In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea
© Mann et al; licensee BioMed Central Ltd. 2012
Received: 19 January 2012
Accepted: 27 April 2012
Published: 27 April 2012
Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell.
Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes.
The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs.
Molluscan shells are extraordinarily stable biocomposites of calcium carbonate and an organic matrix consisting of polysaccharides and proteins. The organic matrix, although constituting a very minor fraction of the biocomposite by weight, is thought to be of utmost importance for the construction of the biocomposite and its final properties because it controls crystal nucleation, crystal growth, crystal shape and choice of calcium carbonate polymorph [1, 2]. Previously established methods to identify new mollusc shell matrix proteins, such as isolation by chromatography and biochemical characterization or molecular biology approaches, have been complemented recently by mass spectrometry-based proteomic analysis or combination of proteomic and transcriptomic studies [3–11]. However, proteomic approaches depend on the comparison of experimentally determined spectra with theoretical spectra obtained by in silico digestion of proteins and in silico fragmentation of resulting peptides [12, 13]. Therefore protein sequence databases that are as comprehensive as possible, usually derived from genome sequencing, are presently indispensable for high-throughput proteomics. The need for a comprehensive database is highlighted by previously published proteomic studies of shell matrices in various molluscan species [3–11]. These studies relied on translated EST databases contributed by a number of groups [7, 11, 14–18] and usually less than 15 proteins were identified from isolated organic matrices. Sometimes database searches were combined with de novo mass spectrometric sequencing. However, de novo sequencing algorithms, which attempt to interpret spectra independently of a sequence database , are not compatible with high-throughput analysis at present. Transcriptomics, on the other hand, does not identify matrix proteins directly, making additional techniques, such as immunohistochemical localization, necessary to demonstrate the actual location of potential shell matrix proteins. Thus, although previous studies have identified several very interesting new matrix proteins, these studies may fail to show the actual complexity of the shell matrix proteome indicated by proteomic studies of biomineral matrices of organisms with sequenced genomes, such as chicken  or the sea urchin Strongylocentrotus purpuratus[21–23].
The first genome sequence of a mollusc, the limpet Lottia gigantea, was made public recently (http://genome.jgi-psf.org/Lotgi1/Lotgi1.download.html) . In the present report we used a protein sequence database derived from this genome sequence to perform a high-throughput in-depth proteomic analysis of the shell matrix of this marine snail.
The shell of Lottia and related limpets consists of five layers [25, 26], which are divided into 3 outer layers, M + 1, M + 2 and M + 3 and separated from an inner layer M-1 by the intermediate myostracum (M layer). The outermost layer, M + 3, is reported to contain calcite as mineral phase. This layer appears eroded and often disappears altogether around the top of the shell. The M + 2 layer consists of flat prismatic crystals made of aragonite, another common calcium carbonate mineral. The M + 1 and M-1 layers are described to consist of lamellar prisms similarly made of aragonite. Compared to the other layers, the M layer, sandwiched between M + 1 and M-1, is very thin and has a prismatic structure of aragonite. Organic matrix was visible in M + 3 and M + 2, but was not detected in other layers .
Using LTQ Orbitrap Velos high-performance mass spectrometers  in combination with the MaxQuant software package designed for analysis of large high-resolution mass spectrometric data sets [28–30] we identified 311 proteins in the organic matrix of the Lottia shell with very high stringency. This is the first in-depth proteomic study of a mollusc shell matrix.
Materials and methods
The shells of freshly collected limpets were carefully cleaned manually and treated with sodium hypochlorite solution (Merck, Darmstadt; Germany; 6–14% active chlorine) to remove organic surface contaminants. Shells were either treated with hypochlorite for 2 h at room temperature (A), for 2 h with two 5 min ultrasonic treatments at the start of each hour (B), or for 24 h with two 5 min ultrasound bursts as before and one after 24 h (C). The shells were then washed with de-ionized water, dried, and crushed into small pieces using a hammer. The pieces were demineralized in 50% acetic acid (20 ml/g of shell) in a cold room overnight, yielding a dark brown suspension. Acid-soluble and acid-insoluble matrix was separated by centrifugation at 14000gav at 5°C for 1 h. The pellet was washed twice by re-suspension in approximately 20 volumes of 50% acetic acid, centrifugation for 30 min at 14000gav, and lyophilized. The supernatant was dialyzed twice against 10 volumes of 10% acetic acid followed by three times 10 volumes of 5% acetic acid at 4–6°C (Spectra/Por 6 dialysis membrane, molecular weight cut-off 2000; Spectrum Europe, Breda, The Netherlands), and lyophilized.
