key: cord-0258265-h4v3f7rb authors: Brola, TR; Dreon, MS; Qiu, JW; Heras, H title: A highly stable, nondigestible lectin from Pomacea diffusa unveils clade-related protection systems in apple snail eggs date: 2020-06-24 journal: bioRxiv DOI: 10.1101/2020.06.23.167262 sha: 55cc6c56eb47abb8e60b32e1aa90e1e3fb1dea9e doc_id: 258265 cord_uid: h4v3f7rb The acquisition of egg protection is vital for species survival. Poisonous eggs from Pomacea apple snails have defensive macromolecules for protection. Here we isolated and characterized a novel lectin called PdPV1 that is massively accumulated in the eggs of Pomacea diffusa and seems part of its protective cocktail. The native protein, an oligomer of ca. 256 kDa, has high structural stability, withstanding 15 min boiling and denaturing by sodium dodecyl sulphate. It resists in vitro proteinase digestion and displays structural stability between pH 2.0–12.0 and up to 85 °C. These properties, as well as its subunit sequences, glycosylation pattern, presence of carotenoids, size, and global shape resemble those of its orthologs from other Pomacea. Further, like members of the canaliculata clade, PdPV1 is recovered unchanged in faeces of mice ingesting it, supporting an antinutritive defensive function. PdPV1 also displays a strong hemagglutinating activity specifically recognizing selected ganglioside motifs with high affinity. This activity is only shared with PsSC, a perivitelline from the same clade (bridgesii clade). As a whole, these results indicate that species in the genus Pomacea have diversified their eggs defences: Those from the bridgesii clade are protected mostly by non-digestible lectins that lower the nutritional value of eggs, in contrast with protection by neurotoxins of other Pomacea clades, indicating apple snail egg defensive strategies are clade-specific. The harsh gastrointestinal environment of predators would have favoured their appearance, extending by convergent evolution the presence of plant-like highly stable lectins, a strategy not reported in other animals. Summary statement Analysis of key snail egg proteins shows evolutionary defensive trends associated with the phylogenetic position, extending by convergent evolution the presence of plant-like defensive strategies not reported in other animals Pomacea freshwater snails (Caenogastropoda, Ampullariidae), particularly those of invasive 57 species belonging to the canaliculata clade, lay colourful and poisonous egg masses on 58 emergent substrates above the water level, thus exposing eggs to environmental stressful agents 59 and terrestrial predators (Przeslawski, 2004; Hayes et al., 2015) . These defences pay off and as 60 a result their eggs have almost no reported predators except for the fire ant Solenopsis geminata 61 (Yusa, 2001) . We have found that these defences are provided by proteins (perivitellins) (Ituarte et al., 2012 (Ituarte et al., , 2018 , its orthologues from the invasive species P. canaliculata and P. 76 maculata (PcOvo and PmPV1, respectively) lack lectin activity. 77 Many lectins have been experimentally identified in molluscs, mostly in eggs or in organs 78 involved in the synthesis of egg components. Probably, the best-known gastropod egg lectin is 79 Helix pomatia agglutinin, which nowadays has important biomedical applications as a 80 histopathological biomarker of tumours displaying differential glycosylation patterns (Sanchez 81 et al., 2006) . In ampullariids, lectin activity has been reported in the eggs of Pila ovata 82 (Uhlenbruck, Steinhausen and Cheesman, 1973) and Pomacea urceus (Baldo and Uhlenbruck, 83 1974) and assumed to be part of the innate immune system against microbial invasions (Prokop, 84 Uhlenbruck and Kohler, 1968). However, the only ampullariid lectin isolated and functionally 85 characterized so far is the PV1 carotenoprotein PsSC from Pomacea scalaris eggs. This lectin 86 role immediately suggests an unexpectedly functional divergence within Pomacea egg 87 carotenoproteins (Ituarte et al., 2018) , but this hypothesis needs further comparative analysis. 