Glassin, a histidine-rich protein from the siliceousskeletal system of the marine sponge Euplectella,directs silica polycondensationKatsuhiko Shimizua,1, Taro Amanob, Md. Rezaul Barib,2, James C. Weaverc, Jiro Arimab, and Nobuhiro Morib

aDivision of Regional Contribution and Lifelong Learning, Organization for Regional Industrial Academic Cooperation, Tottori University, Tottori 680-8550,Japan; bDepartment of Agricultural, Biological and Environmental Science, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan; and cWyssInstitute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 01238

Edited by Galen D. Stucky, University of California, Santa Barbara, CA, and approved July 17, 2015 (received for review April 9, 2015)

The hexactinellids are a diverse group of predominantly deep seasponges that synthesize elaborate fibrous skeletal systems ofamorphous hydrated silica. As a representative example, membersof the genus Euplectella have proved to be useful model systemsfor investigating structure–function relationships in these hierar-chically ordered siliceous network-like composites. Despite recentadvances in understanding the mechanistic origins of damage tol-erance in these complex skeletal systems, the details of their syn-thesis have remained largely unexplored. Here, we describe apreviously unidentified protein, named “glassin,” the main constit-uent in the water-soluble fraction of the demineralized skeletalelements of Euplectella. When combined with silicic acid solutions,glassin rapidly accelerates silica polycondensation over a pH rangeof 6–8. Glassin is characterized by high histidine content, and cDNAsequence analysis reveals that glassin shares no significant similar-ity with any other known proteins. The deduced amino acid se-quence reveals that glassin consists of two similar histidine-richdomains and a connecting domain. Each of the histidine-rich do-mains is composed of three segments: an amino-terminal histidineand aspartic acid-rich sequence, a proline-rich sequence in the mid-dle, and a histidine and threonine-rich sequence at the carboxylterminus. Histidine always forms HX or HHX repeats, in which mostof X positions are occupied by glycine, aspartic acid, or threonine.Recombinant glassin reproduces the silica precipitation activity ob-served in the native proteins. The highly modular composition ofglassin, composed of imidazole, acidic, and hydroxyl residues, favorssilica polycondensation and provides insights into the molecularmechanisms of skeletal formation in hexactinellid sponges.

fusion materials | Porifera | biomineral | silicon dioxide |organic–inorganic composite

The hexactinellids are a circumglobal group of predominantlydeep sea sponges. Exhibiting a diverse array of morphologies,

the skeletal systems of hexactinellids, which are composed ofamorphous hydrated silica (the other silica-forming sponge class isthe Demospongiae), range in structural complexity from looseaggregations of individual skeletal elements (spicules) to complex,hierarchically ordered lattices. Hexactinellids have evolved theability to colonize either rocky substrates or soft sediments andexhibit remarkable skeletal modifications to accomplish these feats(1). For example, the sediment-dwelling hexactinellids producebundles of long fibrillar anchor spicules that form robust holdfaststructures. These anchor spicules exhibit surprising flexibility anddamage tolerance and have served as useful model systems forinvestigating high performance silica-based organic-inorganic bi-ological composites (2–4). In one representative genus, Euplectella(Fig. 1), the constituent spicules are assembled into a highly regularcylindrical lattice that exhibits multiple levels of structural hierar-chy spanning from the nanoscale to the macroscale. The lattice iscomposed of two interpenetrating grid-like networks of nonplanarcross-like spicules that are reinforced with diagonal spicule bundlesand consolidated by a laminated silica cement. Each of the constituent

spicules contains a central axial filament of protein that directs thespicule’s core geometry, and the main load-bearing spicules exhibita laminated architecture consisting of concentric silica cylindersseparated by thin organic interlayers (5).Recent investigations with individual hexactinellid spicules have

demonstrated excellent mechanical performance (1, 2), exceedingthose of man-made glass rods with similar dimensions, and exhibitintriguing optical wave-guiding properties (6). Although the ultra-structural and mechanical properties of spicules from hexactinellidslike Euplectella have been well documented, characterization of themacromolecular components of the spicules and their roles in spiculeformation has largely remained unexplored. Identification and char-acterization of these occluded organic molecules is an initial step inunderstanding the molecular mechanisms of spicule formation, andfurthermore, the lessons learned from such studies could be appliedtoward the development of new synthesis routes to silica-basedcomposite materials under environmentally benign conditions (7).Previously, with Morse and colleagues, we demonstrated that

the organic axial filaments from the siliceous spicules of thedemosponge Tethya aurantia are composed of a group of closelyrelated proteins called “silicateins” (8). The silicateins share highsequence similarity with cathepsin L and other members of thepapain-like family of cysteine proteases and the larger super-family of catalytic triad-containing hydrolases. In vitro, silicateinspromote the hydrolysis and polycondensation of silicon alkoxidesto yield silica at neutral pH through a similar mechanism to that

