International Journal of Molecular Sciences

Article Lupinus albus γ-Conglutin, a Protein Structurally Related to GH12 Xyloglucan-Specific Endo-Glucanase Inhibitor Proteins (XEGIPs), Shows Inhibitory Activity against GH2 β-Mannosidase

Stefano De Benedetti † , Elisabetta Galanti †, Jessica Capraro , Chiara Magni and Alessio Scarafoni *

Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, 20133 Milano, Italy; [email protected] (S.D.B.); [email protected] (E.G.); [email protected] (J.C.); [email protected] (C.M.) * Correspondence: [email protected] Those authors contributed equally to this work. †


Received: 10 September 2020; Accepted: 30 September 2020; Published: 3 October 2020


Abstract: γ-conglutin (γC) is a major protein of Lupinus albus seeds, but its function is still unknown. It shares high structural similarity with xyloglucan-specific endo-glucanase inhibitor proteins (XEGIPs) and, to a lesser extent, with Triticum aestivum endoxylanase inhibitors (TAXI-I), active against fungal glycoside hydrolases GH12 and GH11, respectively. However, γC lacks both these inhibitory activities. Since β-galactomannans are major components of the cell walls of endosperm in several legume plants, we tested the inhibitory activity of γC against a GH2 β-mannosidase (EC 3.2.1.25). γC was actually able to inhibit the enzyme, and this effect was enhanced by the presence of zinc ions. The stoichiometry of the γC/enzyme interaction was 1:1, and the calculated Ki was 1.55 µM. To obtain further insights into the interaction between γC and β-mannosidase, an in silico structural bioinformatic approach was followed, including some docking analyses. By and large, this work describes experimental findings that highlight new scenarios for understanding the natural role of γC. Although structural predictions can leave space for speculative interpretations, the full complexity of the data reported in this work allows one to hypothesize mechanisms of action for the basis of inhibition. At least two mechanisms seem plausible, both involving lupin-γC-peculiar structures.

Keywords: seed proteins; γ-conglutin; enzyme inhibitors; plant cell wall-degrading enzymes; plant defense; Lupinus albus

1. Introduction Glycoside hydrolases (GHs) are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides [1]. The GHs have been classified into more than 100 families [2]. Each family contains proteins that are related by sequence and, in consequence, share common structural properties [3]. The classification of GH families into larger groups, termed “clans”, has been proposed [4,5]. A clan is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanisms of action [6]. Thus, knowledge of three-dimensional structure and the functional assignment of catalytic residues is required for classification into clans. The various biological functions of GHs are several and include the degradation of different plant cell wall polysaccharides [7,8]. The primary walls of plant cells are essentially composed of cellulose, pectin and combinations of hemicelluloses [9]. An important hemicellulose in most plants is xyloglucan, but glucuronoxylan, arabinoxylan, glucomannan and galactomannan are also found in different proportions in the primary

