Linking Adhesive and Structural Proteins in the Attachment Plaque of Mytilus californianus

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090–11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-l-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins. The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090–11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-l-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins. Mussels inhabit wind and wave swept rocky seashores. Such habitats are deathtraps for exposed organisms lacking a secure attachment. Accordingly, mussels have evolved a robust holdfast known as the byssus, which is essentially a specialized 4–5-cm long bundle of collagenous fibers that is proximally rooted in the mussel and distally bonded to foreign surfaces underwater by flattened attachment plaques (1Waite J.H. Holten-Andersen N. Jewhurst S.A. Sun C.J. J. Adhesion. 2005; 81: 297-317Crossref Scopus (297) Google Scholar). Given that engineering durable adhesive bonds between minerals and organic polymers in the presence of moisture remains a serious technological challenge, fundamental insights into the mechanism of holdfast adhesion in mussels and other sessile marine organisms represent a potential data base of bio-inspired solutions to the moisture problem (1Waite J.H. Holten-Andersen N. Jewhurst S.A. Sun C.J. J. Adhesion. 2005; 81: 297-317Crossref Scopus (297) Google Scholar). One popular technique for improving "wet" adhesion on siliceous substrates involves the application of surface-coupling agents or adhesion promoters (2Walker P. Wilson A.D. Nicholson J.W. Prosser H.J. Surface Coatings. 1. Elsevier Applied Science, New York1987: 189-232Google Scholar). Organosilanes are the best known synthetic adhesion promoters and typically designed with specific moieties for silica ligation at one end and reactivity toward the organic polymer at the other (2Walker P. Wilson A.D. Nicholson J.W. Prosser H.J. Surface Coatings. 1. Elsevier Applied Science, New York1987: 189-232Google Scholar). The use of surface-coupling agents to promote adhesion resonates with the adhesive biochemistry of byssal plaques made by mussels. A recent investigation of plaque footprints in Mytilus californianus has revealed a family of protein variants (mcfp-3) with a 3,4-dihydroxyphenyl-l-alanine (Dopa) 2The abbreviations used are: Dopa, 3,4-dihydroxyphenylalanine; Mfps, Mytilus foot proteins; mcfp-5 and mcfp-6, M. californianus foot protein 5 and 6; NBT, nitroblue tetrazolium; MALDI-TOF, matrix-assisted laser desorption and ionization with time-of-flight; GdnCl, guanidine hydrochloride; RACE, rapid amplification of cDNA ends.2The abbreviations used are: Dopa, 3,4-dihydroxyphenylalanine; Mfps, Mytilus foot proteins; mcfp-5 and mcfp-6, M. californianus foot protein 5 and 6; NBT, nitroblue tetrazolium; MALDI-TOF, matrix-assisted laser desorption and ionization with time-of-flight; GdnCl, guanidine hydrochloride; RACE, rapid amplification of cDNA ends. content that approaches 25 mol % (3Zhao H. Robertson N.B. Jewhurst S.A. Waite J.H. J. Biol. Chem. 2006; 281: 11090-11096Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 4Papov V.V. Diamond T.V. Biemann K. Waite J.H. J. Biol. Chem. 1995; 270: 20183-20192Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). With its many Dopa residues, mcfp-3 has been compared with a multifunctional surface-coupling agent (4Papov V.V. Diamond T.V. Biemann K. Waite J.H. J. Biol. Chem. 1995; 270: 20183-20192Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). By forming bidentate coordination complexes with metal centers in metal oxide and mineral surfaces, the Dopa/surface interaction is stronger than any noncovalent interaction (5Dalsin J.L. Lin L. Tosatti S. Vörös J. Textor M. Messersmith P.B. Langmuir. 