Significance

Hexactinellid sponges of the genus Euplectella produce highlyordered and mechanically robust skeletal systems of amorphoushydrated silica. The high damage tolerance of their constituentskeletal elements and the environmentally benign conditions un-der which these sponges form have prompted additional in-vestigations into the characterization of the proteins driving thesynthesis of these materials. In the present report, we describe apreviously unidentified protein, named “glassin,” extracted fromthe demineralized skeletal elements of Euplectella. Glassin is ahistidine-, aspartic acid-, threonine-, and proline-rich protein anddirects silica polycondensation at neutral pH and room temperature.

Author contributions: K.S. and J.C.W. designed research; K.S., T.A., M.R.B., J.A., and N.M.performed research; K.S. and J.C.W. analyzed data; and K.S. and J.C.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the Euro-pean Molecular Biology Laboratory database, the GenBank database, and the DNA DataBank of Japan (accession nos. LC012024–LC012028, and LC010923).1To whom correspondence should be addressed. Email: kshimizu@cjrd.tottori-u.ac.jp.2Present address: School of Agriculture and Rural Development, Bangladesh Open Uni-versity, Gazipur 1705, Bangladesh.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1506968112/-/DCSupplemental.

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of proteases in the hydrolysis of peptide bonds and the moregeneral mechanism of the hydrolases (9–11). Because these ini-tial early discoveries, silicateins have been identified in numer-ous other demosponge species (12–18).More recently, silicatein-like proteins were also identified in the

hexactinellid sponges. Müller et al. (19–21) reported that anti-bodies against silicatein α from the demosponge Suberites domunculacross-reacted with a 27-kDa protein in the giant anchor spicules ofMonorhaphis chuni and with a 24-kDa protein in the spicules ofCrateromorpha meyeri. Mass spectrometric analysis of a fragmentfrom the M. chuni 27-kDa protein demonstrated that it containedsequences corresponding to the silicateins from demosponges (22).Silicatein sequences deduced from cDNA clones from M. chuni andC. meyeri contain the characteristic catalytic amino acid triad, serine-histidine-asparagine (19, 21). The silicatein gene was also identifiedfrom Aulosaccus schulzei, although here, a catalytic cysteine wasconfirmed instead of the usual serine (23). A partial silicatein cDNAfrom Euplectella aspergillum has also been archived [(GenBank ac-cession no. FR748156; Müller et al. (2011)], although a detaileddescription is not yet available.In addition to the silicateins, other organic molecules proposed

to be involved in spicule formation have been identified fromhexactinellid sponges. Travis et al. (24) reported that the demin-eralized skeletons of Euplectella sp. contained collagen, cellulose-like filaments, hexosamine-containing organics, and proteinscontaining relatively large amounts of aspartic acid, glutamicacid, glycine and histidine. Ehrlich et al. (25) isolated a fibrousmaterial from the anchoring spicules of the hexactinellid spongeHyalonema sieboldi, and demonstrated that the material wascomposed of collagen with an unusual glycine-(3-hydroxypro-line)-(4-hydroxyproline) motif, which in vitro, exhibited silicapolycondensation and templating activities. Ehrlich and Worch(26) also reported chitin in hexactinellid sponges includingFarrea occa and E. aspergillum as an organic component of theirsilicious skeletal systems.Inspired by these previous studies, we investigated the spicule-

associated proteins from the mineralized skeletal frameworkof Euplectella. From these analyses, we describe a previouslyunidentified silica-occluded protein, named “glassin,” as themain constituent in the water-soluble fraction extracted from

demineralized spicules, and through a series of in vitro assays,investigate its potential role in biogenic silica formation.

ResultsFollowing Euplectella skeletal dissolution with buffered hydro-fluoric acid, the resulting organic material was centrifuged forseparation into water-soluble and insoluble fractions. When 2 gof silica skeletal material (dry weight) was used as starting ma-terial, the water-soluble fraction yielded 0.2 mg of protein.When the water-soluble protein fraction was dissolved in 100 mM

sodium phosphate buffer at pH 6.0 and mixed with a freshly pre-pared metastable silicic acid solution, a silica precipitate could becollected by centrifugation, while even after 30 min, no silica wasformed in the protein-free control under otherwise identical ex-perimental conditions. The amount of precipitate formed was di-rectly proportional to protein concentration (Fig. 2A) with silicaprecipitation occurring at pH values ranging from 6.0 to 8.0, withthe maximum product yield occurring at pH 7.0 (Fig. 2B). Little orno precipitate was formed below pH 5.0 and heat denaturationof the protein solution did not affect the activity. No significantprecipitate formation was observed when tetraethoxysilane (whichrequires hydrolysis before condensation) was used as a silicaprecursor. This latter finding is significant, because tetraethox-ysilane and related conjugates of silicon and heavy metals arereadily hydrolyzed by silicatein to form the corresponding silica,silsesquioxanes, or metal oxides (9, 11).The silica precipitates formed by the water-soluble protein

