The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, pollicipes pollicipes.

International Journal of Molecular Sciences Article The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes Dany Domínguez-Pérez 1,†, Daniela Almeida 1,†, Josef Wissing 2, AndréM. Machado 1, Lothar Jänsch 2, Luís Filipe Castro 1,3, Agostinho Antunes 1,3 , Vitor Vasconcelos 1,3 , Alexandre Campos 1and Isabel Cunha 1,* 1CIIMAR–Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua General Norton de Matos s/n, Terminal de Cruzeiros do Porto de Leixões, 4450-208 Matosinhos, Portugal; [email protected] (D.D.-P.); [email protected] (D.A.); [email protected] (A.M.M.); [email protected] (L.F.C.); [email protected] (A.A.); [email protected] (V.V.); [email protected] (A.C.) 2Cellular Proteomics Research, Helmholtz Centre for Infection Research, Inhoffenstraße. 7, 38124 Braunschweig, Germany; Josef.W[email protected] (J.W.); Lothar[email protected] (L.J.) 3Biology Department, Faculty of Sciences, University of Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal *Correspondence: [email protected]; Tel.: +351-22-340-1800; Fax: +351-22-339-0608 †These authors contributed equally to this work. Received: 19 February 2020; Accepted: 1 April 2020; Published: 5 April 2020   Abstract: Adhesive secretion has a fundamental role in barnacles’ survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia. Keywords: underwater adhesion; cement protein; Q-Exactive; proteogenomic; MaxQuant; iBAQ; protein expression Int. J. Mol. Sci. 2020,21, 2524; doi:10.3390/ijms21072524 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 2524 2 of 20 1. Introduction Sessile marine organisms are bioinspiring for the development of new biomimetic materials and mechanisms. Barnacles’ attachment is very plastic, as they live holdfast to a variety of substrates, under a variety of hydrologic regimes, in oceanic or coastal habitats, submersed or intermittently immersed in intertidal zones, in protected overhangs, crevices, in the deep sea, or directly exposed to strong waves [ 1 , 2 ]. Adhesive secretion has been evolutionarily optimized, since firm and permanent attachment to the substrate is crucial for goose barnacles’ survival, keeping them in an adequate position on the rocks to meet their oxygen and food requirements, grouped in turfs with hard plates facing outside, to protect their soft stalks from predators, and close enough to mate to conspecifics (Figure 1a) since their fecundation is internal [ 3 ]. The practical interest in understanding the adhesion of barnacles is related to industrial applications, including the development of antifouling strategies, induction on demand of larvae settlement and fixation for aquaculture, and mimicking the nature-inspired chemistry in new functional materials, such as underwater industrial glues, surgical and dental composites and glues, and biocompatible scaffolds and coatings, which function under humid or wet conditions [ 4 – 8 ]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 22 understanding the adhesion of barnacles is related to industrial applications, including the development of antifouling strategies, induction on demand of larvae settlement and fixation for aquaculture, and mimicking the nature-inspired chemistry in new functional materials, such as underwater industrial glues, surgical and dental composites and glues, and biocompatible scaffolds and coatings, which function under humid or wet conditions [4–8]. Figure 1. The goose barnacle Pollicipes pollicipes (Crustacea: Cirripedia) (a) attached to the rocky shore forming conspecific clusters alongside mussels; (b) schematic drawing showing sections of the peduncle, highlighting the cement apparatus, formed by clusters of adhesive-secreting cells that together constitute the gland (gl.), located in the upper central core embedded in an apical layer of connective tissue, just beneath the capitulum. Gland secretion passes through a network of ducts that carry the adhesive to the base of the peduncle forming the cement (Cem.). The original model of adult barnacle’s underwater adhesion [9] describes that the cement is secreted to the space between the animal cuticle and the external substrate, containing mainly two types of proteins, interfacial and bulk proteins. The formers are hydrophilic and located at the periphery of the cement complex, providing surface coupling with exterior surfaces through non-covalent interactions, and also interaction with interior hydrophobic bulk proteins. This model has been updated in the last years [10–12], including the notion that surface coupling relies on catechol groups, through various adhesive and cohesive forces, and that catechol, quinone, and lysine-based cross-linking are also involved in protein nanofibers’ curing and proper holdfast to the adhesion surface [10]. However, in barnacles, the catechol groups responsible for β-amyloid fibrils’ crosslinking of bulk proteins do not have a peptidyl origin, differently from other marine organisms, e.g., mussels and sandcastle worms; non-peptidyl catechol precursors and lysine are incorporated among bulk cement protein (CP52k and CP100k) fibrils through enzymatic reactions that involve peroxidases and lysyl oxidase [10], providing structure and mechanical characteristics to the composite. Six barnacle-specific cement proteins (CPs) have been identified, four of which are thought to be interface proteins, CP19k, −20 k, −43 k, and −68 k, and two bulk proteins, CP52k