Int. J. Mol. Sci. 2020, 21, 7305; doi:10.3390/ijms21197305 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7305 2 of 15 and secondary walls of many botanical families [10]. Galactomannans are found as a major component of the endosperm cells in the seeds of several Leguminosae, including lupin [11,12]. Some pathogenic microorganisms are able to secrete GHs to penetrate plant cell walls [13]. As a response, plants produce GH inhibitor proteins (GHIPs) [14]. γ-conglutin (γC) accounts for about 4% of total seed protein, which contributes 35–40% to the dry seed weight of the leguminous plant Lupinus albus [15]. In the last decade, this protein has been receiving increasing interest because of its demonstrated capacity to lower blood glucose levels in humans and animals when orally administered [16,17]. This allows one to hypothesize its use as an agent for the treatment of patients suffering from prediabetes [18]. All of this aside, the natural biological role of γC is still far from clear. Although it has been considered, for a long time, a classical seed-storage protein, more recent studies broaden its functions to include a possible role in defense against pathogenic microorganisms [19,20]. γC is a homo-hexameric glycoprotein, in which each monomer of 45 kDa is made up of two disulfide-bonded polypeptides of about 29 and 17 kDa [15,21] that originate from a single precursor protein synthesized during seed development and processed by post-translational proteolysis [22]. The protein is glycosylated in the 29 kDa subunit [15,23]. The protein undergoes association–dissociation transition between the hexameric and monomeric forms according to the pH conditions. The monomeric form predominates at slightly acidic pHs [24]. Although γC is stored in the cotyledon protein bodies of mature quiescent seeds, the protein has been detected in the extracellular apoplastic regions of germinating seeds [25]. In addition, γC is able to bind divalent metal ions, especially Zn2+ and Ni2+ [26], and phospholipids [27]. Two genes encoding γC have been identified in Lupinus albus, but only one is quantitatively expressed and accumulated in the developing seeds [22,28]. γC shares high structural similarities with two families of GH inhibitors, namely, xyloglucan-specific endo-β-1,4-glucanase inhibitors (XEGIPs) and Triticum aestivum endoxylanase inhibitors (TAXI-I). While XEGIPs inhibit the hydrolytic activity of a xyloglucan-specific β-1, 4-endo-glucanase (XEG) isolated from Aspergillus aculeatus and belonging to the GH12 family [29,30], TAXI-Is are inhibitors of GH11 members [31]. γC lacks the typical inhibitory activity against representative fungal GH11, GH12 and polygalacturonase, although its de novo expression can be elicited by chitosan [19,20]. XEGIPs have been found to be widespread in dicots. They have been detected in the medium of cultured tomato cells [30] and carrot calli [32], and isolated from the nectar of ornamental tobacco [33]. Moreover, it has been demonstrated they that are capable of protecting potatoes from disease caused by Phytophthora infestans [34], and they were found to be up-regulated in apples in response to infection by Botryosphaeria dothide [35] and in Humulus lupus [36]. In cereals, three types of GHIPs occur in a fairly coordinated fashion throughout grain development and germination, the Triticum aestivum L. endoxylanase inhibitors (TAXI-I-like) being the most represented [31,37]. Sequence alignments and structural studies showed that in XEGIPs and the TAXI-I protein, two functional domains are responsible for the inhibitory capacity [21,38]. Both are located in the C-terminal regions of the proteins. The first is located between cys10 and cys11, and defines a surface-exposed region known as inhibitory loop 1 (IL1), where a conserved arginine in XEGIP-like proteins, or a conserved leucine in TAXI-I, are involved in binding with the respective GH12 or GH11. In γC, instead, IL1 is missing due to a deletion of about five amino acids [19,21]. This is likely the cause of an unfavorable local spatial conformation of the protein for the correct interaction with the enzyme [20], leading to the lack of inhibitory capacity of γC and other similar legume proteins, including soybean Bg7S [39]. The disulfide bridge between cys9 and cys12 defines another functional region called inhibitory loop 2 (IL2), where the key amino acids are an arginine residue in XEGIPs or a histidine in TAXI-I and γC. The sequence of the IL2 loop of γC is more similar to the sequence of the IL2 loop of TAXI-I than to the one of XEGIPs. Studies on γC mutants [20] demonstrated that the presence of IL1 is not strictly required to manifest inhibition, even if the inserted amino acid stretches enhanced the Int. J. J. Mol. Sci. Sci. 20202020,, 2121,, x 7305 FOR PEER REVIEW 3 of 15 necessary not only to manifest the inhibitory competence but also to drive the specificity toward the activity.respective Moreover, target GH. the structure of IL2 is the essential element that is very likely necessary not only to manifestIn the the present inhibitory work competence, we undertook but also lab toexperiments drive the specificity and in silico toward predictions the respective aiming target to define GH. and characterizeIn the present the work, inhibitory we undertook activity labof γ experimentsC extracted andfrom in L. silico albus predictions seeds. γC aimingwas tested to define against and a characterizeGH2 β-mannosidase the inhibitory (EC 3.2.1.25 activity) and of γa CGH5 extracted xyloglucan from-specificL. albus seeds.endo-βγ-1,4C was-glucanase tested against(EC 3.2.1.151 a GH2). β-mannosidase (EC 3.2.1.25) and a GH5 xyloglucan-specific endo-β-1,4-glucanase (EC 3.2.1.151). 2. Results and Discussion 2. Results and Discussion Enzymes whose activity could be influenced by γC have been elusive for a long time [19]. A greatEnzymes number of whose findings activity indicate could that be influenced the target byof γCγC is have almost been certainly elusivefor a GH a long enzyme. time [ 19In ].previous A great numberwork, the of findingspossible indicateinhibitory that activity the target of γCof γ wasC is almosttested certainlyunsuccess afully GH enzyme.against GH11In previous and GH12 work, the[19,20 possible], all included inhibitory in GH activity clan C of [γ4]C. Despite was tested their unsuccessfully low sequence againstidentityGH11 (only the and three GH12 amino [19,20 acids], all includedessential infor GH catalysis clan C [ 4are]. Despite completely their low conserved sequence across identity all (only member the threes [4 amino0]), the acids overall essential three for- catalysisdimensional are completelystructures for conserved all the known across GH all memberss grouped [ 40in]), this the clan overall are remarkably three-dimensional similar structures(Figure 1, forcyan all and the knownpurple GHsmodels grouped). Conversely, in this clan in arethe remarkablypresent work similar, we focused (Figure1 ,our cyan attention and purple on enzymes models). Conversely,having different in the structural present features work, we than focused GH11s our and attention GH12s, onnot enzymes yet explored having for ditheirfferent susceptibility structural featuresto γC inhibition. than GH11s The and choice GH12s,s were not a yet β- exploredmannosidase for their (GH2) susceptibility and a xyloglucan to γC inhibition.-specific Theendo choices-β-1,4- wereglucanase a β-mannosidase (GH5), both (GH2)members and of a xyloglucan-specificGH clan A. The catalytic endo-β domain-1,4-glucanase of the (GH5),enzymes both from members this clan of GHhas a clan (β/α) A. 8 The-barrel catalytic fold, also domain called of a the TIM enzymes-barrel fold from [4 this]. The clan first has enzyme a (β/α )[4 8-barrel1] was selected fold, also because called aβ- TIM-barrel(1→4)-linked fold pol [ysaccharides4]. The first enzymecontaining [41 ]mannose was selected are major because componentsβ-(1 4)-linked of the polysaccharidesendosperm cell → containingwalls in seeds mannose of several are majorlegume components plants, including of the endospermsoybeans and cell lupin wallss [ in11] seeds. We thus of several hypothesized legume plants,that a potential including inhibitory soybeans activity and lupins from [11 the]. Welupin’ thuss γC hypothesized could be exerted that a potentialtowards enzymes inhibitory targeting activity fromthe major the lupin’s constituentγC could of its be cellexerted wall. towards The second enzymes enzyme targeting was theconsidered major constituent because ofthe its described cell wall. Thesubstrate second specificity enzyme wasis similar considered to that because of the GH12 the described xyloglucan substrate-specific specificity endo-glucanase is similar [29, to42 that], against of the GH12which xyloglucan-specificcarrot EDGP and tomato endo-glucanase XEGIP are [29 active,42], againstas inhibitors which [ carrot30,39]. EDGPFrom andthe structural tomato XEGIP point are of activeview, the as inhibitors selected enzymes [30,39]. From are very the structuraldifferent pointfrom GH11 of view, and the GH12 selected enzymes enzymes, but are similar very di toff erenteach fromother GH11(Figure and 1, green GH12 and enzymes, pink models but similar). to each other (Figure1, green and pink models).

Figure 1. StructureStructure comparison comparison of of representative representative members members of GH of GH families. families. For each For each structure structure,, the PDB the PDBcode codeand the and enzyme the enzyme names names are reported are reported below below each each model model.. GH11 GH11 and andGH12 GH12 are reported are reported with with the therespective respective inhibitor inhibitorss (gray) (gray) as asthey they appear appear in inPDB PDB accessions accessions.. Catalytic Catalytic amino amino acids acids are are shown shown in spacefillspacefill representation.

β β GH2 β-mannosidase-mannosidase from fromCellulomonas Cellulomonas fimi [ fimi43] and[43 GH5] and xyloglucan-specific GH5 xyloglucan endo--specific-1,4-glucanase endo-β-1,4- fromglucanasePaenibacillus from Paenibacillussp. [44] activities sp. [44 were] activities assayed were by incubating assayed by each incubating enzyme each with enzyme chromogenic with γ substrates,chromogenic as describedsubstrates in, as Materials described and in Methods,Materials inand the Methods, presence in or the absence presence of C, or atabsence an initial of molarγC, at ratioan initial of 1:1 molar (Figure ratio2). Onlyof 1:1 in (Figure the first 2) case. Only did in the the presence first case of did the the lupin presence protein of reduce the lupin the enzyme protein activity,reduce the to about enzyme 73% activity of its maximum, to about activity,73% of itsin the maximum adopted activity experimental, in the conditions, adopted experimental whereas no significantconditions,e whereasffects were no observedsignificant when effects it waswere tested observed against when the it GH5 was tested enzyme. against the GH5 enzyme.