fraction were collected via centrifugation and observed by scan-ning electron microscopy. The resulting silica product exhibited afine granular texture composed of silica nanoparticles measuring20–30 nm in diameter, a morphology typical of silica formedvia sol-gel processes (Fig. 2C). In addition, the process ofsilica polycondensation in the reaction mixture was followedusing transmission electron microscopy, which revealed that the

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Fig. 1. The hexactinellid sponge Euplectella. (A and B) Dried silica skeletonof E. aspergillum (A) and live specimen of E. curvistellata (B). The fibrousholdfast structure of the live specimen is missing, which is likely a result ofdamage incurred during collection. (Scale bars: 1 cm.)

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Fig. 2. Silica precipitation activity by the water-soluble fraction obtainedfrom the demineralized skeletons of Euplectella. (A) Silica yield increases as afunction of protein concentrations. (B) Effect of pH on silica precipitation. Inthe presence of the water-soluble fraction (solid squares), precipitates wereformed at pH values ranging from 6.0 to 8.0, with the maximum productionof silica at pH 7.0. Little or no precipitate formed below pH 5.0, and noprecipitate was observed at any pH in the absence of the water-solublefraction (open squares). Heat denaturation of the protein solution did notaffect the activity (a solid circle at pH 6.0). Each reported value is the averageof three independent experiments and in all cases, the SD was less than 10%of the mean. (C) Scanning electron micrograph of the precipitated silicawhich exhibits a distinctly granular substructure. (D and E) Transmissionelectron micrographs of silica in the reaction mixtures incubated for 1 min(D) and 5 min (E). Silica nanoparticles form network-like aggregates and thesize of the particles increases over time. (Scale bars in C–E: 50 nm.)

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silica nanoparticles formed network-like aggregates and thatthe size of the particles increased over time (Fig. 2 D and E).The proteins in the water-soluble fraction isolated from

E. aspergillum were analyzed by SDS-containing polyacrylamidegel electrophoresis (SDS/PAGE (Fig. 3A). After staining withCoomassie Brilliant Blue R250 (CBB), the main constituentprotein exhibited an apparent molecular mass of 23 kDa (Fig. 3A,lane 2), which we named “glassin.” Similar results were also obtainedwhen the water-soluble fraction prepared from E. curvistellata wasanalyzed (Fig. 3A, lane 3). In contrast, the SDS-extractable proteinyield from the insoluble fraction following silica demineralizationwas low and, as a result, was not further investigated as part of thepresent study.Protein posttranslational modification, specifically glycosylation,

was tested with peptide-N-glycosidase and O-glycosidase, whichremove N-linked and O-linked carbohydrate moieties linked toproteins at the amines of asparagine and hydroxyls of serine/thre-onine, respectively. After enzymatic treatment, the samples weresubjected to SDS/PAGE (Fig. 3B). The electrophoretic mobility ofglassin increased following treatment with O-glycosidase but notpeptide-N-glycosidase F, suggesting that glassin is O-glycosylated.Amino acid analysis of the water-soluble protein fraction from

demineralized Euplectella skeletons (which is primarily glassin) isshown in Table 1. For comparison, the calculated amino acidcomposition from the sequence of the mature peptide of T. aurantiasilicatein α (8) and glassin (as described below) are also included.The high compositional similarity between glassin and the watersoluble protein fraction extracted from demineralized Euplectellaskeletons combined with the relatively simple electrophoretic pro-file revealed from SDS/PAGE indicates that glassin is the dominantwater-soluble spicule-associated protein from Euplectella.The most striking compositional feature of glassin is its high

histidine content, at more than 30% of the total amino acids. Asconfirmed through site-directed mutagenesis studies (10), histidineis an essential active site amino acid in the silica-precipitating ac-tivity of silicatein α, in which the histidine content is only 1%.Proline and threonine are also abundant in glassin, at 13% and11%, respectively. Hydroxyls play important roles in the inductionof polycondensation by silicatein α (9, 10, 27), which contains 16%