SDS-PAGE was done using pre-cast 4–12% Novex Bis-Tris gels in MES buffer with reagents and protocols supplied by the manufacturer (Invitrogen, Carlsbad, CA). Samples were suspended in 30 μl sample buffer/200 μg of organic matrix and heated to 95°C for 5 min. Sample buffer-insoluble matrix was removed by centrifugation in an Eppendorf bench top centrifuge for 5 min at 13000 rpm. Gels were loaded with 30 μl of matrix sample supernatant per lane and stained with colloidal Coomassie (Invitrogen) after electrophoresis. The protein standard used for molecular weight estimation was Novex Sharp, pre-stained (Invitrogen). Gels were sliced into 12 sections for in-gel digestion with trypsin . The eluted peptides were purified on C18 Stage Tips .
Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the EASY-nLC system (Proxeon Biosystems, Odense, Denmark; now Thermo Fisher) with 15 cm capillary columns of an internal diameter of 75 μm filled with 3 μm Reprosil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). The gradient consisted of 5–30% acetonitrile in 0.5% acetic acid at a flow rate of 250 nl/min for 85 min, 30–60% acetonitrile in 0.5% acetic acid at a flow rate of 250 nl/min and 60–80% acetonitrile in 0.5% acetic acid at a flow rate of 250 nl/min for 7 min. The eluate was electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) through a Proxeon nanoelectrospray ion source. The Orbitrap Velos was operated in a HCD top 10 mode essentially as described [Olsen et al., 2009] at a resolution of 30,000 for full scans and of 7,500 (both at m/z 400) for MS/MS scans.
Data analysis was performed with MaxQuant (v126.96.36.199) [28, 29], a computational proteomics platform based on the Andromeda search engine  (http://www.maxquant.org/), using the Lotgi1_GeneModels_Filtered Models1_aa.fasta.gz protein sequence database comprising 23,851 gene models at present (http://genome.jgi-psf.org/Lotgi1/Lotgi1.download.html) , together with the corresponding reversed database and the sequences of common contaminants, including human keratins from IPIhuman. Carbamidomethylation was set as fixed modification. Variable modifications were set as oxidation (M), N-acetyl (protein) and pyro-Glu/Gln (N-term). Initial peptide mass tolerance was set to 7 ppm and fragment mass tolerance was 20 ppm. Two missed cleavages were allowed and the minimal length required for peptide identification was seven amino acids. The peptide and protein false discovery rates (FDR) were both set to 0.01. The maximal posterior error probability (PEP) for peptides, which is the probability of each peptide to be a false hit considering identification score and peptide length [28, 29], was set to 0.01. The Re-quantify and Second Peptide  options were enabled. At least two MaxQuant group sequence-unique peptides with a score >100 were required for protein identification. Furthermore, identifications were only accepted if the peptides were identified in at least two replicates within the respective group A, B or C. Identifications with only two unique peptides were manually validated considering the assignment of major peaks, occurrence of uninterrupted y- or b-ion series of at least 4 consecutive amino acids, preferred cleavages N-terminal to proline bonds, the possible presence of a2/b2 ion pairs and immonium ions, and mass accuracy. The ProteinProspector MS-Product program (http://prospector.ucsf.edu/) was used to calculate the theoretical masses of fragments of identified peptides for manual validation. BLAST and FASTA searches against non-redundant databases (all organisms) were performed using the programs provided by NCBI (http://www.ncbi.nlm.nih.gov/blast) and EBI http://www.ebi.ac.uk/Tools/sss/. Domains were predicted with InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) and PROSITE (http://prosite.expasy.org/). For sequence alignments we employed Kalign (http://www.ebi.ac.uk/Tools/msa/kalign/) and ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Sequence repeats were predicted using RADAR (http://www.ebi.ac.uk/Tools/Radar/index.html). The abundance of proteins was estimated by calculating the exponentially modified protein abundance index (emPAI) . Observable peptides were determined and counted with Protein Prospector (http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi? form = msdigest) using zero miss-cleavages, a peptide mass of 700–2800, and a minimal peptide length of seven amino acids. Observed unique parent ions with a minimal length of seven amino acids and a mass between 700–2800 used for emPAI calculation included ions with up to two miss-cleavages, modifications specified for MaxQuant analysis (see above), different charges, and neutral losses . Proteins with emPAI ≥9 were referred to as major proteins in this report.