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Figure 2. Enzyme residual activity in the absence (white bars) and in the presence of γC at a molar ratio of 1:1 (gray bars). GH2 and GH5 stand for β-mannosidase from Cellulomonas fimi and for xyloglucan-specific-endo-β-1,4-glucanase from Paenibacillus polymyxa, respectively. Residual activity FigureFigure 2. EnzymeEnzyme r residualesidual activity activity in in the the absence absence (white (white bars) bars) and and in in the the presence presence of of γCγC at at a a molar was calculated as (AE-AEI)/AE × 100, where AE is the measured enzyme activity (mmol/min) and ratioratio of of 1:1 (gray bars).bars). GH2 GH2 and and GH5 GH5 stand stand for for ββ--mannosidasemannosidase from from CellulomonasCellulomonas fimi fimi andand for for AEI is the activity of the enzyme in the presence of γC. See text for experimental details. Each point xyloglucanxyloglucan-specific-endo--specific-endo-ββ--1,4-glucanase1,4-glucanase from from PaenibacillusPaenibacillus pol polymyxaymyxa,, respectively. respectively. Residual Residual activity activity wasis the calculated mean of asthree (AE-AEI) assays./AE 100, where AE is the measured enzyme activity (mmol/min) and AEI was calculated as (AE-AEI)/AE× × 100, where AE is the measured enzyme activity (mmol/min) and γ AEIis the is activitythe activity of the of enzymethe enzyme in the in presence the presence of C. of See γC text. See for text experimental for experimental details. details. Each pointEach point is the This is the first time we have been able to describe an inhibitory activity for L. albus γC. ismean the mean of three of assays.three assays. Encouraged by this result, we focused on the further characterization of GH2 β-mannosidase’s This is the first time we have been able to describe an inhibitory activity for L. albus γC. Encouraged inhibitingThis is activity. the first time we have been able to describe an inhibitory activity for L. albus γC. by this result, we focused on the further characterization of GH2 β-mannosidase’s inhibiting activity. EncouragedAt first , bythe thisinhibit resultory, a wectivity focused was tested on the at two further different characterization pHs, regardless of GH2 of the β optimal-mannosidase enzyme’s At first, the inhibitory activity was tested at two different pHs, regardless of the optimal enzyme inhibitingreaction conditions. activity. The native quaternary structure of γC is determined by the transition from a reaction conditions. The native quaternary structure of γC is determined by the transition from a hexamericAt first to, the monomeric inhibitory state activity, according was tested to theat two acidity different of the pHs medium, regardless [24]. Theof the results optimal repor enzymeted in hexameric to monomeric state, according to the acidity of the medium [24]. The results reported in reactionFigure 3 conditions.show β-mannosidase The native’s quaternaryresidual activity structure at pH of 4.8 γC (where is determined γC is completely by the transitionin the monomeric from a Figure3 show β-mannosidase’s residual activity at pH 4.8 (where γC is completely in the monomeric hexamericform) and topH monomeric 6.0 (within state the optimal, according range to thefor enzymeacidity of activity the medium and where [24]. theThe γC results monomeric reported form in form) and pH 6.0 (within the optimal range for enzyme activity and where the γC monomeric form Figuredisappears 3 show to βform-mannosidase oligomers).’s residualThe residual activity activities at pH 4.8observed (where at γC pH is completely4.8 and 6.0 werein the very monomeric similar , disappears to form oligomers). The residual activities observed at pH 4.8 and 6.0 were very similar, and form)and small and pHvariations 6.0 (within may the depend optimal on range the oligomerization for enzyme activity state and of γC where or the the different γC monomeric charge sform that small variations may depend on the oligomerization state of γC or the different charges that proteins disappearsproteins assume to form at oligomers).different pHs. The However, residual activitiesthe observed observed differences at pH 4.8were and not 6.0 statistically were very differentsimilar, assume at different pHs. However, the observed differences were not statistically different (p 0.05). and(p ≤ small0.05). variations may depend on the oligomerization state of γC or the different charge≤s that proteins assume at different pHs. However, the observed differences were not statistically different (p ≤ 0.05).

FigureFigure 3.3. ResidualResidual activityactivity of GH2GH2 ββ-mannosidase-mannosidase incubated in the presence of γγCC at didifferentfferent pHs,pHs, d usingusing pNP-pNP-ββ--D-mannopyranoside-mannopyranoside asas thethe substrate.substrate. ActivitiesActivities determineddetermined atat pHpH 4.84.8 andand 6.06.0 werewere notnot statisticallystatistically didifferentfferent ( (pp ≤ 0.05). Residual enzyme activity is expressed as percent percentageage activity compared Figure 3. Residual activity≤ of GH2 β-mannosidase incubated in the presence of γC at different pHs, withwith thethe enzymeenzyme alone.alone. EachEach pointpoint isis thethe meanmean ofof threethree assays.assays. using pNP-β-D-mannopyranoside as the substrate. Activities determined at pH 4.8 and 6.0 were not statisticallyWe then tested different the (p e ff≤ ects0.05). of Residual three metal enzyme ions activity (Cu2 is+ ,expressed Ni2+ and as Zn percent2+) thatage haveactivity been compared previously We then tested the effects of three metal ions (Cu2+, Ni2+ and Zn2+) that have been previously shownwith to the produce enzyme controversial alone. Each point effects is the on mean the GH2 of three enzyme’s assays. activity. It has been indeed shown that shown to produce controversial effects on the GH2 enzyme’s activity. It has been indeed shown that many mannosidases, including those belonging to the GH2 group, may be sensitive to metal ions. The many mannosidases, including those belonging to the 2+GH22+ group, may2+ be sensitive to metal ions. results,We however,then tested are the debated, effects sinceof three the samemetal ionions can (Cu positively, Ni and or Zn negatively) that have influence been thepreviously activity The results, however, are debated, since the same ion can positively or negatively influence the shownof different to produce enzymes controversial [45–48]. Intriguingly, effects on theγC GH2 was enzyme shown’ tos activity be able. It to has interact been withindeed metal shown ions, that in activity of different enzymes [45–48]. Intriguingly, γC was shown to be able to interact with metal manyparticular, mannosidases those investigated, including [26 those]. The belonging presence to of the the GH2 ions, group in most, may cases, be ledsensi totive a decrease to metal inions. the ions, in particular, those investigated [26]. The presence of the ions, in most cases, led to a decrease Thesolubility results of, thehowever protein,, are when debated present, since in molar the excesssame ion and can according positively to the or pH negativ of theely incubation influence bu theffer. in the solubility of the protein, when present in molar excess and according to the pH of the activityHowever, of 1different mM Zn2 enzyme+ did nots [ a4ff5ect–48 the]. Intriguingly solubility of, γC the was protein shown at pH to valuesbe able between to interact 4.5 andwith 6.3 metal [26]. ions , in particular, those investigated [26]. The presence of the ions, in most cases, led to a decrease in the solubility of the protein, when present in molar excess and according to the pH of the

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incubation buffer. However, 1 mM Zn2+ did not affect the solubility of the protein at pH values Webetween therefore 4.5 incubatedand 6.3 [26 the]. βWe-mannosidase therefore incubated with or without the β-mannosidaseγC, in the presence with ofor diwithoutfferent metal γC, in ions, the atpresence the final of concentration different metal of ions, 1 mM at (Figure the final4). concentration of 1 mM (Figure 4).