88 In addition to PV1s, the conspicuous pink-reddish eggs of the invasive species have a 89 neurotoxic perivitelline lethal to mice (Heras et al., 2008; Giglio et al., 2016) that is absent in 90 the noninvasive species P. scalaris which has pale pinkish eggs. This prompted us to study 91 whether the different defensive roles of perivitellins against predation was associated with their 92 phylogeny (Hayes, Cowie and Thiengo, 2009) Uppsala, Sweden) as described by Ituarte et al (Ituarte et al., 2018) . Purification process of the 147 protein (hereafter named PdPV1) was checked by polyacrylamide gel electrophoresis (PAGE) 148 as described below. Protein content was determined using BSA as standard (Lowry et al., 1951) 149 in an Agilent 8453 UV/Vis diode array spectrophotometer (Agilent Technologies). PsSC was 150 prepared as described elsewhere (Ituarte et al., 2008) . System (Bio Rad Laboratories, Inc.). High molecular mass standards were run in the same gels. To assay denaturant conditions, 4-20% SDS-PAGE containing 0.1% SDS was carried out; 156 samples were denatured at 100 °C for 10 min with and without reducing agent (2-ME). Gels 157 were stained with Coomassie Brilliant Blue R-250 Pomacea diffusa PVF was also analysed by 158 native PAGE and protein bands were quantified by calibrated scanning densitometry using the 159 ImageJ software (Bourne and Bourne, 2010). Size and global shape 162 The molecular weight of PdPV1 was estimated by 4-20% Native-PAGE using high molecular 163 weight standard and by SEC as previously described in Ituarte et al. (Ituarte et al., 2008) , 164 calibrated with thyroglobulin, ferritin, catalase, aldolase and ovoalbumin as MW standards. Protein global shape was determined by Small Angle X-ray scattering (SAXS). Assays were 166 performed at the D02A-SAXS2 line, in the Laboratorio Nacional de Luz Sincrotron, Campinas 167 (SP, Brazil). The scattering pattern was detected using a MARCCD bidimensional charge-168 coupled device assisted by Fit2d software (Hammersley, 2016) . The experiments were 169 performed using a wavelength of 1.448 Å. The distance between the sample and the detector 170 was 1044 mm, allowing a Q-range between 0.012 and 0.25 Å -1 (Dmax =260 Å). Temperature 171 was controlled using a circulating water bath, and kept to 25 °C. Each individual run was 172 corrected for sample absorption, photon flux, buffer scattering, and detector homogeneity. At 173 least three independent curves were averaged for each single experiment, and buffer blank 174 scattering was subtracted. To rule out a concentration effect in the data, SAXS experiments 175 with a protein concentration range of 3.0-0.2 mg/mL were performed. Data was analysed using 176 the ATSAS package 2.6.0 (Petoukhov et al., 2012) . The low-resolution model of PdPV1 was 177 obtained from the algorithm built with DAMMIN and DAMMIF programs (Svergun, 1999) . The average of the best 10 models fitting the experimental data was obtained with DAMMIF. Data were analyzed by one-way analysis of variance (ANOVA). When p values were < 0.05, 344 the significance between groups was estimated by the Tukey's test. Structure 348 PdPV1 was purified and isolated by ultracentrifugation and HPLC. After NaBr gradient 349 ultracentrifugation of the egg soluble fraction, two protein fractions were obtained: a colourless 350 fraction of ~1.22 g/mL and a coloured fraction of ~1.24 g/mL (Fig 1A) . When the coloured 351 fraction was subjected to SEC, two peaks were observed (Fig 1B) . The larger one was a 352 carotenoprotein we named PdPV1, while the other is a PdPV1 aggregate, having both peaks 353 the same apoprotein composition in SDS PAGE (Fig 1B inset) . Native (non-denaturing) PAGE 354 of PVF proteins of P. diffusa eggs (Fig 1C) showed that PdPV1 is the most abundant at 382 nm ( Fig 1D) . However, unlike the other apple snail carotenoproteins, its intensity (and 362 hence the egg coloration) is very weak. Total carbohydrate content of PdPV1 represents 8.9 % 363 (by wt), which probably resulted in an inaccurate molecular weight estimation by native PAGE. The protein glycosylation pattern was determined by lectin dot-blot (Table 1) The internal sequences, together with three N-terminal aminoacid sequences (Table S1) Interestingly, PdPV1 higher affinity scores were against sialylated structures that featured a GalNAcb1-4Gal motif. PdPV1 affinity for these GalNAc-Gal would explain the presence of 414 aggregated PdPV1 lectin observed in the chromatographic analysis (Fig.1B) because these 415 glycans, which predominate in the glycan decoration of PdPV1 (Table 1) Table 2 . only at pH 2.0 the protein partially alters its conformation as seen by an intensity decrease in 427 the carotenoid absorbing ( Fig 3A) and a blue shift in the 4 th derivate of the spectra (Fig 3B) . 