Results and discussion
Matrix isolation and characterization by SDS-PAGE
Proteomic analysis of matrix fractions
Altogether 569 proteins were identified in matrices obtained after different hypochlorite treatments. To obtain a representative, high-confidence, shell matrix proteome of Lottia gigantea, we assembled a consensus proteome comprising all database entries identified in all three types of samples (Figure 3). The consensus proteome of the acid-soluble fraction included 204 proteins and the consensus proteome of the acid-insoluble fraction contained 242 proteins. Given an overlap of 135, this summed up to a total of 311 Lottia database entries containing shell matrix protein sequences. However, these numbers should not be regarded as final because some database entries may eventually turn out to contain the sequence of more than one protein and some protein sequences may be divided among several database entries. Furthermore, the identifications not comprised in the consensus proteome are by no means to be considered as false positives but may be true shell matrix components. In most cases these were minor proteins and their absence or presence in different fractions may be due to experimental variability or the still limited dynamic range of mass spectrometers. Additional files 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 contain protein and peptide details, such as accession numbers of proteins sharing group-unique peptides, scores, masses, peptide sequences, and distribution in gel slices ( Additional files 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14). Unlike Additional file 1 and Additional file 2 ( Additional file 1 and Additional file 2), Additional files 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 contain data of all peptides and proteins identified within the set thresholds for MaxQuant searches (including identifications with one sequence-unique peptide), irrespective of whether they were accepted after manual inspection or not.
Both consensus proteomes contained intracellular proteins. In the soluble proteome these amounted to approximately 15% ( Additional file 1). The acid-insoluble fraction contained approximately 36% ( Additional file 2). Many of these proteins, such as the endoplasmatic reticulum and Golgi apparatus residents, may be by-products of secretion processes. Others may be releases into the extrapallial fluid by damaged or decaying cells of the epithelium lining the mantle cavity. Once in the extrapallial fluid, they have free access to the growing shell surface, may bind there, and may eventually be overgrown by further calcium carbonate deposition in shell growth periods. As true intra-crystalline components, although probably without any function, they may not be removed even by rigorous hypochlorite cleaning. Because the acid-insoluble consensus proteome contained more of these intracellular components, one may conclude that many of them were already structurally modified and aggregated before incorporation into the growing shell. Proteins of previously known intracellular location were also found in other invertebrate skeletal matrices analyzed in depth using similar proteomic technology [22–24]. However, it is rather unlikely that matrix components with a well-defined intracellular location have any function in the shell. However, specific functional shell matrix proteins may be found among the major matrix proteins and those with recognized or predicted extracellular location.
Uncharacterized Lottia matrix proteins with unusual amino acid composition and short sequence repeats
Previously uncharacterized major Lottia shell matrix proteins with unusual primary sequence features
14% P, 11% T, 6 repeats of ~30aa, starting with MITPE; pI: 4.7; 319aa
25% Q, 10% E, 17% P, 12% V, 10% N 10% L; 6 short repeats: k/qQQPxVELNKQQP; pI 5.2; 182aa
38% Q, 11% L, 10% P; 5 ~70aa repeats containing shorter repeat motifs like NQQQ and KQQQ; pI: 10.5; 322aa
20% G, 12% P; pI: 9.7; 137aa
11% P; Q-rich C-term (aa210–240); pI: 9.7; 258aa
26% E, 13% L,12% T; pI: 4; starting with aa156 8x SNLLQQPDa/tTQqLa/tTNeQQQ; (Figure 6 )
17% D, 16% A; EFh, pI: 3.8; 643aa; 12 ca30aa repeats similar to AxVDNxxMADMIDTxQDxxEDAADNMADNIDTAQDAQ between aa32–453
13% S; frequent doublets (SS, QQ, TT, YY, NN); G/E block aa322–337; pI: 4.4; 357aa
23% Q, 13% N, 13% S; aa130–702: 31 x 14aa repeats similar to QSNQQFNxxQSNQQF; pI: 7.1; 1184aa
~10% of P, N and G; in aa107–170 10x GAMP/GSMP; pI: 9.6; 563aa
19% P in aa50–400 and 35% P in aa778–882; pI: 9.5; 882aa
aa17–126: 17% R + K, 12% P, 11% L; pI: 11; 126aa
16% R, 11% S; pI: 11.7; 160aa; R/H-rich from aa103–150
19% G, 12% P; aa433–481: 27% M; pI: 4.6; 481aa; R/H-rich C-term half
aa26–230: 18% P; pI: 4.2; 230aa; acidic blocks in N-term half
A/P-rich motif aa150–170; H-rich motif aa171–185; pI: 8.8; 219aa
31% D, 10% E; pI: 3.6; similar to aspein?