FigureFigure 4.4. IncubationIncubation ofof GH2GH2β β-mannosidase-mannosidase in in the the absence absence (white (white bars) bars) and and in in the the presence presence of ofγC γC at at a 2+ 2+ 2+ molara molar ratio ratio of of 1:1 1:1 (gray (gray bars), bars) with, with 1 mM 1 mM Ni Ni,2+ Zn, Zn2+and and Cu Cu2+.. Residual enzyme activityactivity isis expressedexpressed asas percentagepercentage activityactivity comparedcompared withwith thethe enzymeenzyme alonealone byby usingusing pNP-pNP-ββ--dD-mannopyranoside-mannopyranoside asas thethe substrate.substrate. EachEach pointpoint isis thethe meanmean ofof threethree assays.assays.

2+ 2+ TheThe presencepresence ofof NiNi2+ andand ZnZn2+ ionsions alone did not aaffectffect the the activity activity of of ββ-mannosidase-mannosidase inin the the 2+ adoptedadopted experimentalexperimental conditions,conditions, whereaswhereas Cu Cu2+ markedlymarkedly decreaseddecreased itsits functionality.functionality. WhenWhen γγCC waswas 2+ added,added, the the residual residual activity activity was was lower lower in all in cases. all cases. The activityThe activity in the presencein the presence of Ni wasof Ni only2+ w slightlyas only 2+ 2+ aslightlyffected, affected but it dropped, but it dropped drastically drastically when Zn whenwas Zn added2+ was added (about (about 55%). Thus,55%). Thus, the presence the presence of Zn of increasesZn2+ increases the inhibitory the inhibitory activity activity of γ ofC byγC aboutby about 20%. 20%. The The metal metal ion ion could could possibly possibly be be coordinatedcoordinated byby specificspecific aminoamino acidacid residuesresidues promotingpromoting conformationalconformational changeschanges inin γγCC regionsregions involvedinvolved inin thethe interactioninteraction withwith thethe enzyme,enzyme, suchsuch asas thethe inhibitoryinhibitory looploop IL2,IL2, and,and, inin turn,turn, favoringfavoring thethe inhibitoryinhibitory 2+ competencycompetency (Supplementary(Supplementary FigureFigure S1). S1). TheThe resultsresults obtainedobtained withwith CuCu2+ ions are difficult difficult to interpret, sincesince thethe enzymeenzyme itselfitself isis veryvery sensitivesensitive toto itsits presencepresence inin thetheincubation incubation medium.medium. ItIt isis worthworth notingnoting thatthat thethe purifiedpurifiedγ γCC usedused throughoutthroughout thethe experimentsexperiments describeddescribed herehere diddid notnot carrycarry anyany kindkind ofof metalmetal ions,ions, asas confirmedconfirmed byby inductivelyinductively coupledcoupled plasma plasma (ICP)-MS. (ICP)-MS. Similarly,Similarly, as as reported reported for for GH2 GH2 enzymes, enzymes, the the eff effectect of of metal metal ions ions on on GH11 GH11 and and GH12 GH12 seems seems not not to 2+ followto follow a general a general rule. rule. A GH11 A GH11 enzyme enzyme from fromAspergillus Aspergillus tamarii tamariiwas stronglywas strongly inhibited inhibited by 5 mMby 5 Cu mM 2+ 2+ andCu2+ Zn and ,Zn while2+, while Ni Niincreased2+ increased enzyme enzyme activity activity [49 [].49 Rawat]. Rawat et et al. al. [50 [5],0], instead, instead, reported reported on on a a GH12GH12 2+ 2+ endo-glucanaseendo-glucanase fromfromAspergillus Aspergillus niger nigerstrongly strongly inhibited inhibited by by Cu Cu2+,, while while the the Zn Zn2+ inhibitory eeffectffect waswas onlyonly partialpartial at theat thesame same concentration. concentration. The activity The activity of a GH12 of acidic a GH12 endo-glucanase acidic endo from-glucanaseGloeophyllum from 2+ 2+ trabeumGloeophyllumwas not trabeum affected was by not Zn affectedand Cu by Zn, even2+ and at Cu 502+ mM, even concentrations at 50 mM concentration [51]. Juturus and [51] Wu. Juturu [52] reviewedand Wu [5 the2] review effectsed of metalsthe effect ons xylanasesof metals on belonging xylanases to belonging different families to different (GH10, families GH11 (GH10, and GH39), GH11 +2 +2 +2 +2 +2 +2 +2 alland negatively GH39), all a ffnegativelyected by Hg affected, Fe by, CoHg+2,, Fe Mn+2, Co, Ag+2, Mn, Pb+2, Agand+2, Pb Cu+2 and, but Cu they+2, but provided they provid no cluesed no 2+ 2+ aboutclues Znaboutand Zn2+ Ni and. Ni Testing2+. Testing the e fftheect effect of a possible of a possible inhibitory inhibitory activity activity of γC againstof γC against GH12 orGH12 GH11 or enzymesGH11 enzymes in the presencein the presence of metal of ions metal was ions out was of theout scope of the of scope this work.of this work. 2+ WeWe finally finally aimed aimed to determineto determine some some kinetic kinetic parameters, parameters in particular,, in particular the Ki,, of the the K Zni, of-mediated the Zn2+- γmediatedC inhibition γC ofinhibition GH2, since of GH2 this ion, since was this the ion most was eff ectivethe most in enhancing effective in the enhanc inhibitorying the activity. inhibitory The inhibitionactivity. The features inhibition were determined features were as usual determined by monitoring as usual the hydrolysis by monitoring of the chromogenic the hydrolysis substrate of the pNP-chromogenicβ-d-mannopyranoside, substrate pNP- thisβ-D- timemannopyranoside, with increasing this concentrations time with increasing of γC, in theconcentrations presence of 1of mM γC, 2+ Znin the. Thepresence experimental of 1 mM data Zn2+ are. The plotted experimental in Figure data5. The are x-intercept plotted in value Figure of 5 the. The traced x-intercept tangent value line of of thethe besttraced fit curvetangent indicated line of anthe enzyme best fit/inhibitor curve indicated stoichiometry an enzyme/inhibitor of 1.14, a value very stoichio closemetry to a theoretical of 1.14, a enzymevalue very/inhibitor close to ratio a theoretical of 1:1. enzyme/inhibitor ratio of 1:1.