and CP100k, [13]. Barnacle-specific CPs are those proteins present in the cement that share no homology with any other marine adhesive proteins [13,14] or any other proteins at all. The cement apparatus of the goose barnacle Pollicipes pollicipes (Crustacea: Cirripedia) is found at the top of the peduncle core, formed of clusters of a single type of adhesive-secreting unicellular gland, mostly just below the mantle cavity, and a network of ducts that coalesce and carry the adhesive to the base of the peduncle (Figure 1b) [15]. In ripened individuals, some adhesive-secreting cells are intermingled with the ovary, but most of them are between the ovary and the capitulum. The presence of large nucleoli in the nucleus and the large amounts of Figure 1. The goose barnacle Pollicipes pollicipes (Crustacea: Cirripedia) ( a ) attached to the rocky shore forming conspecific clusters alongside mussels; ( b ) schematic drawing showing sections of the peduncle, highlighting the cement apparatus, formed by clusters of adhesive-secreting cells that together constitute the gland (gl.), located in the upper central core embedded in an apical layer of connective tissue, just beneath the capitulum. Gland secretion passes through a network of ducts that carry the adhesive to the base of the peduncle forming the cement (Cem.). The original model of adult barnacle’s underwater adhesion [ 9 ] describes that the cement is secreted to the space between the animal cuticle and the external substrate, containing mainly two types of proteins, interfacial and bulk proteins. The formers are hydrophilic and located at the periphery of the cement complex, providing surface coupling with exterior surfaces through non-covalent interactions, and also interaction with interior hydrophobic bulk proteins. This model has been updated in the last years [ 10 – 12 ], including the notion that surface coupling relies on catechol groups, through various adhesive and cohesive forces, and that catechol, quinone, and lysine-based cross-linking are also involved in protein nanofibers’ curing and proper holdfast to the adhesion surface [ 10 ]. However, in barnacles, the catechol groups responsible for β -amyloid fibrils’ cross-linking of bulk proteins do not have a peptidyl origin, differently from other marine organisms, e.g., mussels and sandcastle worms; non-peptidyl catechol precursors and lysine are incorporated among bulk cement protein (CP52k and CP100k) fibrils through enzymatic reactions that involve peroxidases and lysyl oxidase [ 10 ], providing structure and mechanical characteristics to the composite. Six barnacle-specific cement Int. J. Mol. Sci. 2020,21, 2524 3 of 20 proteins (CPs) have been identified, four of which are thought to be interface proteins, CP19k, − 20 k, − 43 k, and − 68 k, and two bulk proteins, CP52k and CP100k, [ 13 ]. Barnacle-specific CPs are those proteins present in the cement that share no homology with any other marine adhesive proteins [ 13 , 14 ] or any other proteins at all. The cement apparatus of the goose barnacle Pollicipes pollicipes (Crustacea: Cirripedia) is found at the top of the peduncle core, formed of clusters of a single type of adhesive-secreting unicellular gland, mostly just below the mantle cavity, and a network of ducts that coalesce and carry the adhesive to the base of the peduncle (Figure 1b) [ 15 ]. In ripened individuals, some adhesive-secreting cells are intermingled with the ovary, but most of them are between the ovary and the capitulum. The presence of large nucleoli in the nucleus and the large amounts of rough endoplasmic reticulum in the cytoplasm of these cells suggest an intense protein synthesis. The cytoplasm of the adhesive-secreting cells also features numerous small electron-dense secretory vesicles, which stain positively for proteins (tetrazonium) and polysaccharides (PASs) but not for the presence of lipids (Sudan black) in histological studies [ 15 ]. Contrarily, on barnacles’ cyprids, the adhesive is reported to be a bi-phasic system containing lipids and phosphoproteins, the two distinct phases contained in two different granule kinds, in the cyprid cement gland cells [ 16 ]. Additionally, post-translational modifications do not seem to play a role in adult barnacles’ adhesion, except for the glycosylation of MrCP52k [ 17 ], which is in line with the positive PAS dying observed of the electron-dense secretory vesicles in the gland cells’ cytoplasm. This study focused on the P. pollicipes adhesion system (Figure 1), a species that lives in the Easten Atlantic strongly exposed intertidal rocky shores and cliffs, from the south-west British Isles and France to Morocco [ 18 ]. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using label-free LC-MS high-throughput proteomic analysis combined with bioinformatics approaches. The proteogenomic analyses applied allowed the identification of known CPs both in the proteome and gland transcriptome, as well as a group of unknown proteins in the cement proteome. These unknown proteins lacked annotation or conserved domains and were detected as being highly expressed in the cement proteome, gland proteome, and gland transcriptome. In addition, the analysis of some physico-chemical features, such as the molecular weight, isoelectric point, hydrophobicity, amino acid relative composition, secondary structure composition, and disorder of proteins, allowed the conclusion that some of those unannotated proteins could be considered as a new group of surface-coupling cement proteins. We expect that the thorough quantitative description of the proteins found in these two samples, of a pedunculate instead of an acorn barnacle, will provide clues to support recent barnacles’ cement adhesion model updates and help to shed complimentary ideas to design the whole picture of adhesion in Cirripedia. 