hydroxy amino acids (which is similar to glassin). However, thre-onine is dominant in glassin (11% threonine and 5% serine),whereas serine is rich in silicatein α (12% serine and 4% threo-nine). Cysteine is not found in glassin, although cysteine formsdisulfide bonds that constrain the 3D structure of silicatein α andrepresents 3% of their total amino acid content. The content ofhydrophobic residues (alanine, valine, leucine, isoleucine, andphenylalanine) is also very low in glassin, in contrast to the highhydrophobic amino acid content in silicatein α. These resultsdemonstrate that the amino acid composition of the Euplectellaglassin is significantly different from that of silicatein α, suggestingthat the two proteins are not closely related.Partial amino acid sequences of glassin were analyzed with a

peptide sequencer and the amino-terminal peptide was identified asR(H or P)GHHGHH. A fragment generated by digestion withtrypsin had the sequence HDHHDHHHDHAPPXPPXVPP, whereX could not be identified. The sequence should follow a lysine orarginine (trypsin cleavage sites), so glassin should contain the peptide(K or R)HDHHDHHHDHAPPXPPXVPP. Both the amino-ter-minal and trypsin-derived peptide sequences of glassin from freshlycollected live specimens were identical to those of glassin derivedfrom commercially available dry skeletons. These sequences wereused to design degenerate oligonucleotide primers for amplificationof the corresponding cDNA by reverse transcription–PCR, startingwith RNA obtained from a live specimen. We obtained a 1338-bpcDNA clone, RT-PCR 1, encoding the 3′ region of the gene (Fig.S1). Then, rapid amplification of cDNA ends (RACE) was per-formed to obtain upstream sequence information. 5′-RACE pro-duced three clones, 5′-RACE 1/clones 1–3, all of which containedone potential amino-terminal sequence of glassin, RPGHHGHH;5′-RACE 1/clone 2 contained a stop codon in the coding region ofRT-PCR 1. A second round of 5′-RACE was performed with gene-specific primers using sequence information from 5′-RACE 1/clones1–3. The second round clone, 5′-RACE 2/clone 1, covered the otherpotential amino-terminal sequence, RHGHHGHH.When RT-PCRwas performed with primers encoding the region upstream ofRHGHHGHH and the region downstream of the stop codon ofRT-PCR 1, a product encoding the entire sequence could not beobtained. Instead, a clone, RT-PCR 2, was obtained by RT-PCRwith primers encoding upstream of RHGHHGHH and downstream

A B

Fig. 3. SDS/PAGE analysis of the Euplectella skeletal proteins. (A) The water-soluble fractions (1 μg) were subjected to SDS/PAGE, and the proteinswere visualized with CBB staining: lane 1, protein standards with molecularmasses indicated at the left margin; lane 2, the water-soluble fraction from askeleton of E. aspergillum; lane 3, the water-soluble fraction from a skeletonof E. curvistellata. An open triangle points to the dominant constituents ofthe water-soluble fractions. (B) The water-soluble fraction was incubatedwith peptide-N-glycosidase F or O-glycosidase and then analyzed by SDS/PAGE. Lane 1, protein molecular weight standards; lane 2, no glycosidase(control); lane 3, peptide-N-glycosidase; lane 4, O-glycosidase. The mobilityof glassin increased following incubation with O-glycosidase. Incubationwith peptide-N-glycosidase F did not significantly change the mobility ofglassin. The open triangle indicates the position of intact glassin and thefilled triangle indicates glassin after O-glycosidase treatment. Lettered ar-rows indicate peptide-N-glycosidase F (P) and O-glycosidase (O).

Table 1. Percentage of amino acid composition from theEuplectella skeleton soluble-protein fraction, Euplectella glassin,and T. aurantia silicatein α

Amino acid Soluble fraction, % Glassin, %* Silicatein α, %*

Aspartic acid 10.34 11.54 11.21Threonine 11.26 11.23 3.74Serine 4.81 3.74 12.15Glutamic acid 2.66 2.34 8.88Glycine 8.16 7.96 12.62Alanine 5.02 4.22 10.28Valine 3.88 2.34 7.011/2-Cystine 0.00 0.00 2.80Methionine 0.00 0.00 2.34Isoleucine 1.04 0.94 4.21Leucine 2.49 2.34 4.67Tyrosine 0.60 0.47 7.94Phenylalanine 0.55 0.94 2.34Lysine 4.48 3.74 3.74Histidine 30.17 35.56 0.93Arginine 1.05 0.94 2.80Proline 13.49 11.70 2.34Total 100.00 100.00 100.00

*Calculated from the amino acid sequences of Euplectella glassin (Fig. 4A)and T. aurantia silicatein α mature peptide (8).