428 No changes in tryptophan fluorescence emission spectra were recorded (Fig 3C) , indicating 429 that the protein remains properly folded up to pH 2.0, where a slight red shift of the emission 430 maximum was observed. However, changes at pH 2.0 might be consider small changes in 431 protein structure because SAXS experiments showed that the particle shape remains globular 432 in the pH 2.0-12.0 range (Fig 3D) with no changes of its Rg except for a small reduction at pH 433 12.0 (Fig 3E) . Regarding thermal stability, no structural perturbations in the conditions assayed 434 could be detected in PdPV1 neither by absorption (Fig 4A and B ) nor fluorescence 435 spectroscopy (Fig 4C) , indicating protein stability up to 85 °C. These results are supported by 436 SAXS although at 85°C a partial loss of protein globularity occurs (Fig 4D) associated with a 437 slight Rg increase (Fig 4E) . Moreover, extreme thermal treatment of PdPV1 (boiled for 15 min) 438 did not affect its oligomer integrity as seen by native PAGE (Fig 4F inset) . electrophoretic mobility when incubated with SDS; they need to be heated to be disassembled 447 into their subunits. In this regard, PdPV1 did not changed it migration pattern after incubation 448 with SDS. Only when PdPV1 was boiled in the presence of SDS the oligomer disassembled 449 into its 6 subunits as evidenced by SDS PAGE (Fig 5B) . 450 The particle was also very resistant to enzymatic digestion, withstanding a simulated 451 gastrointestinal digestion for 2 h. The integrity of the protein after gastric and duodenal phases 452 was analysed by SDS-PAGE showing no significant alterations in its electrophoretic migration 453 (Fig 6A) . Furthermore, when the protein was administered to mice, the protein was recovered 454 almost intact in the faeces as shown by Native PAGE and WB analysis indicating that PdPV1 455 can pass through the digestive system without altering its structure (Fig 6B and C) . Subunit names in parenthesis correspond to the general nomenclature adopted for Ampullariid Group O(H)-Like Activities in Extracts from the Molluscs Pomacea paludosa and Pomacea 555 urceus', Vox Sanguinis ImageJ', Fundamentals of Digital Imaging in Medicine Biosynthesis in the Albumen Gland-Capsule Gland Complex 559 Limits Reproductive Effort in the Invasive Apple Snail Pomacea canaliculata Validation by qPCR of reference genes 562 for reproductive studies in the invasive apple snail Pomacea canaliculata Global shape and pH stability of ovorubin, an oligomeric protein 565 from the eggs of Pomacea canaliculata Novel Animal Defenses against Predation: A Snail Egg 568 Neurotoxin Combining Lectin and Pore-Forming Chains That Resembles Plant Defense and 569 Bacteria Attack Toxins Astaxanthin binding and structural stability 571 of the apple snail carotenoprotein ovorubin Metabolism of ovorubin, the major egg 574 lipoprotein from the apple snail The role of the proteinase inhibitor ovorubin 577 in apple snail eggs resembles plant embryo defense against predation Colorimetric Method for Determination of Sugars and Related 580 Substances The eggs of the apple snail Pomacea maculata are defended by 582 indigestible polysaccharides and toxic proteins FIT2D : a multi-purpose data reduction , analysis and 584 visualization program Insights from an integrated view of the biology of apple snails 587 (caenogastropoda: Ampullariidae) A global phylogeny of apple snails: 589 Gondwanan origin, generic relationships, and the influence of outgroup choice 590 (Caenogastropoda: Ampullariidae) First egg protein with a neurotoxic effect on mice Lectins Methods and Protools The importance of intrinsic disorder for protein 597 phosphorylation AmpuBase: A transcriptomic database of eight species of apple snails 599 (Gastropoda: Ampullariidae)', Gigascience Understanding the transition from water to land: Insights from 602 multi-omic analyses of the perivitelline fluid of apple snail eggs Egg perivitelline fluid proteome of a freshwater snail 605 (Caenogastropoda): insight into the transition from aquatic to terrestrial egg deposition Isolation and characterization of a novel perivitellin from the eggs of 608 Carbohydrates and glycoforms of the major egg perivitellins from 611 Agglutinating Activity and Structural Characterization of Scalarin, 615 the Major Egg