42% Q in aa281–630; G/L/A-rich region aa631–928; pI: 9.2; 928aa
aa120–247: 20% P, 16% A, 10% Q; pI: 9.7; 247aa
15% P, 15% T; pI:5.7; 557aa
aa171–270: 33% G, 25% T, 15% P, 14% Q; 16 x GGQPs/tT; pI: 5.4; 303aa
24% P, 18% Q, 10% N; pI: 8.9; 729aa; aa57–376: 17 repeats of 16aa, similar to NNxa/vQPPxxQxxYQPt/p
19% P, 10% A, 10% V, 10% R; pI: 10; 317aa
21% Q, 18% P; aa268–356: 4 xAQPGAYQQP(x)2–4 GAYxQQP repeats; pI: 8.4; 440aa
22% P, 13% Q, 10% A; Q-rich regions: ~aa61–160 and ~ aa721–990; P-rich: ~aa280–600 and ~780–970¸pI: 8.8; 1035aa
aa61–232: 32% D + E, 12% N; pI: 3.7; 323aa; (Figure 4 )
13% A, 11% R, 11% L; K/R/A-rich C-terminus (aa185–219); pI. 10.3; 219aa
16% G, 12% M, 10% Q; G blocks in N-term half; pI: 9.9; 145aa
20% G, 18%M, 12%A, 10% L; pI: 11.2; 186aa; some similarity to shematrins
13% T, 12% S, 10% P; blocks of T from aa185–240; pI: 9.7; 609aa
22% G, 12% N; pI:9.5; 191aa; some similarity to GAAP_HALAI (Figure 5 )
19% P, 15% S; 12% G; 9 x g/dSQPGIYP and 4 x imperfect; pI: 4.5; 173aa
23% N, 15% P, 15%T, 11% S; 7 repeats similar to TPxxxNNVNPGSETPxTxNNVNPGSE and 2 incomplete; pI: 3.8; 234aa
Lottia matrix proteins with possible sequence homologs in other shells
Kunitz-type protease inhibitor KCP_HALAI
36–38% (1.6E–9 – 5.2E-6)
nacrein B2/B3/A1/B4; aa421–633 very acidic, with similarity to such proteins as aspein
(4.1E-6 – 3.3E-5)
Proteins with possible homologs in other shells
Dermatopontin, ependymin-like and gigasin-2-like proteins
A protein similar to the ependymin-related proteins recently discovered in Haliotis asinina shells  was found in Lotgi1|233583, a minor protein of the acid-insoluble consensus proteome ( Additional file 1 and Additional file 2). It was also similar to an unpublished Haliotis discus protein submitted to databases by Kang et al. (2006) under the name X-box binding protein with the accession number B6RB39 ( Additional file 15 Figure SA). The function of ependymin and related proteins is unknown at present.
Entry Lotgi1|235548 contained a protein sequence partially (~aa170-540) similar to the recently discovered Crassostrea gigas shell protein gigasin-2 (Cgigas-IMSP-2)  and the related proteins EGF-like domain containing protein-1 and −2 from Pinctada maxima [Jackson et al., 2009] ( Additional file 15 Figure SB). Lotgi1|235548 was a minor protein in both, acid-soluble and acid-insoluble, consensus proteomes ( Additional file 1 and Additional file 2).
One of the most important enzymes in biomineralization events is carbonic anhydrase, which catalyzes the formation of hydrogen carbonate from CO2 and water. The first carbonic anhydrase isolated from a mollusc shell and characterized at the molecular level was nacrein . This protein, which was isolated from the nacreous layer of Pinctada fucata shells, contained two carbonic anhydrase domains separated by a Gly-X-Asn repeat domain. The same protein was also identified in the prismatic layer . Since then nacrein-like proteins or nacrein-encoding genes have been identified in several other molluscs [4, 7, 10, 57, 58].
