages us to investigate further polysaccharides as new class of ARG inhibitors. 18 Additionally, polysaccharides have already been described as metabolites with leishmanicidal activity (Adriazola et al., 2014; Amaral et al., 2015; Kangussu-Marcolino et al., 2015; Valadares et al., 2011, 2012). The usage of a drug conjugated of Amphotericin B and arabinogalactan reduced toxicity and showed better efficacy against Leishmania sp. as effect of the polysaccharides on the immune response (Ehrenfreund-Kleinman, Domb, Jaffe, Benni PT Leshem, & Golenser, 2005; Nishi et al., 2007). RI The immune response showed by polysaccharides previously related is due to their interaction with macrophages cells inducing pathways and processes to prevent leishmania SC infection. Among the host cell responses stimulated by polysaccharides, are the increase of reactive oxygen intermediates (ROI) and nitric oxide (NO) (Amaral et al., 2015; Kangussu- U Marcolino et al., 2015; Schepetkin & Quinn, 2006). The metabolic pathway of nitric oxide N synthase (NOS) that generate reactive oxygen species is regulated by TH1 and TH2 cytokines as A microbicidal response. However, Leishmania trick the immune response activating TH2 cytokine M and increasing arginase expression for their growth and survival (Blaña-Fouce et al., 2012; ED Colotti & Ilari, 2011; Kropf et al., 2005; Roberts et al., 2004). In this view, arginase inhibition PT by FG-Aa could be an additional clue on how polysaccharides act as immunomodulators. 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Wolfrom (Eds.), Methods in Carbohydrate Chemistry (pp. 211-215). New York: Academic Press. Yang, N., Li, D., Feng, L., Xiang, Y., Liu, W., Sun, H., & Wang, D. (2009). Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom PT Agrocybe aegerita. Journal of Molecular Biology, 387, 694-705. Ye, L., Zhang, J., Zhou, K., Yang, Y., Zhou, S., Jia, W., Hao, R., & Pan, Y. (2008). Purification, RI NMR study and immunostimulating property of a fucogalactan from the fruiting bodies of A CC E PT ED M A N U SC Ganoderma lucidum. Planta Medica, 74, 1730-1734. 24 Table 1. 1H and 13C NMR chemical shifts of heterogalactan from A. aegerita a,b. Units 1 2 3 4 5 6 a b 13 104.10 5.09 71.28 3.82 72.42 3.90 74.60 3.85 69.85 4.18 18.42 1.25 →2,6)--Galp-(1→ (Residue B) 13 100.87 5.04 80.53 3.83 71.28 4.08 72.47 4.07 72.00 4.14 70.03 3.98 3.64 →6)--Galp-(1→ (Residue C) 13 101.02 4.99 71.16 3.87 72.35 3.89 72.54 4.01 71.65 4.20 69.48 3.89 3.70 C 1 H C H 1 C H 1 Assignments were based on 1H, 13C, HSQC-DEPT, and HSQC-TOCSY examinations. (b) The values of chemical shifts were recorded with reference to TMS as internal standard. A CC E PT ED M A N U SC RI (a) PT -Fucp-(1→ (Residue A)