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of the stop codon of 5′-RACE 1/clone 2. The sequence of clone RT-PCR 2 was consistent with that of 5′-RACE 1/clone 2.Fig. 4A shows the amino acid sequence of glassin deduced from

clone RT-PCR 2. Both of the two candidates for the N-terminalpeptide sequence, RHGHHGHH and RPGHHGHH, are con-tained in the deduced sequence, RHGHHGHH being locatedbefore RPGHHGHH and represents the N-terminal sequence ofthe mature glassin polypeptide. The sequence also containsKHDHHDHHHSHAPPSPPTVPP. The 10th amino acid wasidentified as aspartic acid by peptide sequencing, but as serine inthe sequence deduced from the gene. Unidentified amino acids inthe tryptic fragment are assigned as serine and threonine. Mis-identified or unidentified amino acids in peptide sequencing werelikely attributable to posttranslational modifications of serine andthreonine as suggested by the SDS/PAGE experiment showingan apparent molecular weight reduction following O-glycosidasetreatment of glassin (Fig. 3B).The sequence from the amino-terminal arginine to the stop co-

don contains 215 aa, and its molecular mass is calculated as 23 kDa,matching the apparent molecular mass of the protein observed bySDS/PAGE. The sequence is characterized by abundant histidine,reaching 35% of the total amino acids. Proline, threonine, andaspartic acid are also rich in the sequence (12%, 11%, and 10%,respectively), consistent with the amino acid analysis results.The histidines always form H(G/D/H/S/A/T/P/N) or HH(G/D/T)

repeats. The overall sequence consists of two histidine-rich re-petitive sequences (91 and 96 aa long, respectively) connected by a22-aa linker sequence. Alignment of the two repeated sequencesreveals that 90% of the corresponding amino acids are identical(Fig. 4B). Each repetitive sequence can be divided into threedomains: (i) a histidine and aspartic acid (HD)-rich domain,(ii) a histidine and threonine (HT)-rich domain, and (iii) a proline(P)-rich domain (Fig. 4C). In the HD-rich domain, a doublet HD

and a triplet HHD is often seen and HHD is sometimes replacedby HHG or HGK. In the HT-rich domain, HTHATVP is repeatedand followed by HTHATHT. The connecting sequence betweenthe two repeats consists of acidic and hydrophobic amino acidswith no regularity. Protein database analysis reveals that glassinshares no significant similarity with any known proteins. All ofthe above mentioned sequences have been deposited in theEuropean Molecular Biology Laboratory database, the GenBankdatabase, and the DNA Data Bank of Japan. Accession numbersare as follows: RT-PCR 1, accession no. LC012024; 5′-RACE1/clone 1, accession no. LC012025; 5′-RACE 1/clone 2, acces-sion no. LC012026; 5′-RACE 1/clone 3, accession no. LC012027;5′-RACE 2/clone 1, accession no. LC012028; and RT-PCR 2,accession no. LC010923.Recombinant glassin containing a His tag at the amino ter-

minus was expressed in Escherichia coli, and purified using aHisGraviTrap column from cells extracted with Fast Break CellLysis reagent (Fig. S2). Expression and purification of recombinantglassin was confirmed by SDS/PAGE and Western blot analyseswith an anti-glassin antibody (Fig. 5 A and B). Recombinant glassin(29 kDa as calculated from the deduced amino acid sequence)exhibited an apparent molecular mass of 35 kDa in SDS/PAGE andWestern blots. When recombinant glassin was added to a meta-stable solution of silicic acid, silica promptly precipitated, as wasseen with native glassin (Fig. 5C). These results demonstrate thatthe protein sequence of the recombinant glassin is sufficient toaccelerate silica precipitation and that glycosylation is not requiredfor the observed activity.

DiscussionIn the present study, we identified a water-soluble protein, glassin,extracted from the demineralized siliceous skeleton of Euplectella.Glassin is a protein characterized by alternating histidine andaspartic acid or threonine sequences, shares no significant se-quence similarity with any other known proteins, and acceleratessilica polycondensation from silicic acid at near neutral pH.Although glassin is the dominant constituent protein in the

water soluble fraction from Euplectella skeletal material, we alsoobtained water-insoluble materials, which were not characterizedas part of the present study due to their low yield of SDS-extractableproteins. Previously, the presence of collagen was suggested in thiswater-insoluble fraction based on amino acid analysis (24), whereaschitin was identified as the dominant nonprotein organic compo-nent (26). Further detailed characterization of this water-insolublefraction and its effect on the induction of silica polycondensation