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FigureFigure 5.5. Titration curve of GH2 β--mannosidasemannosidase with γγCC.. Increasing concentrationsconcentrations ofof γγCC were added toto aa fixedfixed concentrationconcentration ofof enzymeenzyme (11.8(11.8 µμM).M). ResidualResidual enzymeenzyme activityactivity isis expressedexpressed asas percentagepercentage activityactivity compared compared with with the the enzyme enzyme alone, alone using, using pNP- pNPβ-d--mannopyranosideβ-D-mannopyranoside as the as substrate the substrate (0.8 mM), (0.8 atmM) pH, 6.0,at pH in 6.0 the, presencein the presence of 1 mM of Zn1 mM2+. EachZn2+. pointEach point is the is mean the mean of three of assays.three assays. Error Error bars havebars beenhave omittedbeen omitted for better for better clarity. clarity. The best The fit best curve fit (blackcurve (black full line) full has line) R2 has= 0.9913. R² = 0.9913. The x-intercept The x-intercept value ofvalue the tracedof the traced tangent tangent line (dash line line)(dash indicates line) indicates an inhibitor an inhibitor//enzymeenzyme stoichiometry stoichiometry of 1.14. of 1.14.

KKii was estimated estimated according according to to C Cerer et etal. al. [53 [],53 who], who proposed proposed a method a method to calculate to calculate Ki valuesKi values from fromexperimentally experimentally determined determined IC50IC50 valuesvalues for for enzyme enzyme inhibitors inhibitors and and for for binding binding reactions reactions between macromolecules,macromolecules, includingincluding proteinsproteins andand ligands.ligands. TheThe IC50IC50 waswas determined accordingaccording toto thethe equation 2 of the best fit curve of Figure5 (y = 22.342x 2 70.059x + 97.699). The half of the maximum enzyme of the best fit curve of Figure 5 (y = 22.342 x − − 70.059 x + 97.699). The half of the maximum enzyme activityactivity inin thethe adopted adopted experimental experimental condition condition was was reached reached when when the the molar molar ratio ratio [γC] [γC/[GH2]]/[GH2] was 0.99.was The resulting K was 1.55 0.08 µM, assuming a competitive mechanism of action of the inhibitor. 0.99. The resultingi Ki wa±s 1.55 ± 0.08 μM, assuming a competitive mechanism of action of the Thisinhibitor. inhibition This inhibition mechanism mechanism seems the seems most plausible, the most givenplausible, the mechanismgiven the mechanism of action of of the action other of GH the inhibitorsother GH [inhibitors30,54] and [30,5 in light4] and of thein light in silico of the predictions in silico predictions and analysis and described analysis below.described below. ToTo obtainobtain further further insights insights and and to to outline outline the the possible possible rationale rationale behind behind the the interaction interaction between betweenγC andγC andβ-mannosidase, β-mannosidase an, in an silico in silico structural structural bioinformatic bioinformatic approach approach was followed.was followed. To this To purpose, this purpose, an L. albusan L.γ albusC 3D γC model 3D was model created was using, created as using a template,, as a thetemplate crystal, the structure crystal of structureγC from L. of angustifolius γC from L. (PDB:angustifoliu 4PPH)s (PDB [21], which: 4PPH shares) [21], 88.73%which shares sequence 88.73% identity. sequence The predicted identity. structureThe predicted is a homo-hexamer, structure is a likehomo the-hexamer, template, like with the an template, expected with accuracy an expected value (global accuracy model value quality (global estimation model quality (GMQE); estimation range 0–1) of 0.82 and a qualitative model energy analysis (QMEAN) Z-score of 1.08, indicating a good (GMQE); range 0–1) of 0.82 and a qualitative model energy analysis (QMEAN− ) Z-score of −1.08, agreementindicating a between good agreement the computed between model the computed structure andmodel structures structure of and similar structures sizes experimentallyof similar sizes determined.experimentally The determined. structure of The each structure monomer of highlyeach monomer overlaps highly the structure overlaps of theγC structure from L. angustifolius of γC from withL. angustifolius a root mean with square a root deviation mean square (RMSD) deviation of 0.152 (RMSD) Å; the of lowest 0.152 homology Å; the lowest is in homology the loop between is in the aminoloop between acids 255 amino and 272, acids unmodeled 255 and 272, in the unmodeled template, containingin the template, the cleavage containing site of the the cleavage precursor site that of originatesthe precursor the largethat originates and small the subunits large and of γ smallC. subunits of γC. WeWe performed performed all all the the following following in silicoin silico analyses analyses using using the structure the structure of the homolog of the homolog GH2 enzyme GH2 fromenzymeTrichoderma from Trichoderma harzianum (ThMan2A) harzianum (ThMan2A) [55], since the [5 3D5], structure since the of 3D the GH2structureβ-mannosidase of the GH2 from β- Cellulomonasmannosidase fimi fromused Cellulomonas for our wet fimi experiments used for our has wet been experiments not yet determined. has been Thenot yet superposition determined of. T thehe ThMan2Asuperposition structure of the (PDB: ThMan2A 4CVU) with structure a 3D predictive(PDB: 4CVU) model, with created a 3D on predictive purpose with model SwissModel, created [ 56on] usingpurpose the withCellulomonas SwissModel fimi [5GH26] usingβ-mannosidase the Cellulomonas sequence fimi GH2 (UniprotKB: β-mannosidase Q9XCV4) sequence [43] as (UniprotKB the query,: indicatedQ9XCV4) an [43 excellent] as the query, agreement indicated between an excellent the two models agreement (RMSD between= 0.987 the Å) two (Supplementary models (RMSD Figure = 0.987 S2). Thus,Å) (Supplementary we decided to performFigure Sdocking2). Thus, analyses we decided assigning to the perform ThMan2A docking structure analyses as the assigning receptor andthe theThMan2A monomer structure of the L.as albusthe receptorγC model and as the the monomer ligand molecule, of the L. wherealbus γC necessary, model as without the ligand any molecule, restraint, followingwhere necessary, each program’s without default any restraint, input instructions. following each The program monomer’s ofdefaultγC was input chosen instructions. as a docking The molecule.monomer Theof γC results was are chosen reported as a indocking Figure 6 molecule and are relative. The results to the are best reported docking in obtained Figure with6 and each are piecerelative of software,to the best i.e., docking the result obtained showing with the each highest piece score of software, calculated i.e. including, the result electrostatic showingand the vanhighest der Waalsscore energycalculated contributions, including sorted electrostatic by each and computation van der Waals as the energytop solution. contribution For betters, sorted representation by each andcomputation clarity, ThMan2A as the top is representedsolution. For with better the representation coloring attributed and byclarity Nascimento, ThMan2A et al. is [r55epresented], highlighting, with inthe blue, coloring the catalytic attributed domain by Nascimento (residues 347–737),et al. [55], whilehighlight structuraling, in blue domain, the 1 catalytic (residues domain 26–221) (residues is pink, 347–737), while structural domain 1 (residues 26–221) is pink, domain 2 (residues 222–346) is yellow, domain 4 (residues 738–849) is orange and domain 5 (residues 850–942) is green. The AutoDock Vina

Int. J. Mol. Sci. 2020, 21, 7305 7 of 15

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 15 domain 2 (residues 222–346) is yellow, domain 4 (residues 738–849) is orange and domain 5 (residues 850–942)software isdocked green. Thethe substrateAutoDock β Vina-galactomannan software docked (GM) the, the substrate main storageβ-galactomannan polysaccharide (GM), the in many main storagelegume polysaccharide species, in several in many conformations legume species, into inthe several active conformations site; only the into one the with active the best site; onlydocking the oneenergy with is theshown. best docking energy is shown.

