Cement Proteins of the Tube-building Polychaete Phragmatopoma californica

The mineralized tube of the sandcastle worm Phragmatopoma californica is made from exogenous mineral particles (sand, shell, etc.) glued together with a cement secreted from the “building organ” on the thorax of the worm. The glue is a cross-linked mixture of three highly polar proteins. The complete sequences of Pc-1 (18 kDa) and Pc-2 (21 kDa) were deduced from cDNAs derived from previously reported peptide sequences (Waite, J. H., Jensen, R., and Morse, D. E. (1992) Biochemistry 31, 5733–5738). Both proteins are basic (pI ∼10) and exhibit Gly-rich peptide repeats. The consensus repeats in Pc-1 and -2 are VGGYGYGGKK (15 times), and HPAVXHKALGGYG (eight times), respectively, in which X denotes an intervening nonrepeated sequence and Y is modified to 3,4-dihydroxyphenyl-l-alanine (Dopa). The third protein, Pc-3, was deduced from the cement to be about 80 mol % phosphoserine/serine, and the cDNA was obtained by exploiting the presence of poly-serine repeats. Pc-3 consists of a family of at least seven variants with 60–90 mol % serine most of which is phosphorylated in the cement. Pc-1, -2, and -3 contain cysteine some of which reacts to form 5-S-cysteinyl-Dopa cross-links during the setting process. The mineralized tube of the sandcastle worm Phragmatopoma californica is made from exogenous mineral particles (sand, shell, etc.) glued together with a cement secreted from the “building organ” on the thorax of the worm. The glue is a cross-linked mixture of three highly polar proteins. The complete sequences of Pc-1 (18 kDa) and Pc-2 (21 kDa) were deduced from cDNAs derived from previously reported peptide sequences (Waite, J. H., Jensen, R., and Morse, D. E. (1992) Biochemistry 31, 5733–5738). Both proteins are basic (pI ∼10) and exhibit Gly-rich peptide repeats. The consensus repeats in Pc-1 and -2 are VGGYGYGGKK (15 times), and HPAVXHKALGGYG (eight times), respectively, in which X denotes an intervening nonrepeated sequence and Y is modified to 3,4-dihydroxyphenyl-l-alanine (Dopa). The third protein, Pc-3, was deduced from the cement to be about 80 mol % phosphoserine/serine, and the cDNA was obtained by exploiting the presence of poly-serine repeats. Pc-3 consists of a family of at least seven variants with 60–90 mol % serine most of which is phosphorylated in the cement. Pc-1, -2, and -3 contain cysteine some of which reacts to form 5-S-cysteinyl-Dopa cross-links during the setting process. The California sandcastle worm, Phragmatopoma californica (Fewkes), is a premier sand mason (1Sisson R.F. Nat. Geographic. 1986; 169: 252-255Google Scholar). In common with other sabellariid polychaetes, it exhibits an almost frenzied diligence in the collection, sorting and placement of sand grains for the construction and repair of its tubular home 3R. J. Stewart, personal communication.3R. J. Stewart, personal communication.. Although each worm builds primarily the tube in which it resides, a colony of worms can coordinate its efforts to erect massive boulder-like concretions that play a pivotal role in reef ecology (3Vovelle J. Arch. Zool. Exp. Gen. 1965; 106: 1-187Google Scholar, 4Chisholm J.R.M Kelley R. Nature. 2001; 409: 152-153Crossref PubMed Scopus (11) Google Scholar). The cement used by Phragmatopoma and related sabellariids to bind together grains of sand has been of interest for some time in that it adheres irreversibly to wet mineral surfaces and is used with extraordinary speed and economy. Perhaps 4–7 “spot welds,” each about 100 μm in diameter, are used to hold each sand grain (diameter 500 μm) in place in the natural concrete (3Vovelle J. Arch. Zool. Exp. Gen. 1965; 106: 1-187Google Scholar, 5Jensen R. Morse D.E. J. Comp. Phys. 1988; 158B: 317-324Crossref Scopus (83) Google Scholar). Phragmatopoma cement consists of proteins and significant levels of phosphate, calcium, and magnesium (6Gruet Y. Vovelle J. Grasset M. Can. J. Zool. 1987; 65: 837-842Crossref Google Scholar, 7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). Two of the cement proteins, Pc-1 and Pc-2, 4The abbreviations used are: PcPhragmatopoma cement proteinDIGdigoxigeninESIelectrospray ionizationms/mstandem mass spectrometryRACErapid amplification of cDNA endsDopa3,4-dihydroxyphenyl-l-alanine.4The abbreviations used are: PcPhragmatopoma cement proteinDIGdigoxigeninESIelectrospray ionizationms/mstandem mass spectrometryRACErapid amplification of cDNA endsDopa3,4-dihydroxyphenyl-l-alanine. known from an earlier partial characterization (8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar), resemble the byssal adhesives of mussels (9Waite J.H. Integ. Comp. Biol. 2002; 