2005; 21: 640-646Crossref PubMed Scopus (396) Google Scholar) and, unlike noncovalent interactions, not diminished by the large dielectric constant of water (6Israelachvili J.N. Intermolecular and Surface Forces. Academic Press, London1985: 12-23Google Scholar). If multiple Dopa side chains represent the surface-ligating moiety in mcfp-3, then there must also be another moiety that is specialized for reactivity with other plaque proteins. The present investigation was undertaken to determine whether there is a sidedness to the reactivity of mussel adhesive proteins with surfaces. In other words, if Dopa moieties of adsorbed proteins interact with the mineral surface, what interactions define their binding to other proteins in the adhesive plaque? Of the two new proteins detected in the footprints (mcfp-5 and mcfp-6), mcfp-6 is a thiol-rich protein that may mediate coupling of the surface proteins with those in the plaque by cysteinyldopa cross-links. MALDI-TOF Mass Spectrometry—MALDI-TOF examination of footprints and purified proteins was performed using a Voyager DE mass spectrometer (Applied Biosystems, Foster City, CA). The MALDI matrix was prepared by dissolving sinapinic acid (10 mg/ml) in 50% acetonitrile. Footprints of byssal plaques were screened for proteins by MALDI-TOF mass spectrometry. The technique as currently practiced involves the following: glass cover slips were collected shortly after plaque deposition. The cover slips were cleaned of slime and debris by washing thoroughly with Q-water after which the plaques were scraped off using a clean single-edge razor. The glass surface with the footprint residue was dried and mounted onto a MALDI sample plate with double stick tape. Two microliters of matrix mixture (30% sinapinic acid) were applied to the footprint and air-dried before subjecting to pulsed laser irradiation (4Papov V.V. Diamond T.V. Biemann K. Waite J.H. J. Biol. Chem. 1995; 270: 20183-20192Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Singly and doubly charged peaks for apomyoglobin from horse (16,952.56, and 8476.78, respectively, for average masses) were used as internal calibrants. The purified footprint proteins derived thereof were dissolved in this matrix solution to give a final concentration between 1 and 10 pmol/μl. About 1 μl of this solution was applied to the target plate and allowed to evaporate. The sample spots were irradiated using an N2 laser (LSI, Inc., Cambridge, MA) with a wavelength of 337 nm, pulse width of 8 ns and operated at a repetition rate of 5 Hz. MALDI ionization generates protonated singly and doubly charged ions for the footprint proteins, which were accelerated using either 20 or 25 kV accelerating voltage. Protein Isolation from Adhesive Plaques—M. californianus were collected locally from Goleta pier at Santa Barbara, CA and immediately transferred to shallow tanks in the laboratory and maintained with running sea water. To collect the plaques for protein isolation, mussels were tethered onto plastic plates. After a couple of hours, mussels started deposition of plaques on the plastic plate. New plaques deposited within a 24-h period were delaminated using a clean single edge razor blaze and rinsed extensively with Milli-Q water to remove any salts. Collected plaques either were used immediately or stored at –80 °C for future protein isolation. Once 2 g of plaques were accumulated, they were homogenized on ice with 20 ml of 5% acetic acid and 8 m urea with protease inhibitors using a small hand-held tissue grinder (Kontes, Vineland, NJ). Supernatant was harvested by centrifugation for 40 min at 20,000 × g and 4 °C using SS-34 rotor. Ammonium sulfate was then added to give a final concentration of 20% for protein precipitation. After centrifugation, the supernatant was collected and dialyzed against 4 liters of Milli-Q water overnight at 4 °C. Dialysis resulted in mcfp-3 precipitation, which was removed by centrifugation. The resulting supernatant was dialyzed against 5% acetic acid overnight and concentrated by ultrafiltration (Amicon) to 1–2 ml. For each run, only 200 μl of the concentrated crude extract subjected to gel filtration on a Shodex-803 column (5 μm, 8 × 300 mm). The column was equilibrated and eluted with 5% acetic acid in 0.2% trifluoroacetic acid, and monitored at 280 nm (7Ohkawa K. Nishida A. Yamamoto H. Waite J.H. Biofouling. 