2. Results 2.1. Protein Identification The shotgun proteomics approach employed to profile the proteome of the cement gland and cement of the stalked barnacle Pollicipes pollicipes (Figure 1) allowed the identification of 11,755 peptide sequences in the gland (Table S1) and 2880 peptide sequences in the cement (Table S2). After filtering (contaminants and REV_ removal), a total of 5654 proteins clustered in 1616 proteinGroups remained in the gland proteome (Table S3), and 1568 proteins clustered in 489 proteinGroups in the cement (Table S4). Altogether, 6644 unique proteins were identified, with 778 proteins shared between the gland and cement (Figure 2a). Of all the proteins identified, 3404 and 588 were found in the three replicates of the gland samples and cement, accounting for 889 and 152 proteinGroups, respectively (Table S5 and Table S6). Among them, 3739 were unique proteins whilst 253 were found in all replicates analyzed (Figure 2b). The original MaxQuant output files containing all proteinGroups without filtering can be found in Table S7 for the gland and in Table S8 for the cement. Int. J. Mol. Sci. 2020,21, 2524 4 of 20 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 22 rough endoplasmic reticulum in the cytoplasm of these cells suggest an intense protein synthesis. The cytoplasm of the adhesive-secreting cells also features numerous small electron-dense secretory vesicles, which stain positively for proteins (tetrazonium) and polysaccharides (PASs) but not for the presence of lipids (Sudan black) in histological studies [15]. Contrarily, on barnacles’ cyprids, the adhesive is reported to be a bi-phasic system containing lipids and phosphoproteins, the two distinct phases contained in two different granule kinds, in the cyprid cement gland cells [16]. Additionally, post-translational modifications do not seem to play a role in adult barnacles’ adhesion, except for the glycosylation of MrCP52k [17], which is in line with the positive PAS dying observed of the electron-dense secretory vesicles in the gland cells’ cytoplasm. This study focused on the P. pollicipes adhesion system (Figure 1), a species that lives in the Easten Atlantic strongly exposed intertidal rocky shores and cliffs, from the south-west British Isles and France to Morocco [18]. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using label-free LC-MS high-throughput proteomic analysis combined with bioinformatics approaches. The proteogenomic analyses applied allowed the identification of known CPs both in the proteome and gland transcriptome, as well as a group of unknown proteins in the cement proteome. These unknown proteins lacked annotation or conserved domains and were detected as being highly expressed in the cement proteome, gland proteome, and gland transcriptome. In addition, the analysis of some physico-chemical features, such as the molecular weight, isoelectric point, hydrophobicity, amino acid relative composition, secondary structure composition, and disorder of proteins, allowed the conclusion that some of those unannotated proteins could be considered as a new group of surface-coupling cement proteins. We expect that the thorough quantitative description of the proteins found in these two samples, of a pedunculate instead of an acorn barnacle, will provide clues to support recent barnacles’ cement adhesion model updates and help to shed complimentary ideas to design the whole picture of adhesion in Cirripedia. 2. Results 2.1. Protein Identification The shotgun proteomics approach employed to profile the proteome of the cement gland and cement of the stalked barnacle Pollicipes pollicipes (Figure 1) allowed the identification of 11,755 peptide sequences in the gland (Table S1) and 2880 peptide sequences in the cement (Table S2). After filtering (contaminants and REV_ removal), a total of 5654 proteins clustered in 1616 proteinGroups remained in the gland proteome (Table S3), and 1568 proteins clustered in 489 proteinGroups in the cement (Table S4). Altogether, 6644 unique proteins were identified, with 778 proteins shared between the gland and cement (Figure 2a). Of all the proteins identified, 3404 and 588 were found in the three replicates of the gland samples and cement, accounting for 889 and 152 proteinGroups, respectively (Table S5 and Table S6). Among them, 3739 were unique proteins whilst 253 were found in all replicates analyzed (Figure 2b). The original MaxQuant output files containing all proteinGroups without filtering can be found in Table S7 for the gland and in Table S8 for the cement. (a) (b) Figure 2. Venn diagram of the proteins identified with MaxQuant freeware in the cement and gland proteome of the barnacle P. pollicipes. Unique and shared proteins between (a) all proteins identified in the cement (PPCIM) Figure 2. Venn diagram of the proteins identified with MaxQuant freeware in the cement and gland proteome of the barnacle P. pollicipes. Unique and shared proteins between ( a ) all proteins identified in the cement (PPCIM) and gland proteome (PPGL); and ( b ) all proteins identified in the three biological replicates of the cement (R3_CIM) and gland proteome (R3_GL). 