Figure 6. DockingDocking results results from from all all the the tested tested software. software. ThMan2A ThMan2A catalytic catalytic domain domain is colored is colored in blue, in blue,whereas whereas structural structural domain domain 1 is pink, 1 is pink,domain domain 2 is yellow, 2 is yellow, domain domain 4 is orange 4 is orange and domain and domain 5 is green 5 is green[55]. C [55atalytic]. Catalytic amino amino acids acidsE489 E489 and E594 and E594are represented are represented as white as white spheres, spheres, and and the the substrate substrate β- βgalactomannan-galactomannan is isrepresented represented as as white white sticks. sticks. Refer Refer to to text text for for ThMan2A ThMan2A coloring coloring details. L. albus γγCC isis representedrepresented inin tantan color.color. PanelPanel ((A)) shows ThMan2A alone;alone; thethe othersothers showshow thethe complexcomplex withwith γγCC as predictedpredicted by thethe pyDockWEBpyDockWEB (B), ClusProClusPro 2.02.0 ((C),), PRISMPRISM 2.02.0 ((DD),), GRAMM-XGRAMM-X ((E),), HDOCKHDOCK ((F)) andand FRODOCK ((GG)) software.software. Atoms of the catalytic residues in the active site are white colored in spacefillspacefill representationrepresentation toto highlighthighlight thethe localizationlocalization ofof thethe acid-baseacid-base E489E489 andand thethe nucleophilenucleophile E594E594 [[5555].].

ItIt isis remarkableremarkable thatthat fivefive algorithmsalgorithms forfor molecularmolecular docking,docking, out of thethe sixsix tested,tested, predictpredict thethe interactioninteraction betweenbetween didifferentfferent portionsportions ofof thethe lupinlupin conglutinconglutin gammagamma modelmodel andand the ThMan2A active site region,region, thusthus indicatingindicating that accessaccess for thethe substratesubstrate toto thethe catalyticcatalytic residuesresidues couldcould potentiallypotentially bebe hindered and limited by the presence of γγCC.. DiDifferentfferent on-lineon-line tools identifyidentify didifferentfferent regionsregions ofof γγCC as responsibleresponsible forfor thesethese interactions;interactions; thus, thus, it it is is di difficultfficult to to hypothesize hypothesize a univocala univocal potential potential mechanism mechanism of actionof action only only on on the the basis basis of theof the reported reported data. data. However, However, it must it must be noted be noted that that ClusPro ClusPro 2.0 (Figure 2.0 (Figure6C), which6C), which allowed allow theed refinement the refinement of the of selectionthe selection of docking of docking structures structures by weighingby weighing the the contribution contribution of electrostaticof electrostatic and vanand der van Waals der forces, Waals indicated forces, indicated residues Q341,residues K357 Q341, and K358 K357 of andγC as K358 responsible of γC foras theresponsible interaction. for Q341the interaction. is conserved Q341 across is conserved sequences across of γC sequences homologs inof otherγC homologs legume species,in other whereas legume K357species, and whereas K358 are K357 peculiar and K358 exclusively are peculiar to L. albus exclusivelyγC. to L. albus γC. Conversely, the the analysis analysis performed performed with with HDOCK HDOCK (Figure (Figure7) shows 7) ashows possible a interactionpossible interaction mediated bymediated residues by R426 residues and R428 R426 from andγ R428C, directed from towardsγC, directed the ThMan2A towards the active ThMan2A site. These active amino site. acids These lie withinamino aacids protruding lie within loop a resemblingprotruding XEGIPs’ loop resembling inhibitory XEGIPs’ loop 1 IL1, inhibitory in which loop the inhibitory1 IL1, in which activity the is borneinhibitory by the activity critical is residue borne R322.by the Furthermore, critical residue XEGIPs’ R322. inhibitory Furthermore, loop IL2’sXEGIPs’ competence inhibitory is dependentloop IL2’s oncompetence a conserved is dependent arginine residueon a conserved (R403) [a20rginin,38]. eγ residueC, similarly (R403) to [20, its3 homolog8]. γC, similarly from soybeans, to its homolog Bg7S, lacksfrom these soybean criticals, Bg7S, residues lacks in these IL1 and critical IL2, residues thus lacking in IL1 inhibitory and IL2, activity thus lacking towards inhibitory members activity of the GH12towards family members [39]. of However, the GH12 the family R426 [39 and]. However R428 of γ, Cthe possess R426 and the R428 potential of γC to possess mediate the inhibitory potential activityto mediate towards inhibit membersory activity of thetowards GH2 members family. Finally, of the theGH2 docking family. analysisFinally, the of thedocking whole analysis molecule of tothe ThMan2A whole molecule domain to 3 ThMan2A indicates thatdomain the interaction3 indicates couldthat the be interaction mediated bycould helix be H4Nmediated of γC, by which helix isH4N composed of γC, which of alternate is composedα-helical of and alternate 310-helical α-helical segments and 3 and10-helical confers segments to this a regionnd confer a deformeds to this region a deformed curved shape peculiar only to γC [21]. In conclusion, despite residue R428 being conserved in all γC homologs, sequence analysis shows that R426 is conserved only in legumes and in pepper amino acid sequences [57].

Int. J. Mol. Sci. 2020, 21, 7305 8 of 15 curved shape peculiar only to γC[21]. In conclusion, despite residue R428 being conserved in all γC homologs, sequence analysis shows that R426 is conserved only in legumes and in pepper amino acid sequences [57]. Int.Int. J.J. Mol.Mol. Sci.Sci. 2020,, 21,, xx FORFOR PEERPEER REVIEWREVIEW 8 of 15

FigureFigure 7.7. DockingDocking ofof γγCC (tan(tan color)color) andand ThMan2AThMan2A (catalytic(catalytic domain:domain: blue)blue) asas predictedpredicted byby HDOCKHDOCK software.software. The The picture picture is is aa selectedselected areaarea showingshowing thethe regionregion ofof thethe activeactiveactive site;site;site; ThMan2AThMan2A domaindomain 11 isiiss omittedomitted forfor betterbetter representation.representation. TheThe banana-shapedbanana--shapedshaped ααα-helix--helix ofof γγCC isis coloredcolored inin lightlight green.green. RedRed andand whitewhite ballsballs areare thethe catalyticcatalytic residues,residues, whereaswhereasβ β-galactomannan--galactomannan (GM) (GM)(GM) is isis reported reportedreported as as sticks. sticks.