Protein of the Snail Pomacea scalaris (d'Orbigny, 1832) A lectin of a non-invasive apple snail as an egg defense against 618 predation alters the rat gut morphophysiology Non-digestible proteins and protease inhibitors: Implications for 621 defense of the colored eggs of freshwater apple snails Role of glycosylation in nucleating protein 624 folding and stability MEGA X: Molecular evolutionary genetics analysis across 627 computing platforms Protein measurement with the Folin phenol reagent A first insight intocstress-induced neuroendocrine and immune 632 changes in the octopus Eledone cirrhosa Structural basis of protein kinetic stability: Resistance to 634 sodium dodecyl sulfate suggests a central role for rigidity and a bias toward β-sheet 635 structure Genetic exchange between two freshwater apple snails, Pomacea 637 canaliculata and Pomacea maculata invading East and Southeast Asia Gel-staining techniques Preparative ultracentrifugation and analytic 642 ultracentrifugation of plasma lipoproteins Convergent evolution of plant and animal embryo defences 645 by hyperstable non-digestible storage proteins The major egg reserve protein from 648 the invasive apple snail Pomacea maculata is a complex carotenoprotein New developments in the ATSAS program package for 652 small-angle scattering data analysis Lectins as plant defense proteins A New Source of Antibody-Like 657 Substances Having Anti-Blood Group Specificity: A Discussion on the Specificity of Helix 658 A review of the effects of environmental stress on embryonic 660 development within intertidal gastropod egg masses Biochemical and structural analysis of Helix pomatia agglutinin A hexameric lectin with a novel fold Precision mapping of the human O-GalNAc glycoproteome 665 through SimpleCell technology First proteome of the egg perivitelline fluid of a freshwater gastropod 668 with aerial oviposition Restoring low resolution structure of biological macromolecules from 671 solution scattering using simulated annealing An incomplete anti-B 674 agglutinin in the eggs of the prosobranch snail Pila ovata Predation on Eggs of the Apple Snail Pomacea Canaliculata Ampullariidae) By the Fire Ant Solenopsis Geminata Predation on the apple snail GalNAcb1-4(Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3)Galb1 GalNAcb1-4(Neu5Aca2-8Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3)Galb1-4Glcb-Sp0 Gala1-4Galb1-3GlcNAcb1-2Mana1-6(Gala1-4Galb1-3GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp19 Results are expressed as the mean value of relative fluorescence units (RFU) ± 1SD, N = 4 The complete glycan array data is A) Protein (orange circles) and hydration density (blue triangles) profile of P. diffusa egg 705 soluble fraction; (i) uncoloured and (ii) coloured fraction. B) Size exclusión chromatogram of 706 ii coloured fraction, (1) Agregate, (2) PdPV1. Inset: SDS-PAGE of 1 and 2 peaks showing the 707 same apoprotein composition. C) Native PAGE of the egg soluble fraction MWM: Molecular weight markers. D) Absorption spectrum of purified PdPV1. E) Ab-709 initio model of SAXS data of PdPV1 by DAMMIF (representative of best model cluster) Figure 2: Phylogenies of PV1perivitellins subunits versus morphological phylogenies of 712 A) Maximum-likelihood phylogram obtained with RAxML under the best partition model for 714 Values at nodes represent maximum-likelihood bootstrap 715 percentages under the best partition model using RAxML (BPRAxML) and IQ-TREE (BPIQ-716 TREE) and clade posterior probabilities under the best partition model using MrBayes 717 (PPMrBayes) and the CAT-GTR mixture model using PhyloBayes (PPPhyloBayes) 2009) indicating species, invasiveness and some of the perivitelline defenses present in 720 each. (C) Cross reactivity of PdPV1 with polyclonal antibodies anti-PsSC (left panel) Effect of pH on PdPV1 structural stability A) Absorption spectra. B) Fourth derivative absorption spectra. C) Intrinsic fluorescence 725 emission spectra and D) Kratky plots of PdPV1 at pH 2.0 (blue) and 12.0 (violet). E) Gyration 726 radii at different pH values Effect of temperature on PdPV1 structural stability A) Absorption spectra. B) Fourth derivative absorption spectra. C) Intrinsic fluorescence 730 emission spectra. D) Kratky plots obtained from SAXS data at 25 ºC (blue) and 85 ºC (violet) E) Gyration radii at different temperatures. F) Absorption spectra of unboiled (blue) and