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Fig. 4. Amino acid sequence of glassin. (A) The entire amino acid sequenceof glassin deduced from the DNA sequence of clone RT-PCR 2 (Fig. S1). Thesequence consists of 233 aa, abundant in histidine (cyan), aspartic acid(magenta), proline (tangerine), and threonine (green). The amino acid se-quences with lines below the letters that match information obtained fromdirect peptide sequencing of the native glassin. The solid lines indicate po-tential amino terminal sequences R(H or P)GHHGHH. The sequence indicatedby a dashed line corresponds to that obtained from tryptic peptides. (B) Re-peat sequences in glassin. The glassin sequence contains two repeat se-quences, amino acids 19–109 and 132–227. The sequences 19–65 of the firstrepeat and 132–183 of the second repeat, indicated by arrows marked HD-rich, are characterized by alternating histidine(s) and aspartic acid or lysine.The sequences 67–82 of the first repeat and 185–200 of the second repeat,indicated by arrows marked P-rich, are abundant in proline. The sequences83–109 of the first repeat and 201–227 of the second repeat, indicated byarrows marked HT-rich, are characterized by repeats of histidine and thre-onine. Identical amino acids between the first and the second repeats areshaded. (C) Schematic of the domain structure of glassin. HD-rich, P-rich, andHT-rich domains are represented by blue, yellow, and green boxes. The openbox represents the domain connecting the first and second repeats.

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Fig. 5. Production of recombinant glassin and its silica precipitating activity.(A) SDS/PAGE analysis of recombinant proteins. Each lane contained 10 μL ofsamples as follows: lane 1, bacterial lysate; lane 2, proteins that did not bind tothe HisGraviTrap column; lane 3, the first fraction of proteins eluted from thecolumn with 500 mM imidazole; lane 4, the second eluted fraction; and lane 5,the third eluted fraction. (B) Western blot analysis with a rabbit anti-glassinantibody. The contents of samples were the same as in A. (C) Silica precipitationin the presence of recombinant glassin. Silica precipitates were obtained bymixing metastable silicic acid with purified recombinant glassin [R, the secondfraction (lane 4) in A and B] or the native water-soluble fraction from demin-eralized skeletal material from Euplectella (N). The experiments were performedat pH 6.0, and product yields were determined by the molybdate blue assay.

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when combined with glassin may provide new insights into theroles of these specific components in hexactinellid sponge spiculeformation.Glassin is quite different from silicatein α, which we and our

colleagues previously identified from the siliceous spicules of thedemosponge, T. aurantia (8, 9). The silicateins are assembledinto a linear fiber (28, 29) that is found within the core ofdemosponge spicules and catalyze silica formation via a hydro-lysis pathway from organically functionalized silicic acid pre-cursors at neutral pH (8, 9, 11). In addition to their discovery inseveral other demosponge species (12–18), silicateins or silica-tein-like proteins have also been reported from hexactinellidsponges such as M. chuni and C. meyeri (19–21). If silicateinexists in Euplectella, as is suggested based on partial cDNA data[GenBank accession no. FR748156; Müller et al. (2011)], thenthe two proteins (glassin and silicatein) may either act co-operatively during silica biosynthesis in hexactinellids or mayplay distinct functional roles. For example, silicatein may be in-volved in establishing spicule symmetry and primary minerali-zation and glassin may be associated with secondary spiculecementation and subsequent skeletal consolidation.It is noteworthy that the band of native silica-purified glassin

is broad and diffusive in SDS/PAGE, whereas the recombinantglassin exhibits a very well defined band. This could be explained bythe fact that the silica-purified glassin may be a mixture of closelyrelated polypeptides, an idea supported by the observation thatglycine and proline can be substituted in the native glassin at po-sition 2. Silafins, the group of polypeptides directing silica for-mation in diatoms, are also a mixture of related polypeptides,producing a similarly broad band pattern in SDS/PAGE (30).The high histidine content in a protein involved in biogenic silica

formation is not surprising considering that artificial histidine-richpeptides have often been used as catalysts for silica polyconden-sation. For example, 20-kDa histidine homopolypeptides and evenmuch smaller decahistidine homopolypeptides possess the ability toform silica at near neutral pH from either organically substitutedsilanes or solutions of metastable silicic acid (31, 32). In addition,site-directed mutagenesis showed that the active site histidine andserine in silicatein α play essential roles in the in vitro hydrolysisand polycondensation of silicon alkoxides (10), and subsequentstudies confirmed that the coexistence of hydroxyl or acidic aminoacid residues with histidine residues increases their catalytic activityin silica polycondensation (32). Phage display selection of silica-precipitating peptide sequences independently confirmed that thepresence of hydroxyl- and imidazole-containing amino acids werecritical for the induction of silica polycondensation (33). Kunoet al. (32) recently demonstrated that a diblock copolypeptide ofhistidine and aspartic acid (H5D5) and an alternating polypeptideof histidine and aspartic acid ((HD)5) were more effective in hy-drolysis of trimethylethoxysilane than histidine homopolypeptide,and that (HD)5 was more effective than H5D5 because of a “chargerelay effect.” Accordingly, alternating histidine and aspartic acid/threonine motifs in glassin may play a similar functional role in itsobserved activity.The induction of silica polycondensation from silicic acid is not a

simple process because supersaturated silicic acid solution containsvarious silicic acid species and particles of different sizes, each ofwhich behaves differently (34). As a result, the precise mechanismof action of glassin has yet to be fully elucidated. Perry and co-workers proposed that the imidazole group may accelerate silicicacid condensation from this medium by forming hydrogen bondswith silicic acid, increasing silica precipitation activity throughelectrostatic interaction with oligomeric (poly)silicic acids (35).Hydrogen bond formation and electrostatic interaction betweenimidazole groups in glassin and specific silicic acid species may bemodified by adjacent acidic (aspartic acid) or hydroxyl (threonine)residues, as predicted through computational simulation (36).