Intriguingly,Intriguingly,Intriguingly, a aa set setset ofofof predictionspredictionspredictions mademademade withwithwith thethe FRODOCK softwaresoftwaretware indicatedindicated thatthat residueresidue N131N131 mightmight lielie closeclose tototo thethethe activeactiveactive sitesitesite ofofof thethethe enzyme.enzyme.enzyme. TheThThe residueresidue isis part ofof thethe uniqueunique NN-glycosylation--glycosylation consensusconsensus motifmotif onon γγC.C. ThisThis suggestssuggests thatthat thethe glycosylglycosyl moietymoiety ofof γγCγCC maymay directlydirectly interactinteractinteract withwith thethe activeactive site of the enzy enzymeme also also thank thankss toto to itsits its flexibflexib flexibility.iilitylity.. ItIt It hashas has beenbeen been shownshown shown thatthat that thethe the glycosylicglycosylic glycosylic portionportion portion ofof ofγCγ containsC contains mannose mannose residues residues at at its its extremity extremity [23 [23],],], thusthus thus presentingpresenting presenting toto to thethe the enzymeenzyme enzyme aa structure structure thatthat resemblesresembles itsits naturalnatural substratesubstrate (Figure(Figure(Figure8 8).).).

FigureFigure 8.8. DockingDocking prediction of γγCC interaction with GH2 β--mannosidase-mannosidase using FRODOCK software software... One of the glycosylations described by Schiarea et al. [23] (Man2 (Fuc) GlcNAc2) was added to N131 One of thethe glycosylationsglycosylations describeddescribed byby SchiareaSchiarea etet al.al. [[2323]] (Man(Man22 (Fuc)(Fuc) GlcNAcGlcNAc22) was added to N131 withwith thethethe GlyProtGlyProt tooltool andand isis shownshown inin stickstick representation.representation.. γγCC isis tantan colored,colored, whereaswhereas ββ-mannosidase--mannosidase structuralstructural domainsdomainsdomains areareare coloredccolored as as reportedreportedreported in inin Figure Figure6 .6..

The glycosyl moiety of γC lieslies atat thethe surfacesurface of each monomer [[21]],, thusthus makinging intriguingintriguing a mechanism of inhibition involvinvolvinging thisthis post--translationaltranslational modification. modification. However, this hypothesis seems to be unlikely since tthe number of potential glycosylation sites inin proteinsproteins homologoushomologous toto γCγC isis highly variable.. IIn thethe EDGP sequence,, indeed,indeed, therethere areare fourfour glycosylationglycosylation consensus motifs;; inin NEC4,, six;; inin XEGIP,, fivefive;; and inin thethe TAXI--II proteinprotein,, onlyonly oneone.. Glycosylation seems to have very

Int. J. Mol. Sci. 2020, 21, 7305 9 of 15

The glycosyl moiety of γC lies at the surface of each monomer [21], thus making intriguing a mechanism of inhibition involving this post-translational modification. However, this hypothesis seems to be unlikely since the number of potential glycosylation sites in proteins homologous to γC is highly variable. In the EDGP sequence, indeed, there are four glycosylation consensus motifs; in NEC4, six; in XEGIP, five; and in the TAXI-I protein, only one. Glycosylation seems to have very limited effects on TAXI-I inhibitory activity [31]. However, we leave this hypothesis open and worthy of further experiments, considering that the structure of the target enzyme GH2 β-mannosidase is very different from that of the GH11 and GH12 endo-xylanases.

3. Materials and Methods

3.1. Reagents All reagents were obtained from Sigma-Aldrich (Milan, Italy), if not otherwise specified. β-mannosidase from Cellulomonas fimi and xyloglucan-specific endo-β-1,4-glucanase from Paenibacillus sp. were obtained from Megazyme (Bray, Wicklow, Ireland), with catalog numbers E-BMOSCF and E-XEGP, respectively.

3.2. γ-Conglutin Purification γC was purified from lupin seeds (Lupinus albus, cv. Multitalia) to homogeneity as previously described by Scirè et al. [27], lyophilized and stored at 4 ◦C in sealed vials. Before use, the protein was 1 dissolved to a concentration of about 3 mg mL− in the buffer necessary for subsequent experiments. The solution was then centrifuged for 5 min at 12,000 rpm and spectrophotometrically quantified at 280 nm according to [58].

3.3. Enzyme Activities GH2 β-mannosidase activity was measured in freshly prepared 100 mM sodium maleate buffer, 1 pH 6.0, containing BSA 1 mg mL− and 80 mM pNP-β-d-mannopyranoside (Megazyme, Bray, Wicklow, 1 Ireland), using 0.15 U of enzyme (specific activity: 13 U mg− ). The final volume was 3 mL. Samples were incubated at 35 ◦C for 15 min. The reaction was stopped using 0.5 mL of 5 M NaOH. The amounts of p-nitrophenol produced following enzyme activities were monitored spectrophotometrically at 410 nm. Control samples were set up by using distilled water instead of enzyme solution. Alternatively, 100 mM sodium acetate buffer, pH 4.8, was used. For inhibition assays, 0.15 U of β-mannosidase was preincubated with increasing amounts of γC in order to obtain different molar enzyme/γC ratios as indicated in the text, for 10 min at room temperature in 0.1 mL of incubation buffer. Then, the volume was adjusted to 3 mL with the same buffer containing the substrate as described above. When required, the sodium maleate incubation buffer was prepared by adding metals (ZnCl2, NiCl2 or CuCl2) at final concentrations of 1 mM. GH5 xyloglucan-specific endo-β-1,4-glucanase was assayed according to [29] in 100 mM sodium 1 acetate buffer (100 mM), pH 5.5, at 40 ◦C, using 2 U of enzyme (specific activity: 70 U mg− ) and 2 mg of beechwood xyloglucan. The final volume was 1 mL. The amount of reducing sugars produced following enzyme activity after 30 min of incubation was assessed using p-hydroxy-benzoic acid hydrazide according to [59]. In the inhibition assays, the enzyme was preincubated at room temperature with 150 µg of γC in order to obtain a molar enzyme/γC ratio of 1:1. Enzyme residual activities were calculated as (AE-AEI)/AE 100, where AE is the measured × enzyme activity (mmol/min) and AEI is the activity of the enzyme in the presence of γC.