boiled 732 (violet) PdPV1, inset: Native 4-20% PAGE. Lane b: PdPV1 boiled for 15 min Structural stability of PdPV1 A) Chemical stability evaluated by the unfolding induced by GnHCl. B) Kinetic stability 737 evaluated with an SDS-resistance assay. The same PdPV1 sample was unheated (u) or boiled 738 (b) in the presence of SDS for 10 min immediately prior to be loaded into the gel. LMW and 739 HMW: low and high molecular weight markers Figure 6: In vitro and in vivo digestibility of PdPV1 C+, C-: positive and negative 744 controls of BSA digestion with and without pepsin, respectively. Intestinal phase. PdPV1 745 incubated with trypsin for 60 (60) and 120 (120) min; C+ and C-positive and negative controls 746 of BSA digestion with (C+) and without (C-) addition of trypsin Diagram of experimental protocol showing oral administration of PmPV1 and 748 faeces collection times. (ii) Analysis of faecal proteins by Native PAGE. MW: Molecular 749 weight markers Subunit sequences of PdPV1 Putative signal sequences are in italics 755 and underlined. Potential phosphorilation sites are in bold underlined, potential N-and O-glycosylation 756 sites are in bold and on black squares respectively. A conserved sequence is marked in bold red Multiple sequence alignment of PdPV1 subunits Figure 1: Purification and structure of PdPV1 A) Protein (orange circles) and hydration density (blue triangles) profile of P. diffusa egg 762 soluble fraction; (i) uncoloured and (ii) coloured fraction. B) Size exclusión chromatogram of 763 ii coloured fraction E) Ab-initio model of SAXS data of PdPV1 by DAMMIF (representative of best 767 model cluster) Figure 2: Phylogenies of PV1perivitellins subunits versus morphological phylogenies of 769 A) Maximum-likelihood phylogram obtained with RAxML under the best partition model for 772 Values at nodes represent maximum-likelihood bootstrap 773 percentages under the best partition model using RAxML (BPRAxML) and IQ-TREE (BPIQ-774 TREE) and clade posterior probabilities under the best partition model using MrBayes 775 (PPMrBayes) and the CAT-GTR mixture model using PhyloBayes (PPPhyloBayes) 2009) indicating species, invasiveness and some of the perivitelline defenses present in 778 each. (C) Cross reactivity of PdPV1 with polyclonal antibodies anti-PsSC (left panel) Effect of pH on PdPV1 structural stability A) Absorption spectra. B) Fourth derivative absorption spectra. C) Intrinsic fluorescence 785 emission spectra and D) Kratky plots of PdPV1 at pH 2.0 (blue) A) Absorption spectra. B) Fourth derivative absorption spectra. C) Intrinsic fluorescence 792 emission spectra. D) Kratky plots obtained from SAXS data at 25 ºC (blue) and 85 ºC 793 (violet). E) Gyration radii at different temperatures. F) Absorption spectra of unboiled (blue) 794 and boiled (violet) PdPV1, inset: Native 4-20% PAGE. Lane b: PdPV1 boiled for 15 min Structural stability of PdPV1 A) Chemical stability evaluated by the unfolding induced by GnHCl. B) Kinetic stability 800 evaluated with an SDS-resistance assay. The same PdPV1 sample was unheated (u) or boiled 801 (b) in the presence of SDS for 10 min immediately prior to be loaded into the gel. LMW and 802 HMW: low and high molecular weight markers Figure 6: In vitro and in vivo digestibility of PdPV1 A) Simulated gastrointestinal digestion analyzed by SDS-PAGE. Gastric phase: PdPV1 after 808 incubation with pepsin in SGF for 60 and 120 min (60 and 120): C+, C-: positive and 809 negative controls of BSA digestion with and without pepsin, respectively PdPV1 incubated with trypsin for 60 (60) and 120 (120) min; C+ and C-positive and 811 negative controls of BSA digestion with (C+) and without (C-) addition of trypsin. B) In vivo 812 digestibility of PmPV1. (i) Diagram of experimental protocol showing oral administration of 813 T4: control 815 and faeces collected after 2,3 and 4 h, respectively. C) Immune-detection by Western PdPV1 in faeces collected after 4h mice were gavaged with buffer (C) or with 0.6 mg PdPV1 Subunit sequences of PdPV1 A) Deduced aminoacid sequences of the six PdPV1 subunits. Putative signal sequences are in 822 italics and underlined. Potential phosphorilation sites are in bold underlined O-glycosylation sites are in bold and on black squares respectively. A conserved sequence is 824 marked in bold red. B) Multiple sequence alignment of PdPV1 subunits 545 No competing interests declared.