Other proteins with a possible or established link to biomineralization
Similar to calcineurin
Minor protein; possibly intracellular
Major protein in acid-soluble shell proteoime; possibly intracellular
Minor protein, 4 chitin-binding peritrophin A domains and 4–6 SRCR (scavenger receptor-related) domains
Major protein, secreted; 2–3 chitin-binding peritrophin A domains
Sequence contains predicted secretion signal sequence followed by two chitin-binding peritrophin A domains
Major protein in acid-soluble, minor in acid-insoluble consensus proteome; chitin-binding_3 domain
Major protein in acid soluble proteome; 10 chitin-binding perotrophin A domains organized in two blocks separated by four Pro-rich extensin-like motifs (aa470–600; 29% Pro, 16% Thr, 12% Gln, 12% Asn)
Major protein in acid-insoluble proteome; several SEA domains; chitin-binding peritrophin domain (aa2140–2200)with some similarity to chitinases
Major protein in acid soluble proteome; four chitin-binding peritrophin A domains preceded by a predictedsecretion signal sequence
Major protein in acid-insoluble proteome; predicted secretion signal sequence, VWA domain and Chitin-binding peritrophin A domain
Lysosomal; chitin degradation; major protein
Minor secreted protein
Extracellular matrix protein; minor
Overlapping fragments; extracellular matrix protein; major in acid-soluble matrix, minor in acid-insoluble matrix; Additional file 15: Figure SF
Proteins with CLECT, IGFBP and WAP domains
Compared to perlucin, the EGF- and insulin-binding protein perlustrin was a minor component of the Haliotis laevigata shell nacre matrix [50, 61]. However, its predicted homolog (Figure 9) Lotgi|174065 was one of the most abundant proteins in the Lottia matrix ( Additional file 1 and Additional file 2). A second perlustrin-like protein (Figure 9), Lotgi1|238970, was less abundant, but still a major protein. To our knowledge no perlustrin-like protein has been found in shells other than Haliotis laevigata and Lottia gigantea.
Pif- and BMSP-like proteins
Several identified Lottia proteins showed similarity to the recently described acidic Pinctada fucata nacre matrix protein Pif  and its Mytilus galloprovincialis homolog BMSP  (Table 2; Additional file 16 and Additional file 17). Pif is synthesized as a large precursor cleaved into two products, Pif97 and Pif80. Pif97 contains a von Willebrand type A (VWA) domain and a chitin-binding peritrophin A domain. Pif80, which does not contain any known domain, induces the formation of aragonite. Similarly, BMSP is cleaved into BMSP120, which contains four VWA domains and a chitin-binding domain, and BMSP100, the calcium carbonate-binding protein. The sequence of Pif80 and BMSP100 were described as completely different . A Pif-related protein was also identified in P. margaritifera.
Lotgi1|140660 and Lotgi1|173138 were highly abundant in the acid-insoluble matrix and moderately abundant in the acid-soluble matrix ( Additional file 1 and Additional file 2). The sequence of Lotgi1|140660 contained two predicted VWA domains, but no signal peptide. Lotgi1|173138 contained no VWA domain, no signal sequence, but a chitin-binding domain. As often observed with major proteins, the peptides were detected in all slices of the gel. However, there was an unequivocal tendency towards slices from the high molecular weight region (see, for instance, Additional file 13) indicating that both entries possibly represented cleavage products of a larger protein. Lotgi1|238526 was one of the most abundant proteins in the acid-insoluble Lottia shell proteome and a much less abundant, but still major, protein of the acid-soluble matrix ( Additional file 1 and Additional file 2). The sequence showed a low similarity to the aragonite-binding part of BMSP. The overall sequence identity was 21%, but in the C-terminal ~100 amino acid-long sequence it rose to 40% ( Additional file 16). Because these three entries occurred at the same abundance level and were more similar to BMSP than to Pif (Table 2), we believe that they belong together and may represent fragments of a possible Lottia BMSP homolog.