2.2. Quantitative Proteomic Analyses Protein expression in the gland and cement was determined on absolute protein abundance using an intensity-based absolute quantification (iBAQ) score calculated by MaxQuant (Table S5 and Table S6, respectively). The most expressed proteins in the gland were associated with muscle and cytoskeleton motility (Figure 3a). The majority corresponded to actin, myosin, troponin, and tropomyosin, among other contractile and structural proteins (Figure 4a). In addition, proteins involved in “adhesion, extracellular matrix and membrane” corresponded approximately to 11% of the total expression (Figure 3a), with elastin being one of the most expressed within this functional group (Figure 4a,c). Interestingly, a group of highly expressed proteins accounting for approximately 2% of the total expression remained uncharacterized or unannotated (Figure 3a). In this sense, the relative expression of unannotated proteins was like that of “laminin” and “heat shock proteins (HSPs)”, and relatively higher than “ribosomal” proteins and “histones” (Figure 4a). Minor components related to the stress response, detoxification, immunity, protein biosynthesis, proteases, proteinase inhibitors, and chemical cues were also detected in the cement gland (Figure 3a, Figure 4a,c). The canonical barnacle’s cement proteins were not detected in the quantitative analyses of the cement gland at the proteomic level, except for CP100k (Figure 4a). On the contrary, the cement proteome was dominated by barnacle’s cement canonical proteins, unannotated proteins, chemical cues, and protease inhibitors (Figure 3b). Among the canonical proteins, the bulk proteins CP52k and CP100k were the most expressed, whilst among the surface-coupling CPs, only CP19k was detected at the proteomic level. Unannotated proteins were also found to be highly expressed, accounting for 23.77 % of the relative abundance (Figure 3b). Moreover, 7 proteinGroups classified as unannotated were listed among the 30 most expressed proteins (Figure 5). Noteworthy, some of those abundant unannotated proteins, such as “Ppollicipes_DN91829_c0_g1_i1.p1”, found in the cement proteome in high quantity (at position 12 in Figure 5), were also found among the 50 most expressed proteins in the gland proteome (Table S5). A new cement protein was identified (DN93583_C0_g1_i1.p2) by homology to the protein PP52k (AQA26375.1) of P. pollicipes [ 19 ] and automatically annotated in the proteome as CP52k like, the expression rate of which was also among the 30 most expressed proteins (at position 19 in Figure 5). According to the PCA analysis performed on the amino acid composition of this protein, it is not a bulk protein since it clusters with G1 proteins (Figure 6), being otherwise a surface couple protein. This protein corresponded to the transcript GGJN01121414.1 of the biosample SAMN08662077, bioproject PRJNA437397. The other cement proteins detected, CP100k, CP52k, and CP19k, were homologous to those previously found, ATB53757.1, ATB53756.1, and ATB53755.1, respectively [19]. In addition, other abundant proteins in the cement proteome were associated with “chemical cues” (Figure 3b). MULTIFUNCin and SIPC were the most represented (Figure 4b, Figure 5), accounting for 12.1% of the total intensity, 8.9% and 3.2%, respectively (Figure 3b). Besides, proteins involved Int. J. Mol. Sci. 2020,21, 2524 5 of 20 in “adhesion, matrix, and membrane” and “protease inhibitors” were also relatively abundant, with approximately 10% and 5% of the relative abundance, respectively (Figure 3b). Some enzymes, such as lysyl oxidase, beta-glucosidase, prophenoloxidase, and phenoloxidase, were also highly expressed (Figure 4b). Minor components found in the quantitative analyses were related to catalytic activity, “cuticle”, “immunity and defense”, “muscle and cytoskeleton motility”, and “protein biosynthesis and modification”, in this order (Figure 3b, Figure 4b,c). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 22 Figure 3. Global protein composition by functional groups in (a) the gland proteome (PPGL); and (b) cement proteome (PPCIM) of the barnacle Pollicipes pollicipes. The proportion of functional groups was based on the absolute protein abundance using the intensity-based absolute quantification (iBAQ) score calculated by MaxQuant. Only those proteins found in the three biological replicates (three valid values) of the two studied samples were selected. In total, 890 proteinGroups were used in the analysis of the gland (PPGL), and 152 in the cement (PPCIM). Figure 3. Global protein composition by functional groups in ( a ) the gland proteome (PPGL); and (b) cement proteome (PPCIM) of the barnacle Pollicipes pollicipes. The proportion of functional groups was based on the absolute protein abundance using the intensity-based absolute quantification (iBAQ) score calculated by MaxQuant. Only those proteins found in the three biological replicates (three valid values) of the two studied samples were selected. In total, 890 proteinGroups were used in the analysis of the gland (PPGL), and 152 in the cement (PPCIM). Int. J. Mol. Sci. 2020,21, 2524 6 of 20 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 22 Figure 4. Relative protein abundance within (a) the gland proteome (PPGL); and (b) cement proteome (PPCIM) of the barnacle Pollicipes pollicipes. Absolute protein abundance of broad protein families, shown