42: 1172-1180Crossref PubMed Scopus (308) Google Scholar) in that they are basic and contain 3,4-dihydroxyphenyl-l-alanine (Dopa) (5Jensen R. Morse D.E. J. Comp. Phys. 1988; 158B: 317-324Crossref Scopus (83) Google Scholar, 7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar, 8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar). Surprisingly, the abundant phosphate was not found to be associated with mineral but rather with serine residues in the cement (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). Indeed, the cement is dominated by phosphoserine and glycine, which together account for nearly 60 mol % of all the residues detected post-hydrolysis. Since the serine content of Pc-1 and -2 is negligible (8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar), the existence of a third serine-rich precursor is postulated. Phragmatopoma cement protein digoxigenin electrospray ionization tandem mass spectrometry rapid amplification of cDNA ends 3,4-dihydroxyphenyl-l-alanine. Phragmatopoma cement protein digoxigenin electrospray ionization tandem mass spectrometry rapid amplification of cDNA ends 3,4-dihydroxyphenyl-l-alanine. The aim of the present research was to identify the serine-rich protein, to obtain full-length sequences of Pc-1 and Pc-2, and to gain some insights into the mechanism of cement solidification. Worm Maintenance for Tube Production—Colonies of P. californica were collected from the intertidal zone near Santa Barbara, CA and maintained in the laboratory with flowing filtered seawater and aeration. To collect tubes, several worms were removed from the colony with 1–2 cm of their original tubes intact and spaced out on a bed of 2-cm-thick clean sand grains in a plastic container (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). Commercial sand (grain size range 400–600 μm) (Sigma) was provided for new growth. The collected tubes were washed extensively with deionized water followed by several rinses of double deionized water, then either freeze-dried or blotted dry on tissue paper for immediate protein processing. PCR and Preparation of cDNA—Total RNA was extracted from the cement gland in the thorax of P. californica with RNase plant mini kit from Qiagen (Valencia, CA) according to the supplier's protocols. First strand cDNA was synthesized from total RNA using Superscript II reverse transcriptase with Adapter Primer, 5′-GGC CAC GCG TCG ACT AGT ACT (T)16 (Invitrogen), and used in subsequent PCR reactions. Based on specific amino acid sequences in Pc-1 and Pc-2 from a previous study (8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar) (i.e. VGGYGYGAK and WGHPAVHK, respectively), the 3′-ends of Pc-1 and Pc-2 were PCR amplified using 3′-rapid amplification of cDNA ends (3′-RACE) with degenerate oligonucleotides (sense Pc-1, 5′-GGN GGN TAY GGN TAY GGN GCN AA-3′; Pc-2, 5′-TGG GGN CAY CCN GCN GTN CAY AA-3′) and an abridged universal amplification primer (antisense 5′-GGC CAC GCG TCG ACT AGT AC-3′, Invitrogen). The PCR reaction was carried out in 25 μl of 1 × Buffer B (Fisher), 5 pmol of each primer, 5 μmol of each dNTP, 1 μl of the first strand reaction mixture, and 2.5 units of Taq polymerase (Fisher) for 32 cycles on a Robocycler (Stratagene). Each cycle consisted of 30 s at 94 °C, 30 s at 52 °C, and 1 min at 72 °C, with a final extension time of 5 min. The PCR products were subjected to 1% agarose gel electrophoresis followed by gel purification and cloned into a PCR TA vector (TOPO TA cloning kit, Invitrogen). Plasmids were transformed into competent Top10 cells for amplification, purification, and sequencing. The insert encoded the COOH-terminal sequence of Pc-1 and Pc-2, respectively, including the 3′-untranslated region. To obtain the 5′-ends of the Pc-1 and Pc-2 cDNA, the GeneRacer kit (Invitrogen) was used to obtain sequence information from full-length transcripts by 5′-RACE. PCR was conducted under the same condition as above with gene specific primers (antisense Pc-1, 5′-TTT CCC CCG AAT TGG TAA TTA ATA CCC-3′; Pc-2, 5′-AGA CTT TGG TGT GAG GAA TTA ATA GCC-3′) and a GeneRacer 5′ primer. A cDNA library was constructed from the mRNA extracted from the cement gland in the thorax of P. californica using the CloneMiner™ cDNA library construction kit (Invitrogen) and adapted for serine-rich protein screening. Initial screening was done by PCR using a degenerate oligonucleotide corresponding to Ser5 (sense 5′-GAA TTC AGY AGY AGY AGY AG-3′ with engineered EcoR I site) and a vector-specific universal primer, T7 5′-AATACGACTCACTATAG-3′. PCR conditions, cloning, and sequencing involved the same strategy outlined above are shown in the supplemental material. After obtaining