The glassin protein exhibits a distinctive structural hierarchy.First, histidine residues always form doublets (HX) or triplets(HHX) with amino acids including aspartic acid, glycine, andthreonine. Second, these doublets and triplets form two distincthistidine-rich domains, the HD and HT domains, which areseparated by the P-domain. Third, the series of HD, P, and HTdomains forms a tandem repeat in the overall glassin molecule.Previous studies of other biosilica associated proteins like the

silicateins and silafins (an unrelated group of proteins shown byKroger and colleagues to mediate silica formation from silicic acid indiatoms) have shown that they require strict 3D structures or com-plex posttranslational modifications for their observed activities (9,30). Glassin, in contrast, is thermally stable (which may be related toits largely random coil architecture revealed by Chou–Fasman sec-ondary structure prediction) and does not require posttranslationalmodification for the induction of silica polycondensation.In conclusion, we have identified a protein, glassin, with re-

markably high histidine content in the siliceous skeleton ofEuplectella, and have demonstrated its capacity to direct silicaprecipitation from metastable silicic acid solutions at neutral pH.Future in vivo studies investigating the localization and expressionprofiles of glassin in living sponges may ultimately provide criticalinsight into the processes of spicule synthesis and consolidation inEuplectella, and the development of environmentally benign bio-mimetic synthesis routes to silica and other inorganic materials.

Materials and MethodsSamples. Dried skeletons of the hexactinellid sponge E. aspergillum were ofPhilippine origin and acquired from commercial sources (Fig. 1A). The topand bottom thirds of each skeleton were removed, so that the specimensconsisted entirely of skeletal tubes, but not the sieve plate or holdfast ap-paratus. Live specimens of Euplectella curvistellata (37) were collected bybeam trawling at a depth of 236 m at 32°30 N, 129°10 E in the East China Seaon March 4, 2012, during an expedition (Expedition 343) on the researchvessel Nagasaki Maru of Nagasaki University (Fig. 1B).

Protein Extraction. Dried skeletal material was soaked in a 5% (vol/vol) solution ofsodium hypochlorite overnight to remove any residual external organic matter,rinsed with Milli-Q (Merck Millipore) purified water five times, and air dried atroom temperature. The resulting skeletal material was treated with 2 M HF/8 MNH4F to dissolve the silica and the remaining solution was dialyzed against 10volumes of Milli-Q water at 4 °C for more than 4 h per dialysis, and the outersolution was changed seven times. The dialysate was centrifuged at 10,000 × g for20 min at 4 °C to separate the supernatant from the insoluble material and thesupernatant was concentrated with Amicon Ultra-15 Ultracel (nominal molecularweight limit, Mr 3,000) Centrifugal Filter Units (Merck Millipore).

Protein Analysis. Protein concentration was determined using a DC protein assaykit (Bio-Rad), with γ-globulin as the standard, according to the manufacturer’sinstructions. SDS/PAGE was performed on the soluble protein fraction fromdemineralized skeletons using NOVEX 10% Bis-Tris gels in the NuPAGE system(Thermo Fisher Scientific) with Mes buffer. Samples were dissolved in 1 × LDSsample buffer (Thermo Fisher Scientific) containing 1%DTT. After electrophoresis,the gels were stained with CBB (EzStain Aqua; ATTO) for protein visualization.Molecular masses of the proteins were estimated using Novex Sharp PrestainedProtein Standards (Thermo Fisher Scientific). Protein glycosylation was analyzedenzymatically: 1 μg of protein from the water soluble fraction was incubated with500 units of Peptide-N-Glycosidase F (New England Biolabs) to determine theexistence of asparagine-linked carbohydrates (O-linked), or 40,000 unitsof O-glycosidase to determine the existence of serine/threonine-linked(O-linked) carbohydrates (New England Biolabs), for 4 h at 37 °C. After in-cubation, the proteins were analyzed by SDS/PAGE as described above. Foramino acid analyses, the soluble fraction was hydrolyzed in 6 M HCl at 110 °Cfor 22 h under vacuum and analyzed with an automated amino acid ana-lyzer (L-8500; Hitachi). Amino-terminal and internal peptide sequences wereanalyzed as follows. The water-soluble fraction with and without trypsin(Promega) treatment were separated by SDS/PAGE, and then electropho-retically transferred to polyvinylidene difluoride (PVDF) (ATTO) membranes.After CBB staining, the PVDF membranes containing the proteins of interestwere processed on an automated Edman-degradation protein sequencer(PPSQ31A; Shimadzu).