2004; 20: 101-105Crossref PubMed Scopus (46) Google Scholar). When purifying mcfp-5 from the adhesive plaques, around 1 gram of the adhesive plaques collected was homogenized in 10 ml of 5% acetic acid buffered with 4 m GdnCl. Supernatant was harvested by centrifugation and filtered through a 0.22-μm microfilter from Millipore. The resulting supernatant was fractionated by reverse phase C8 HPLC using a 260 × 7 mm RP-300 Aquapore (Applied Biosciences) column eluted with a linear gradient of aqueous acetonitrile. Protein Isolation from the Mussel Feet—The phenol gland in the mussel foot where the adhesive precursor proteins are produced and stockpiled was also used for protein isolation. Live mussels were shucked, following which the foot was carefully dissected, arrayed on glass plates, and frozen at –80 °C. In preparation for protein extraction, the mussel feet were removed from the freezer and partially thawed. The outer, pigmented epithelium in mussel feet was flayed away with a scalpel. Underlying phenol glands were easily visualized from the ventral side and dissected. Dissected phenol glands were homogenized to a puree on ice in 5% acetic acid with protease inhibitors (10 μm leupeptin and pepstatin) and centrifuged at 20,000 × g and 4 °C. The pellet (P1) was saved for isolation of mcfp-5, while supernatant (S1) containing mcfp-1, -2, and -6 was acidified with 70% perchloric acid to a final concentration of 1.5%. After centrifugation at 20,000 × g, pellet (P2) was discarded and supernatant (S2) was decanted into a small beaker. Ammonium sulfate was then slowly added to a final concentration of 20% (w/v). The mixture was stirred for 40 min at room temperature and centrifuged at 20,000 × g and 4 °C for 30 min, and the resultant supernatant (S3) was collected and dialyzed against 4 liters of 5% acetic acid overnight using dialysis tubing with a molecular weight cutoff 1000 (Spectrum Industries, Los Angeles) with two changes of dialysis buffer, then freeze-dried. The lyophilized crude extract was resuspended in 2 ml of 5% acetic acid and run on a Shodex-803 column (5 μm, 8 × 300 mm), which was equilibrated and eluted with 5% acetic acid in 0.2% trifluoroacetic acid. Eluant was monitored at 280 nm. Fractions containing pure mcfp-6 were pooled and desalted by reversed phase C8 HPLC (260 × 7 mm RP-300 Aquapore, Applied Biosciences) column, which was eluted with a linear gradient of aqueous acetonitrile. Eluant was monitored continuously at 220 and 280 nm, and collected 1-ml fractions were assayed by amino acid analysis and electrophoresis following freeze-drying. To isolate mcfp-5, the pellet (P1) was homogenized with 5% acetic acid and 8 m urea. After centrifugation, supernatant was discarded and pellet was extracted again with 5% acetic acid containing 4 m GdnCl (8Waite J.H. Qin X. Biochemistry. 2001; 40: 2887-2893Crossref PubMed Scopus (495) Google Scholar). The resulting supernatant was harvested by centrifugation and dialyzed against 4 liters of Milli-Q H2O. Dialysis resulted in proteins precipitated out that contained crude mcfp-5 and was harvested by centrifugation followed by redissolving in 2 ml of 5% acetic acid and 8 m urea. Because it was partially dissolved, the resultant crude extract was filtered through a 0.22-μm microfilter before loading onto a C8 column (260 × 7 mm RP-300 Aquapore, Applied Biosciences) by reverse phases HPLC eluted with a linear gradient of aqueous acetonitrile. Electrophoresis—Routine electrophoresis was done on polyacrylamide gels (7.5% acrylamide and 0.2% N,N′-methylenebisacrylamide) containing 5% acetic acid and 8 m urea (9Waite J.H. Benedict C.V. Methods Enzymol. 