as a single bar, was obtained using an intensity-based absolute quantification (iBAQ) score calculated by MaxQuant. The horizontal scales of bars differ between samples (PPGL and PPCIM), being the highest in the gland proteome. Major functional groups (c) corresponding to the protein families identified are represented by color bars on the right side. Figure 4. Relative protein abundance within ( a ) the gland proteome (PPGL); and ( b ) cement proteome (PPCIM) of the barnacle Pollicipes pollicipes. Absolute protein abundance of broad protein families, shown as a single bar, was obtained using an intensity-based absolute quantification (iBAQ) score calculated by MaxQuant. The horizontal scales of bars differ between samples (PPGL and PPCIM), being the highest in the gland proteome. Major functional groups ( c ) corresponding to the protein families identified are represented by color bars on the right side. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 22 Figure 5. The 30 most expressed proteins in the cement proteome (PPCIM) of the barnacle Pollicipes pollicipes, based on the absolute protein abundance. The expression of the 30 most expressed proteins (X-axis) is represented by the intensity-based absolute quantification (iBAQ) score (Y-axis) calculated by MaxQuant. Question marks indicate those highly expressed proteins with no homology (named as unannotated in X-axis) found in the cement proteome, their proteinGroup id, and the name of the corresponding leading protein. 2.1.1. Unannotated Proteins from the Cement Proteome Proteins without annotation, uncharacterized, or just predicted were found to be abundant in the cement proteome (Figure 3b, Figure 4b, Figure 5). Some of those proteins were also found in the gland at the proteomic (Figure 3a, Figure 4a, Table S5) and transcriptomic level (Table S9). In order to figure out the biological function of such proteins, some additional analyses were performed. A total of 32 proteinGroups were blasted against the non-redundant protein database (nr at NCBI) using automatic adjustment of the BLASTp program. Of these, a total of 17 proteinGroups remained unannotated, without any protein homology description or known conserved domains (Table S10). The results of these analyses were also included in the figures previously shown, and detailed information of the Blast search and protein sequences can be found in Table S10. Afterwards, unannotated and uncharacterized proteins were subjected to principal component analyses (PCA) to cluster with known cement adhesive proteins (Figure 6). PCA was employed to compare the relative residue composition (%) of 21 barnacle-specific cement proteins, obtained in the present study from the P. pollicipes cement proteome, to 38 previously identified, classified, and characterized cement proteins of various acorn barnacle species, gathered from NCBI and the literature, belonging to 8 different barnacle species (P. pollicipes, Amphibalanus amphitrite, A. improvisus, A. eburneus, Fistulobalanus albicostatum, Megabalanus rosa, M. volcano, and Tetraclita japonica). The analysis allowed observation of the clustering patterns of the unannotated proteins with the groups of proteins previously defined [19] (Figure 6). The two first principal components (PC1 and PC2) extracted by the PCA explained 46.4 % of the total data variation (27.54 % and 18.90 %, respectively), allowing observation of the proteins’ grouping as a function of the relative amino acid composition (Figure 6). PC1 discriminated G1 from the other two groups, while PC2 allowed for the separation of CP20k (G2) from the other two groups (G1 and G3). The three CPs from A. eburneus, CP36k, −22k, and −7k, identified by Naldrett and Kaplan [20], did not group with any of the three main CP groups and are putative new cement proteins, similar to the 11 unannotated cement proteins that did not group with G1, G2, or G3. Whether these cement proteins have an adhesive function or other function in the cement multicomplex is still to be determined. Figure 5. The 30 most expressed proteins in the cement proteome (PPCIM) of the barnacle Pollicipes pollicipes, based on the absolute protein abundance. The expression of the 30 most expressed proteins (X-axis) is represented by the intensity-based absolute quantification (iBAQ) score (Y-axis) calculated by MaxQuant. Question marks indicate those highly expressed proteins with no homology (named as unannotated in X-axis) found in the cement proteome, their proteinGroup id, and the name of the corresponding leading protein. Int. J. Mol. Sci. 2020,21, 2524 7 of 20 Unannotated Proteins from the Cement Proteome Proteins without annotation, uncharacterized, or just predicted were found to be abundant in the cement proteome (Figure 3b, Figure 4b, Figure 5). Some of those proteins were also found in the gland at the proteomic (Figure 3a, Figure 4a, Table S5) and transcriptomic level (Table S9). In order to figure out the biological function of such proteins, some additional analyses were performed. A total of 32 proteinGroups were blasted against the non-redundant protein database (nr at NCBI) using automatic adjustment of the BLASTp program. Of these, a total of 17 proteinGroups remained unannotated, without any protein homology description or known conserved domains (Table S10). The results of these analyses were also included in the figures previously shown, and detailed information of the Blast search and protein sequences can