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Silica Precipitation Assay. The assay follows a previous reported protocol (29).A solution of orthosilicic acid was freshly prepared by dissolving tetrame-thoxysilane in 1 mM HCl to a final concentration of 1 M. Proteins to betested for silica precipitation activity were dissolved in 100 mM sodiumphosphate buffer (pH 4–8) to a final volume of 10 μL. Subsequently, 1 μL ofthe 1 M orthosilicic acid solution was added and the samples were incubatedfor 5 min at room temperature. The samples were centrifuged for 5 min at14,000 × g and the pellets were washed three times with Milli-Q water toremove free silicic acid and phosphate. Washed pellets were incubated in10 μL of 0.1 M NaOH at 95 °C for 30 min to dissolve the precipitated silica.Concentrations of silicic acid were determined by the molybdate bluemethod (34). For scanning electron microscopy studies, washed silica pre-cipitates were placed directly on aluminum pin mounts and air-driedat room temperature. The specimens were briefly sputter-coated withplatinum/palladium and imaged with a field emission scanning electronmicroscope (SU8020; HITACHI, Tokyo, Japan). To monitor the silica formationprocess, the reaction mixture was spotted on Formvar-coated copper gridsand after 1 or 5 min incubation, the grid was rinsed with Milli-Q water twice,blotted on filter paper to remove excess water, and air-dried at room tem-perature. The resulting reaction products were observed via transmissionelectron microscopy (JEM-1400; JEOL).

Cloning of cDNA. After collection of living sponges, the cellular materialwas immediately immersed into TRIZOL solution (Thermo Fisher Scientific).Extracted RNA (2 μg) was converted to cDNA with the SuperScript pre-amplification system (Thermo Fisher Scientific) according to the manufac-turer’s instructions, except that the oligo dT-3 sites adaptor primer (TakaraBio) was used instead of oligo dT20. The degenerate oligonucleotides 5′-ARCAY GAY CAY CAY GAY CAY C-3′ and 5′-CAY CAY CAY GAY CAY GCN C-3′were synthesized from the partial protein sequences KHDHHDHH and

HHHDHAP, respectively, which were used for subsequent PCR amplification.PCR products containing the 5′ regions of the glassin cDNA were obtainedby RACE-PCR (38). PCR products were ligated into pCR4 vector (ThermoFisher Scientific), and both strands of the cloned inserts were sequenced. TheChou-Fasman secondary structure prediction was performed with GENETYX-MAC Network Version 17.0.4 (GENETYX).

Recombinant Protein Expression. Glassin cDNA encoding the mature proteinregion was amplified by PCR with Platinum Pfx DNA polymerase (ThermoFisher Scientific) with the primers 5′-CGT CAC GGT CAC CAT GGT C-3′ and5′-TCA GGA AAG AGA CCA GGT GAT G-3′ and was subcloned into pET100(Thermo Fisher Scientific). BL2 Star (DE3) cells (Thermo Fisher Scientific) weretransformed with the plasmid and cultured according to the manufacturer’sprotocol. Recombinant protein expression was induced by 0.1 mM isopropylβ-D-1-thiogalactopyranoside. After incubation for 4 h, the cells were harvestedand then solubilized in Fastbreak cell lysis reagent (Promega). The recombi-nant protein was purified using a His tag protein affinity column (HisGraviTrap;GE). Expression and purification of recombinant glassin was confirmed byWestern blot analysis using a WesternBreeze immunodetection kit (LifeTechnologies) and a rabbit anti-glassin antibody that was raised with asynthetic peptide HTHPLPPHTHATVPHTHA from the glassin sequence.

ACKNOWLEDGMENTS. We thank D. E. Morse (University of California, SantaBarbara) for helpful suggestions and S. Ifuku (Tottori University) andM. Watanabe (Fukuyama University) for technical expertise. We also thankA. Hashimoto, C. G. Satuito, and crew members of the Nagasaki Maru forcollection of the live sponge specimens. This work was supported by Ministryof Education, Culture, Sports, Science and Technology Grants-in-Aid for Scien-tific Research 23107521 and 25107722 [Innovative Areas: Fusion Materials(Area 2206) (to K.S.) and Scientific Research (Grant 15K06581, to K.S. and J.A.)].

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