1984; 107: 397-413Crossref PubMed Scopus (78) Google Scholar). After electrophoresis, gels were stained with Coomassie Blue R-250 (Serva Fine Chemicals) and for Dopa-containing proteins by a redox-cycling method with nitroblue tetrazolium (NBT) in 2 m glycinate buffer (10Paz M. Flückinger R. Boak A. Kagan H.M. Gallop P.M. J. Biol. Chem. 1991; 266: 689-692Abstract Full Text PDF PubMed Google Scholar). To estimate the apparent molecular weight, purified footprint proteins were run on 15% SDS-PAGE. Amino Acid Analysis and Sequencing—Purified protein was hydrolyzed in 6 m HCl with 5% phenol in vacuo at 110 °C for 24 h. The hydrolysate was evaporated at 50 °C under vacuum and to dryness with a small volume of Milli-Q water and followed by methanol. Amino acid analysis was performed according to conditions described earlier with Beckman System 6300 Auto Analyzer (11Waite J.H. Methods Enzymol. 1995; 258: 1-19Crossref PubMed Scopus (66) Google Scholar). O-Phosphoserine was identified by amino acid analysis and estimated after correcting for losses because of hydrolysis (12Stewart R.J. Weaver J.C. Morse D.E. Waite J.H. J. Exp. Biol. 2004; 207: 4727-4734Crossref PubMed Scopus (192) Google Scholar). The N-terminal sequence of purified footprint proteins was determined by automated Edman degradation on a Porton Instruments Microsequencer (Model 2090, Porton, CA). Molecular Cloning—Total RNA was extracted from the phenol gland in M. californianus foot tip using the RNase Plant Mini kit from Qiagen (Valencia, CA). Briefly, one freshly dissected foot tip was used, and Qiagen's protocols were followed after initial tissue disruption under liquid nitrogen with a mortar and pestle. Following that, mRNA was purified from total RNA with the Oligotex mRNA Mini kit from Qiagen. With purified mRNA, a cDNA library was constructed using the CloneMiner™ cDNA Library Construction Kit from Invitrogen. This cDNA library was a readily available source of cDNA. Based on the known N-terminal sequence of mcfp-5 and -6 (Y*DGY*SDGY*Y*P and GGGNY*RGY*, in which Y* denotes Dopa), degenerate primers forward, 5′-TAY GAY GGN TAY AGY/TCN GAY GGN TAY TAY CC-3′, and 5′-GGN GGN GGN AAY TAY CGN/AGR GGN TA-3′, were designed and coupled with an vector-encoded T7 universal primer to amplify the cDNA sequences of mature protein from cDNA library, respectively. PCRs were carried out in 25 μl of 1× Buffer B (Fisher) and 5 pmol of each primer, 5 μmol of each dNTP, 1 μl of first-strand reaction, and 2.5 units of TaqDNA polymerase (Fisher) for 32 cycles on a Robocycler (Stratagene). Each cycle consisted of 30 s at 94 °C, 30 s at 52 °C, and 40 s at 72 °C, with a final extension of 5 min. The PCR products were subjected to 1% agarose gel electrophoresis, purified, and cloned into a pCR4-TOPO vector with TOPO TA Cloning kit from Invitrogen and transformed into competent Top10 cells for amplification, purification, and sequencing. For 5′-end information, the GeneRacer kit from Invitrogen was used to obtain sequence information from full-length transcripts by a 5′-RACE strategy. PCR was performed with gene-specific primers (antisense 5′-ATT TAA CAC GTG TGA CTA ACT GCT ACC-3′ and 5′-AAT ATA GGC TCG CCT TTA GTA ACC-3′, which reversely primes C terminus of mcfp-5 and -6, respectively) and a GeneRacer 5′-primer from Invitrogen (sense 5′-CGA CTG GAG CAC GAG GAC ACT GA-3′). Cysteine Modification—Cysteine in mcfp-6 was modified by alkylation with iodoacetate (13Zhang J.G. Matthews J.M. Ward L.D. Simpson R.J. Biochemistry. 1997; 36: 2380-2389Crossref PubMed Scopus (17) Google Scholar). In brief, purified mcfp-6 was reconstituted in 50 ml of 50 mm ammonia bicarbonate and alkylated with 15 μl of 500 mm iodoacetate at room temperature for 40 min in the dark. Alkylated mcfp-6 was subjected to amino acid analysis post-hydrolysis. Cysteine was detected as carboxymethylcysteine, which ran at 9.5 min by amino acid analysis. Isolation of CysteinylDopa Cross-links—M. californianus footprints on glass coverslips were hydrolyzed in 6 m HCl with 5% phenol in vacuo at 110 °C for 1.5 h. Hydrolysates were flash-evaporated to dryness at 50 °C under vacuum with a small volume of Milli-Q water followed by methanol. The flash-evaporated hydrolysate was taken up in 100 mm sodium phosphate buffer (pH 