Lotgi1|228264 was part of both consensus proteomes but was much less abundant than the presumed BMSP fragments described before ( Additional file 1 and Additional file 2). This protein contained a signal sequence, a VWA domain, and a chitin-binding domain. The difference in abundance to the previously described fragments indicated that this protein was a possible Pif homolog rather than a possible BMSP homolog, although it was as similar to BMSP as to Pif in database searches. Lotgi1|232022 was a minor protein of the acid-insoluble consensus proteome and also occurred in fractions A and C of the acid-soluble matrix. It contained a predicted VWA domain and a chitin-binding domain, but no signal sequence ( Additional file 1 and Additional file 2). The sequence aligned to Pif in the same region as Lotgi1|228264 and may be a minor Pif-related protein of the shell matrix ( Additional file 17). Lotgi1|239574 was a major protein of both consensus proteomes. The sequence contained a secretion signal and a predicted chitin-binding domain. The chitin-binding domain was preceded by a Thr-rich motif (aa300–370; 59% Thr). This arrangement of chitin-binding domain and Thr-rich motif was very similar to Lotgi1|228264 and Lotgi1|232022 ( Additional file 17). Our results indicate that the Lottia shell matrix may contain at least three Pif-related proteins occurring at different abundances. We did not identify the aragonite-binding part of any of these possible Pif homologs. However, the sequence of this part of Pif does not contain a known domain structure and may be poorly conserved between species [Suzuki et al., 2009; 2011], probably rendering identification by database searches difficult.
Both Prosite and InterProScan predict a second chitin-binding domain immediately after the published chitin-binding domain of Mytilus galloprovincialis BMSP and Pinctada fucata Pif. This domain was also predicted in all of the Lottia BMSP- and Pif-related proteins described above. In contrast to the regular invertebrate chitin-binding domain with six cysteines there was a cysteine doublet intercalated between regular Cys3 and Cys4 of the normal pattern ( Additional file 16 and Additional file 17). This was reminiscent of cysteine patterns in plant chitin-binding domains, where a cysteine doublet is inserted between Cys2 and Cys3 [64, 65]. Therefore it is not clear whether these sequence motifs are really chitin-binding domains and consequently they were not considered in the respective figures ( Additional file 16 and Additional file 17).
Lotgi1|237510 was a major protein in the acid-soluble and a less abundant protein in the acid-insoluble consensus proteome ( Additional file 1 and Additional file 2). This protein showed similarity to the recently described chitin-binding protein P86860 of different Mytilus species  (Table 2) but part of it (aa1–100) was also predicted to be similar to Pif in database searches.
Lotgi1|166196 encoded a minor protein of the acid-insoluble consensus proteome that was predicted to contain a secretion signal sequence and a tyrosinase domain. Database searches indicated similarity of ~ aa1–400 of this protein to several molluscan tyrosinases previously shown to occur in shells [7, 52], or to be synthesized by mantle cells [17, 53] indicating the shell as destination ( Additional file 15 Figure SD). In addition the sequence was very similar to other molluscan tyrosinase database entries, the known localization of which are either not in shells or was not reported. The C-terminal half of Lotgi1|166196 contained nine repeats of the type GPPVNP (aa393–462). Tyrosinase was suggested to function in periostracum formation of Pinctada fucata. A second, unrelated, putative tyrosinase was found in Lotgi1|234481, but this protein was of low abundance, did not contain a secretion signal sequence, and was only identified in acid-insoluble fractions A and C.
Lotgi1|171918 contained a sequence with high similarity to the protease inhibitor antistasin. However, the sequence was also similar to aa660–950 of the Haliotis rufescens shell protein lustrin A . Two other entries, Lotgi1|231010 and Lotgi1|237013 matched to aa980–1420 of lustrin A in database searches. However, these matches were not convincing and were probably due to similarities in amino acid composition. Most importantly, the typical cysteine pattern of the lustrin A cysteine-rich repeats was not conserved in all of these Lottia sequences.
Lotgi1|132911 contained a fragment of a Kunitz-type protease inhibitor sequence similar to a recently published Haliotis asinina shell protein (Table 2) . Lotgi1|231009, one of the most abundant proteins in the acid-soluble shell matrix, showed some similarity to the Haliotis asinina protein UP2 (Uncharacterized Protein 2; Table 2; Additional file 15 Figure SE) .
Other proteins of possible interest in biomineralization
Lotgi1|230492 contained a sequence with 30% identity in a ~120aa overlap with Pinctada fucata calcineurin B  and a predicted secretion signal sequence. This protein was implicated in shell regeneration processes recently  and was a major component of the acid-soluble proteome ( Additional file 1).