be found in Table S10. Afterwards, unannotated and uncharacterized proteins were subjected to principal component analyses (PCA) to cluster with known cement adhesive proteins (Figure 6). PCA was employed to compare the relative residue composition (%) of 21 barnacle-specific cement proteins, obtained in the present study from the P. pollicipes cement proteome, to 38 previously identified, classified, and characterized cement proteins of various acorn barnacle species, gathered from NCBI and the literature, belonging to 8 different barnacle species (P. pollicipes,Amphibalanus amphitrite,A. improvisus, A. eburneus,Fistulobalanus albicostatum,Megabalanus rosa,M. volcano, and Tetraclita japonica). The analysis allowed observation of the clustering patterns of the unannotated proteins with the groups of proteins previously defined [19] (Figure 6). The two first principal components (PC1 and PC2) extracted by the PCA explained 46.4 % of the total data variation (27.54 % and 18.90 %, respectively), allowing observation of the proteins’ grouping as a function of the relative amino acid composition (Figure 6). PC1 discriminated G1 from the other two groups, while PC2 allowed for the separation of CP20k (G2) from the other two groups (G1 and G3). The three CPs from A. eburneus, CP36k, − 22k, and − 7k, identified by Naldrett and Kaplan [ 20 ], did not group with any of the three main CP groups and are putative new cement proteins, similar to the 11 unannotated cement proteins that did not group with G1, G2, or G3. Whether these cement proteins have an adhesive function or other function in the cement multicomplex is still to be determined. The PCA situated nine of the unannotated proteins (PP95222, PP91829, PP97608, PP94236, PP93477, PP85975, PP85231, PP88577, and PP91778) in the proximity of G1 (surface-coupling proteins of CP19k, − 43k, − 58k, and − 68k families of cement proteins), but in none of the cases in the proximity of the two other groups, G2 (surface coupling proteins of CP20k family) or G3 (bulk proteins of the families CP52k and − 100k). Of the remaining 11 proteins, one of them, the most expressed of all (PP91716), was situated close to AE36k; 3 little expressed proteins (PP96002, PP93694, and PP91305) were located close to AE7k and AE22k; and the remaining 7 were found scattered among the three groups (G1, G2, and G3). One of the cement proteins picked from NCBI, AA52-3L, was misclassified according to the PCA analysis performed. Based on the relative amino acid composition of this A. amphitrite protein, PCA situates it in G1 (Figure 6), but according to the authors, it is a bulk protein, CP52k like [ 14 ]. In the case of being a bulk protein, it should group with G3 proteins instead of G1. The same situation was observed with the PP52k-L identified and annotated in the present work. These two proteins are smaller in length than CP52k proteins, and their physico-chemical properties also corroborate that they are surface-coupling proteins of G1 (Table 1, Table S11, Table S13). Regarding the other characteristics of cement proteins, these are presented in Table S11 and Table 1, with the former presenting the characteristics of the 33 previously characterized adhesive proteins of various barnacle species, whose sequences were available at NCBI, and the second, the characteristics of 22 proteins found in the P. pollicipes cement proteome that could not be annotated by homology nor a conserved domain found, and one new CP that was automatically annotated as being a CP52k-L. Among the latter, the 10 proteins that clustered with G1 surface-coupling proteins were found to be highly disordered (>65% disorder), presented a great percentage of its structure in the form of loops (>60%), and a very small percentage in the form of α -helices (<10%); more than 60% of their residues were exposed, less than 5 % were intermediate residues, and less than 35% were Int. J. Mol. Sci. 2020,21, 2524 8 of 20 buried; these characteristics together with the high protein disorder estimated, agreed with the G1 protein characteristics. Their isoelectric point, aromatic index, and the aliphatic percentage also fall in the range of those of G1 proteins. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 22 The PCA situated nine of the unannotated proteins (PP95222, PP91829, PP97608, PP94236, PP93477, PP85975, PP85231, PP88577, and PP91778) in the proximity of G1 (surface-coupling proteins of CP19k, −43k, −58k, and −68k families of cement proteins), but in none of the cases in the proximity of the two other groups, G2 (surface coupling proteins of CP20k family) or G3 (bulk proteins of the families CP52k and −100k). Of the remaining 11 proteins, one of them, the most expressed of all (PP91716), was situated close to AE36k; 3 little expressed proteins (PP96002, PP93694, and PP91305) were located close to AE7k and AE22k; and the remaining 7 were found scattered among the three groups (G1, G2, and G3). One of the cement proteins picked from NCBI, AA52-3L, was misclassified according to the PCA analysis performed. Based on the relative amino acid composition of this A. amphitrite protein, PCA situates it in G1 (Figure 6), but according to the authors, it is a bulk protein, CP52k like [14]. In the case of being a bulk protein, it should group with G3 proteins instead of G1. The same situation was observed with the PP52k-L identified and annotated in the present work. These two proteins are smaller in length than CP52k proteins, and