3.4. ICP-MS Samples of lyophilized γC (50 mg) were dissolved in 10 mL of 65% nitric acid and digested in Teflon tubes using a microwave digestor (Anton Paar Multiwave-Eco, Rivoli, Torino, Italy). A power ramp was applied as follows: 200 W was reached over 10 min and maintained for 5 min; then, 650 W Int. J. Mol. Sci. 2020, 21, 7305 10 of 15 was reached over 10 min and maintained for 15 min. After a 20 min cooling time, the samples were diluted 1:40 with Milli-Q water, and the concentrations of Zn, Ni or Cu were measured by inductively coupled plasma (ICP) mass spectroscopy (Bruker AURORA M90 ICP-MS, Milan, Italy), according to [60].

3.5. Inhibition Data Analysis

Ki was estimated according to Cer et al. [53], using the web-server tool [61]. The input data were as follows. Substrate concentration: 800 µM; enzyme concentration: 11.8 µM; enzyme Km: 300 µM, according to [43]; inhibitor concentration: 11.7 µM; IC50: 0.99, calculated according to the equation of the best fit curve of the experimental data plotted as enzyme residual activity vs. the [γC]/[enzyme] ratio. The stoichiometry of γC/enzyme binding was determined according to [62], tracing the tangent line of the titration curve (residual activity vs. [γC]/[enzyme]) at the higher inhibition rate point. The x-intercept value of the tangent line indicates the inhibitor/enzyme stoichiometry.

3.6. Sequence Searches and In Silico Predictions The L. albus γC (UniprotKB: Q9FSH9I) and β-mannosidase (UniprotKB: Q9XCV4) sequences were retrieved from the UniProtKB/Swiss-Prot database (www.expasy.org). The PDB model structure of L. albus γC was created using the Swiss Model homology modelling pipeline [56], a tool available on-line via the ExPASy server at https://swissmodel.expasy.org. The UCSF Chimera software was used for molecular graphics, and surfaces were created with the MSMS package [63]. Docking analyses were performed with the L. albus γC model and β-mannosidase with the best resolution in the PDB repository, namely, the enzyme from T. harzianum ThMan2A (PDB: 4CVU) [55]. These elaborations were performed with different on-line available software: pyDockWEB [64], ClusPro 2.0 [65], PRISM 2.0 [66,67], GRAMM-X Protein-Protein Docking Web Server v.1.2.0 [68], HDOCK Server [69] and FRODOCK [70]. All the tools used compute protein–protein interaction using Fast Fourier Transform and rigid-body structural matching, followed by the refinement of the predicted complexes and global energy calculation. The docking of the substrate galactomannan (PubChem CID: 439336) into the enzyme’s active site was performed with AutoDock Vina, available from UCSF Chimera [71]. The Metal Ion-Binding site prediction and docking server (MIB) was used to analyze the potential Zn2+ binding sites of γC. This tool [72] takes advantage of the fragment transformation method for structural comparison between query proteins and templates, after the selection and the gathering of ion binding residues into the query 3D structure and those within 3.5 Å of the metal ion [73,74]. Glycosylation was added to the γC model using the Glyprot software [75], available as web-server tool [76], which correctly identified the glycosylation residue N131 in the γC sequence.

3.7. Statistical Analysis All determinations were carried out in triplicate. Data reported in the histograms are expressed as the means S.E. Data were analyzed by t-tests. p values < 0.05 were considered to be ± statistically significant.

4. Conclusions In this work, we first describe an inhibitory activity of L. albus γC, an effect sought for a long time in vain. Considering its homology with XEGIPs, attention has been previously focused on investigating its activity towards those enzymes targeted by XEGIPs themselves. However, switching the target towards enzymes potentially more relevant to legumes’ cell wall attacks, namely, enzymes acting on β-galactomannan degradation, enabled us to obtain first insights into possible γC involvement in cellular responses to pathogens, strengthening the possibility that γC is a multi-functional protein. Int. J. Mol. Sci. 2020, 21, 7305 11 of 15

From an evolutionary point of view, the production and accumulation in seeds of huge amounts of a particular protein that would be needed only if certain microbes attacked, is unfavorable. Normally, antimicrobial compounds are conditionally expressed [77]. Thus, a combination of storage and antimicrobial roles for γC is plausible, as described for some seed storage proteins [78] and considering that γC is one of the last seed storage proteins to be degraded during germination [79]. In silico predictions showed that the interaction of γC with GH2 β-mannosidase can occur in proximity to the active site and allowed us to propose that the potential inhibitory activity could be mediated by structures that are peculiar to γC, given the lack of XEGIPs’ IL1 and IL2 characteristics. These findings are consistent with the experimental activities since a stoichiometry of 1:1 can be hypothesized from the titration curve of GH2 β-mannosidase with γC. Interestingly, this inhibitory activity described is mediated by the presence of a metal ion. This finding raises new questions on the inhibitory mechanisms of GHIPs, in particular, those that, to date, have been identified as homologs but have failed to demonstrate inhibition, for example, soybean Bg7S. The role of Zn2+ in enhancing the inhibitory activity of γC deserves further research. By and large, this work describes experimental findings that highlight interesting new scenarios for understanding the natural role of γC. Although structural predictions can leave space for speculative interpretations, the full set of data reported in this work allows one to hypothesize possible and allows one to hypothesize possible mechanisms of action for the basis of inhibition. As a matter of fact, at least two mechanisms seem plausible, both involving elements that are peculiar to the lupin γC structure.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/19/ 7305/s1. Supplementary Figure S1: Surface localization of one of the putative Zn2+ binding site to conglutin gamma, Figure S2: Structure superposition of ThMan2A (cyan) with GH2 model (tan) created by Swiss model based on the amminoacid sequence of Cellulomonas fimi GH2 used in in vitro experiments (Panel A). Close up of the active site with catalytic glutammic acids residues highlighted in stick view (Panel B). RMSD of the two matched structures is 0.987 Å. Author Contributions: Conceptualization, S.D.B., E.G. and A.S.; data curation, S.D.B., E.G. and J.C.; formal analysis, A.S.; investigation, S.D.B., E.G., J.C., C.M. and A.S.; software, S.D.B.; supervision, A.S.; writing—original draft, S.D.B. and A.S.; writing—review and editing, E.G., J.C. and C.M. All authors have read and agreed to the published version of the manuscript. Funding: J.C. was supported by Università degli Studi di Milano (Assegno di Ricerca tipo A, 2018-RPDF-0048). S.D.B. is a research fellow funded by CARIPLO (EC 2018-0983). Acknowledgments: We thank Agroservice S.p.A. (S. Severino Marche, Italy) for kindly providing the L. albus seeds. We are in debt to Marcello Duranti for the helpful suggestions and critical comments bestowed during the experimental work. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

GH Glycoside hydrolase GHIPs Glycoside hydrolase inhibitor proteins GM β-galactomannan GMQE Global model quality estimation IL1 Inhibitory loop 1 IL2 Inhibitory loop 2 pNP-β-d-mannopyranoside para-nitrophenyl-β-d-mannopyranoside QMEAN Qualitative model energy analysis RMSD Root mean square deviation

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