Chitin is a major non-protein component of mollusc shells [67–69] and the inhibition of chitin synthase has dramatic effects on the structure of newly formed larval shell . This water-insoluble polysaccharide was suggested from structural studies to constitute a framework binding silk-like and acidic proteins . Apart from proteins similar to Pif or BMSP described above, we have retrieved several proteins with predicted chitin-binding domains but without significant similarity to known shell matrix proteins in database searches (Table 3). In addition we identified a few putative chitin-degrading enzymes that could play a role in shell construction or repair by modifying the chitin framework (Table 3).
In addition to nacrein-like carbonic anhydrases we identified two putative carbonic anhydrases without obvious similarity to nacrein in sequence similarity searches (Table 3). Lotgi1|205401 was a minor carbonic anhydrase with approximately 40% sequence identity to a Pinctada fucata enzyme recently submitted to databases by H. Miyamoto (E5RQ31_PINFU). Lotgi1|66515 contained another predicted carbonic anhydrase, which was a moderately abundant protein in the acid-soluble matrix proteome ( Additional file 1). The lack of a secretion signal sequence indicated an intracellular origin of this protein. Possible roles for these two carbonic anhydrases in the mineralization process remain unclear at present.
FAM20C, also known as dentin matrix protein 4, was first detected in mouse dentin matrix  and may play a regulatory role in osteogenesis and odontogenesis of the mouse. However, similar proteins have also been detected in invertebrates. The sequence in Lotgi1|156599 was 41% identical to the mouse sequence and more than 60% to an uncharacterized putative Daphnia pulex protein (E9GAB5_DAPPU). The regulatory properties of this protein in vertebrates may implicate this minor shell protein in Lottia shell production.
Osteonectin was first isolated from bone matrix  but was soon recognized to occur in many other tissues as well. Sequence comparisons established identity of osteonectin with the basement membrane protein BM-40  and a serum albumin-binding protein secreted by endothelial cells in culture, later called SPARC . Since then many functions have been proposed for this protein, including a regulatory role in some biomineralization events in mammals . Lottia osteonectin was a major protein in the acid-soluble shell matrix proteome and a minor one in the acid-insoluble fraction ( Additional file 1 and Additional file 2). Lotgi1|109908 contained the C-terminus of the protein, the N-terminus was identified in the first 135 amino acids of Lotgi1|176394 ( Additional file 15 Figure SF). Related proteins were reported from Haliotis discus and Pinctada fucata (unpublished, UniprotKB/TrEMBL accessions F2Z9K1_PINFU and F2Z9K2_HALDI, submitted by H. Miyamoto and F. Asada) and the sequences were included in the sequence alignment ( Additional file 15 Figure SF) together with the human sequence . A possible role in molluscan biomineralization is unknown at present.
The Lottia gigantea shell matrix turned out to contain a rather diverse set of proteins, comparable in complexity to the few other invertebrate shell matrix proteomes analyzed in-depth at present [21–23]. Among the 569 proteins identified by high-resolution mass spectrometry-based proteomics were at least 23 with a clear similarity to previously identified bivalve or gastropod shell matrix proteins. Others showed characteristics shared with previously known shell proteins, such as long stretches of acidic amino acids, of glycine, proline, or other amino acids. This made unequivocal recognition of homology difficult, if not impossible. However, such features as similar amino acid composition or preservation of domain structures may at least suggest functional equivalence. In addition we have identified many previously unknown proteins that may eventually turn out to play an important role as framework components or in regulation of matrix assembly and crystallization of the mineral. Despite the long list of identified proteins we do not expect to have identified all Lottia shell matrix proteins. Some may have been missed because of a lack of specific cleavage sites while others may not be represented adequately in the present draft of the database. Other known proteins may have been identified but were not recognized because of a low preservation of amino acid sequence. Nevertheless, we hope that this set of data, the most comprehensive list of mollusc shell matrix proteins available at present, may provide a starting point for the functional characterization of these proteins by researchers interested in biomineralization processes.
Blue Mussel Shell Protein
Insulin-like growth factor-binding protein
Exponentially modified protein abundance index
False discovery rate
Higher-energy collision-induced decomposition
Polyacrylamide gel electrophoresis
Posterior error probability
Von Willebrand type A
- WAP :
Whey acidic protein.
We thank Fred H. Wilt, Department of Molecular and Cell Biology, University of California, Berkeley, for drawing KM’s attention to the Lottia genome project and for bringing KM and EEG into contact.
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