their physico-chemical properties also corroborate that they are surface-coupling proteins of G1 (Table1, Table S11, Table S13). Regarding the other characteristics of cement proteins, these are presented in Table S11 and Table 1, with the former presenting the characteristics of the 33 previously characterized adhesive proteins of various barnacle species, whose sequences were available at NCBI, and the second, the characteristics of 22 proteins found in the P. pollicipes cement proteome that could not be annotated by homology nor a conserved domain found, and one new CP that was automatically annotated as being a CP52k-L. Among the latter, the 10 proteins that clustered with G1 surface-coupling proteins were found to be highly disordered (> 65% disorder), presented a great percentage of its structure in the form of loops (> 60%), and a very small percentage in the form of α-helices (< 10%); more than 60% of their residues were exposed, less than 5 % were intermediate residues, and less than 35% were buried; these characteristics together with the high protein disorder estimated, agreed with the G1 protein characteristics. Their isoelectric point, aromatic index, and the aliphatic percentage also fall in the range of those of G1 proteins. Figure 6. Principal component analysis based on the amino acids’ relative composition of proteins, considering previously characterized cement proteins and those proteins found during the present study, which was not possible to annotate through BLAST or conserved domains. The proteinGroups previously defined, corresponding to surface couple proteins CP19k, − 43k, − 58k, and − 68k (G1) and CP20k (G2), and bulk proteins CP52k and − 100k [ 19 ]. No unannotated proteins were found close to previously defined groups G2 and G3, but many were leaned to those of G1. The two principal components (PC) extracted explain 45.77% of the total variability of data. Blue – complete unannotated sequences; Red – incomplete unannotated sequences or incomplete cement proteins previously characterized; black – cement proteins (CPs) previously characterized. Int. J. Mol. Sci. 2020,21, 2524 9 of 20 Table 1. Characteristics of the 21 unannotated proteins identified in Pollicipes pollicipes cement proteome, and of a new CP protein annotated as 52k-like (DN93583_C0_g1_i1.p2), ranked according to the intensity-based absolute quantification (iBAQ) value. No. Res – number of residues; MM – molecular mass; pI – isoelectric point ; Neg. res. – negative residues (sum of Asp and Glu); Pos. res. - positive residues (sum of Arg, His and Lys);. In grey are proteins that have clustered with the adhesive proteins of group 1 (G1; surface-coupling proteins). iBAQ Rank Barnacle Specific Leading Cement Proteins No. Res MM(x1000) pI Instability Index a Hydropathy (GRAVY) b% Neg. res. % Pos. res. Aliphatic Index c Aromatic res. (%) Secondary Structure Composition (%) Solvent Accessibility (%) Protein Disorder (%) Loop α-Helix β-Sheet Exposed Interm Buried 3 DN91716_c1_g1_i1.p1 390 45.4 10.4 50.95 −0.142 6 47 88.69 17.5 53.13 25.38 19.49 26.15 7.44 66.41 0.51 4 DN95222_c0_g3_i1.p1 145 15.3 9.1 29.78 −0.768 19 22 64.48 0.7 63.45 10.34 26.21 72.41 3.45 24.14 100.0 5 DN86376_c0_g1_i7.p1 398 42.8 9.57 41.03 0.260 7 11 104.95 6.8 40.7 35.18 24.12 40.20 0.00 59.80 1.78 12 DN91829_c0_g1_i1.p1 269 25.3 4.1 49.53 0.111 28 7 79.93 2.2 92.19 5.58 2.23 64.68 1.86 33.56 69.58 19 DN93583_c0_g1_i1.p2 243 23.7 5.4 34.24 −0.293 11 9 72.3 0.4 91.8 6.1 2.1 68.7 2.1 29.2 91.77 20 DN96375_c1_g2_i4.p1 281 27.9 4.0 21.94 −0.67 51 14 62.17 0.8 77.94 6.05 16.01 54.09 2.85 43.03 45.20 26 DN97608_c0_g1_i4.p1 146 14.4 10.7 20.79 −0.738 9 21 52.95 2.1 92.84 3.42 2.74 71.92 2.74 25.34 100.0 28 DN94236_c1_g1_i5.p1 312 29.3 6.7 21.37 −0.013 21 20 69.01 2.5 97.76 0.00 2.24 90.38 0.32 9.29 93.59 36 DN94814_c0_g2_i2.p1 240 26.3 10.6 24.38 0.12 12 36 101.58 8.3 50.00 43.33 6.67 37.08 7.08 55.83 4.58 38 DN93477_c2_g1_i1.p1 353 37.4 8.5 37.24 −0.532 44 46 79.52 0.6 90.93 1.42 7.65 68.56 3.40 28.05 64.58 48 DN92408_c0_g3_i2.p1 392 43.5 11.5 25.19 −0.148 13 37 89.92 4.3 96.68 0.00 3.32 94.90 0.51 4.59 96.17 57 DN85975_c0_g1_i3.p2 203 19.8 4.8 43.26 −0.159 16 11 55.91 6.9 94.58 5.42 0.00 88.67 1.48 9.85 95.07 60 DN88255_c1_g1_i1.p1 265 30.0 11.3 49.76 −0.206 14 45 96.45 6.9 43.77 41.17 9.06 50.94 7.55 41.51 7.17 79 DN85231_c0_g1_i1.p1 246 23.7 12.4 25.83 0.049 3 20 77.03 1.6 96.34 0.00 3.66 67.48 1.22 31.30 87.80 87 DN88577_c1_g1_i6.p1 105 10.3 6.8 4.04 0.25 5 5 75.24 4.5 60.00 0.00 40.00 60.95 4.76 34.29 62.86 89 DN96067_c1_g1_i3.p1 183 20.2 10.8 19.96 0.138 8 28 106.94 7.6 34.97 19.13 45.9 43.72 8.74 47.54 2.73 95 DN96002_c6_g1_i8.p1 238 26.9 10.0 57.57 −0.786 23 37 57.77 7.6 59.24 12.61 26.15 59.24 4.62 36.13 51.68 102 DN91305_c0_g1_i7.p3 190 20.5 6.9 38.75 −0.453 21 21 69.68 4.2 46.84 14.21 38.95 55.79 5.79 38.42 26.32 103 DN91778_c0_g1_i1.p1 606 59.3 5.0 55.78 −0.332 49 32 67.79 0.9 99.05 0.00 0.50 96.20 0.00 3.80 97.95 106 DN91488_c0_g1_i1.p1 204 22.3 9.0 46.8 −0.23 17 22 68.63 7.4 39.71 53.92 6.37 52.94 2.45 44.61 29.90 108 DN88255_c1_g2_i2.p1 265 29.9 10.8 54.04 −0.186 18 42 101.62 5.7 46.79 37.36 15.85 41.51 5.66 52.83 7.55 141 DN93694_c2_g1_i2.p1 302 30.9 8.6 36.49 −0.364 20 24 70.13 4.0 3.31 96.69 0 56.95 4.97 38.08 84.44 a Instability index (II) - provides an estimate of the stability of the protein in a test tube, depending on the presence of certain dipeptides [ 21 ], the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable. b