the 3′ sequences of Pc-3A and Pc-3B, nondegenerate gene-specific primers were designed to amplify the gene sequence with 5′ RACE strategy. To ensure integrity of each cDNA, antisense primers (Pc-3A-reverse, 5′-C TCA ATG GCC TTG AAC CTA GAA TAC-3′; Pc-3B-reverse, 5′-ACA TAT AAG TCG TGT AAA TCT ATT TCT AAC-3′) were designed within the 3′-untranslated region to amplify the full-length sequence. The PCR products were subcloned into a PCR TA vector and sequenced as described above. In Situ Hybridization—Worms were carefully removed from their tubes, anesthetized in 33% magnesium sulfate, then fixed, dehydrated, and embedded in methylmethacrylate (9:1 BMA with 1% benzoyl peroxide for thermal cure) as described in Warren et al. (10Warren K.C. Coyne K.J. Waite J.H. Cary S.C. J. Histochem. Cytochem. 1998; 46: 149-155Crossref PubMed Scopus (20) Google Scholar). Embedded tissue was microtomed into 2-μm-thick sections and acetone-de-embedded. Sections were then rehydrated, washed with diethylpyrocarbonate-treated Q-H2O, digested with proteinase (10 μg/ml proteinase K) for 4 min at 37 °C, acetylated with 0.1 m triethanolamine, 0.5% acetic anhydride for 3 min, and blocked with blocking solution (1 × Denhardt's solution, 5% dextran sulfate, 0.2 mg/ml sheared herring sperm DNA, 4 × SSC, and 50% formamide) for 2 h at 42°C. DIG-labeled oligonucleotide was then added to the blocking solution, and the tissue sections were hybridized overnight at 42 °C. The DIG-labeled oligonucleotide was prepared by DIG-PCR labeling kit (Roche Diagnostics) using primers sense 5′-ATGAAATCCTTCACTATTTTTGCC-3′ and antisense 5′-AGAGCTGGAACTAGAGCTGTA-3′. Negative control reactions for later in situ hybridization included regular dUTP instead of DIG-labeled dUTP. Hybridization product was visualized by incubating the sections with anti-DIG-AP and subsequently adding color substrates nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as described by the supplier (Roche Diagnostics) and viewed by light microscopy. Parallel whole worms were stained for Pc-1 and -2 using the Arnow reaction (11Waite J.H. Methods Enzymol. 1995; 258: 1-20Crossref PubMed Scopus (66) Google Scholar). Cement Analysis—For routine analysis, cement proteins were hydrolyzed in 6 m HCl and 5% phenol in vacuo for 24 h at 110 °C. Phosphoserine losses were corrected by extrapolation of hydrolysis to zero time (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). Hydrolysates were flash evaporated to dryness at 50 °C and subjected to amino acid analysis on a Beckman 6300 Autoanalyzer using an 85-min elution program for post-translationally modified amino acids (12Waite J.H. Anal. Biochem. 1991; 192: 429-433Crossref PubMed Scopus (27) Google Scholar). Cysteine was detected as carboxymethylcysteine following reduction by dithiothreitol and alkylation with iodoacetate as previously described (13Shevchenko M. M. Anal. PubMed Scopus Google Scholar), and phosphoserine was detected as at min (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). analysis, worm cement proteins on sand grains were hydrolyzed for 1–2 h at described above. and were from flash evaporated cement using residues in 100 phosphate were to the Anal. Biochem. 1986; PubMed Scopus Google Scholar). were washed with of phosphate followed by of 2.5 and of deionized with 5% acetic acid were freeze-dried and subjected to amino acid analysis (12Waite J.H. Anal. Biochem. 1991; 192: 429-433Crossref PubMed Scopus (27) Google Scholar) and electrospray ionization mass spectrometry tandem mass using a to at a of 5 were with and the known peptide sequences from cement and as the for gene-specific degenerate complete sequences were deduced from the cDNAs obtained by reverse of cement gland followed by 5′-RACE. and sequences are shown in 1 and in Both Pc-1 and -2 are basic proteins with at and respectively, that are with by (8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar). variants of Pc-1 were and all a mass of about and of three amino glycine, and the of which is extensively modified to (8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar). three contain repeats of a consensus in which the residues are to Pc-2, (21 kDa) was degenerate of a consensus but with in and sequences most of the is to in the protein (8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar). the of was with the of the Pc-1 and -2 are in the other most abundant amino acid of serine precursor with a acid in mol % 100 by amino acid analysis of P. californica cement and deduced from cDNA for the precursor proteins, Pc-1, Pc-2, and and Pc-3 from the of seven variants for three for The cement an of three are levels of and are from previous and the repeats of and modified to and or as in cement but as cysteine for the deduced of the three levels of and are from previous and the repeats of and modified to and Pc-1 and Pc-3 from the of seven variants for three for The cement an of three are levels of and are from previous (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar, 8Waite J.H. Jensen R. Morse D.E. Biochemistry. 