GRAVY - Grand Average of Hydropathy - The GRAVY value for a peptide or protein is calculated as the sum of hydropathy values [ 22 ] of all the amino acids, divided by the number of residues in the sequence. Values define relative hydrophobicity of amino acid residues, the more positive the value, the more hydrophobic in the amino acids located in that region of the protein. c Aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine). It may be regarded as a positive factor for the increase of thermostability of globular proteins [ 23 ]. Protein disorder – percentage of disordered regions as compared to the total protein sequence length predicted by Meta-Disorder [24]. Int. J. Mol. Sci. 2020,21, 2524 16 of 20 instability index, hydropathy, percentage of positive, negative and aromatic residues, and aliphatic index. Predictions on the secondary structure composition, solvent accessibility, and protein disorder were performed by PredictProtein [ 77 ] (https://www.predictprotein.org/) from protein sequences by Meta-Disorder [ 24 ]. It is a system of neural networks that combines several original prediction methods with the evolutionary profiles and sequence features that correlate with the protein disorder, such as the predicted solvent accessibility and protein flexibility (loops), which were used. Principal component analysis (PCA) was used to analyze the composition of the residue (%) of 21 barnacle-specific cement proteins (unannotated proteins) obtained in the present study, in comparison to 38 cement proteins of various acorn barnacle species (order Sessilia), and three proteins (CP100k, − 52k, and − 19k) of P. pollicipes (order Scalpelliform) deposited at NCBI and the literature. Only 20 amino acids were considered, for aspartic acid and asparagine were analyzed together, as well as glutamic acid and glutamine, since in some cases, CPs’ data delivered by the authors was in this form (one value for each of these two pairs of amino acids). Statistica (version 8.0, StatSoft, Inc.) was used to perform the PCA analyses. 5. Conclusions This work revealed for the first time the whole proteome of the adhesion system of the barnacle P. pollicipes. The gland proteome was found to be dominated by proteins involved in muscle, cytoskeleton, and some uncharacterized proteins, while minor components are involved in the stress response, detoxification, immunity, protein biosynthesis, protease inhibitors, and chemical cues. On the contrary, the cement is mainly composed of the barnacle’s adhesive proteins, unannotated proteins, enzymes, chemical cues, and protease inhibitors. The bulk proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Nonetheless, some of the most expressed proteins found lacked homology to any known protein. However, some of these unannotated proteins were found to be highly expressed both in the gland and the cement proteome, as well as at the transcriptomic level. Considering the lack of homology with known proteins, their amino acid composition, molecular weight, isoelectric point, and other chemical physical properties, we conclude that nine of them are surface-coupling adhesive proteins of G1, related to CP19k, as revealed by principal component analyses. Moreover, the low relative expression of known surface coupling indicates that those unannotated proteins could be playing a fundamental role in the bulk proteins–substrate interface. The other 12 unannotated proteins found in the cement may be adhesive or not, but their characteristics fall outside those of the adhesive protein groups characterized so far in the literature. More analyses must be performed in the future to validate the role of the unannotated proteins. The thorough quantitative description of the proteins analyzed, both in the gland and cement from the barnacle P. pollicipes, provides clues to understand the diversity of adhesive proteins and their function in cirripeds, serving as complementary information to update the barnacles’ cement adhesion model. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/7/2524/ s1. Author Contributions: Conceptualization, I.C. and A.C.; Data curation, D.D.-P., D.A. and A.M.M.; Formal analysis, D.D.-P., D.A. and I.C.; Funding acquisition, I.C.; Investigation, J.W., L.J., A.C. and I.C.; Methodology, A.C. and I.C.; Resources, L.F.C., A.A., V.V. and A.C.; Supervision, A.C. and I.C.; Writing—original draft, D.D.-P., D.A., A.M.M. and I.C.; Writing—review and editing, D.A., L.J., L.F.C., A.A. and V.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the project CY-Sensors (PTDC/BTA-GES/32359/2017) through Fundaç ã o para a Ci ê ncia e a Tecnologia (FCT), Portugal. The Transnational Access program of the European Marine Biology Resource Infrastructure Cluster (EMBRIC), EU project (ID: 654008), funded under H2020 - EU.1.4.1.1 “Developing new world-class research infrastructures”, provided support to produce the proteome and the mission costs at Cellular Proteomics Research, Helmholtz Centre for Infection Research, Braunschweig, Germany (Project No. 2757). It was also supported by CIIMAR which is financed by national funds from FCT/MCTES (UIDB/04423/2020 and UIDP/04423/2020). Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2020,21, 2524 17 of 20 References 1. Foster, B.A. Barnacle ecology and adaptation. In Barnacle Biology; Southward, A.J., Crisp, D.J., Eds.; AA Balkema: Rotterdam, The Netherlands, 1987; p. 443. ISBN 9061916283. 2. 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