1992; 31: 5733-5738Crossref PubMed Scopus (91) Google Scholar) and the repeats of and modified to and as in cement but as cysteine for the deduced of the three in a new The of a cement precursor was deduced by the of Pc-1 and Pc-2 from the of whole cement were made in that the in cement is by Pc-1 mol and Pc-2 mol that in cement is derived from Pc-2, and that Pc-1 and Pc-2 each a third to the The of is by the of in the known to be first to then to and to cross-links products that losses of other amino acids Waite J.H. J. J. Biol. PubMed Scopus Google Scholar, Waite J.H. Biochem. PubMed Scopus Google Scholar, R. Scholar) as the content in cement at mol the of Pc-1 and Pc-2 are and respectively, or about out of The and × 3 mol that a precursor 80 mol % is for the third cement to a protein with mol % phosphoserine from the cement gland or from cement by worms on acid washed sand for the of cement is associated with the and that proteins are to following gel electrophoresis J. in Scholar). In a strategy for sequence of the following were at 80 mol % the of at least of be and the for serine in and -2 was the primers on with sequence from the 3′-untranslated region RACE to the sequences The of range from to in of cement gland cDNA with primers on the serine of Pc-3 variants Pc-3A variants are 50 and 60 mol % serine and contain a highly basic with In the variants the basic amino acids at the and serine levels mol range from to 30 with an of to The mol % for for all seven variants was about mol % which with the 80 mol % from the cement of phosphorylated range from to which place the most proteins Pc-3 proteins are variants associated with the cement gland of P. californica was by in situ hybridization of Pc-3 using a gene-specific sequence in in the region The was to the cement gland in the In a a worm was stained for which as a for Pc-1 and -2 the thorax stained with the Two of the deduced sequences shown in 1 are the cysteine content with a cement of a 2 mol % and a content of mol by the cement to be with and acid hydrolyzed cement subjected to amino acid analysis at residues The is the in of To the of cement by the worms sand was collected and subjected to iodoacetate alkylation with or reduction by carboxymethylcysteine be detected the detected carboxymethylcysteine mol was with the mol found in hydrolyzed To account for the of that Pc-1, -2, and -3 be secreted with and The be by of residues at seawater and the of an protein R. Scholar, Biochem. J. PubMed Scopus Google Scholar, J. Scopus Google Scholar). amino acid analysis, and electrospray ionization mass significant levels of 5-S-cysteinyl-Dopa were detected in of Phragmatopoma cement and The from hydrolyzed cement to has an elution time of about min by amino acid analysis, a mass of by mass and a by tandem following that is with 5-S-cysteinyl-Dopa of the to a previously of 5-S-cysteinyl-Dopa following E. Scholar) and are in is that the and are by their elution on amino acid analysis, the has on tandem the of the and is at 100 and not cement with In are in solution in the cement gland at a the is zero to (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). of are then from the cement gland and to or on each sand The gel each sand grain is the tube and to The is to the of magnesium and with phosphate at seawater (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. 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PubMed Scopus Google Scholar). Both of are involved in to and phosphate The sand of P. californica is to be by the of its cement and the of its are to be for the of of the the of and setting or of the in and Google Scholar). were not for conditions, a is and that is of the as a that is by the seawater be Pc-1, -2, and -3 are together or by to the To et al. (7Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar) a on to a that as basic proteins are with as proteins at a is Scholar, Scholar). in a mixture of with and a from the that to be to 1 and that and or proteins, by of proteins M. E. Google Scholar), be to water from wet In the the and solution to most surfaces Scholar). Pc-1, -2, and -3 resemble Scholar) in highly and in solution, but their on in be that is done at which is to be the of a 1988; PubMed Scopus Google Scholar). 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