7.5), microcentrifuged for 10 min at 15,000 × g to remove insolubles, and applied to a pre-equlibrated phenylboronate column (Affi-Gel Boronate, Bio-Rad). Bound ligands on phenylboronate column were washed extensively with 100 mm phosphate buffer, and desalted by washing with 2.5 mm NH4HCO3 and Milli-Q water to facilitate subsequent amino acid analysis and electrospray ionization mass spectrometry described below. Fractions eluted with 5% acetic acid were lyophilized and subjected to a modified ninhydrin based amino acid analysis. Pure authentic 2- and 5-S-cysteinylDopa standards were donated by K. Wakamatsu (Fujita Health University) and detected by ion exchange amino acid analysis (see "Amino Acid Analysis and Sequencing") with elution times at 47 and 54 min, respectively (14Zhao H. Sun C.J. Stewart R.J. Waite J.H. J. Biol. Chem. 2005; 280: 42938-42944Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 15Zhao H. Waite J.H. Biochemistry. 2005; 44: 15915-15923Crossref PubMed Scopus (52) Google Scholar). Cross-linking in Vitro—About 1 mg of mcfp-6 was resuspended in 400 μl of 100 mm phosphate buffer (pH 7.5) and mixed with mushroom polyphenol oxidase (Sigma, T7755, ≥2,000 units/mg) at an enzyme to protein at a ratio of 1:10 by weight (15Zhao H. Waite J.H. Biochemistry. 2005; 44: 15915-15923Crossref PubMed Scopus (52) Google Scholar, 16Burzio L.A. Waite J.H. Biochemistry. 2000; 39: 11147-11153Crossref PubMed Scopus (399) Google Scholar). The mixture was incubated at room temperature under constant stirring for 1 h. The reaction was stopped by adding 100 μl of 6 m HCl. The resulting mixture was hydrolyzed in 6 m HCl with 5% phenol at 110 °C for 2 h in vacuo. The hydrolysate was flash-evaporated to dryness, redissoved in 1 ml of 50 mm phosphate buffer, and applied to phenylboronate column. Fractions eluted with 5% acetic acid were subjected to amino acid analysis for 5-S-cysteinylDopa. A control reaction was run using boiled enzyme (2 min). Phospholipase A2 Activity Assay—Detection of the phospholipase activity was performed following nondenaturing electrophoresis using a 5.5% polyacrylamide gel containing a lecithin emulsion at 33 mg of lecithin per ml of gel (17Shier W.T. Trotter J.T. Anal. Biochem. 1978; 87: 604-611Crossref PubMed Scopus (13) Google Scholar, 18Durkin J.P. Pickwell G.V. Trotter J.T. Shier W.T. Toxicon. 1981; 19: 535-546Crossref PubMed Scopus (23) Google Scholar). Purified mcfp-6 was resuspended in 1 mm Tris-HCl, 10% glycerol, and 2 mm EDTA at pH 7.4, and then loaded onto the lecithin-containing gel. Electrophoresis was carried out at a constant current of 20 mA at 4 °C for 2.5 h with 2 mm EDTA, 5 mm Tris, and 38 mm glycine pH 8.9. Phospholipase activity in gels was visualized as described by Shier & Trotter (17Shier W.T. Trotter J.T. Anal. Biochem. 1978; 87: 604-611Crossref PubMed Scopus (13) Google Scholar). Briefly, gels were incubated overnight at 37 °C with gentle shaking in a bath containing 100 ml of 0.1 m Tris-HCl, 20 mm CaCl2, and 5 μg/μl melittin diluted by 10% by volume with a 0.12% aqueous solution of rhodamine 6G. Afterward, the gels were extensively washed with Milli-Q water to remove the excess dye. The positive control was cobra phospholipase A2 (Sigma P6139); negative controls were the molecular weight standards (Bio-Rad). MALDI-TOF mass spectrometry was recently exploited for in situ detection of proteins in the adhesive footprints of M. californianus (3Zhao H. Robertson N.B. Jewhurst S.A. Waite J.H. J. Biol. Chem. 2006; 281: 11090-11096Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In the present study we have extended this strategy to analyze footprint proteins in addition to mcfp-3. Mussels readily deposit adhesive plaques onto glass coverslips. Following removal of the adhesive plaques, the matrix-impregnated footprints were directly subjected to MALDI-TOF mass spectrometry. Although footprint spectra are dominated by the signal intensity of the mcfp-3 protein family characterized earlier, two new proteins were apparent by zooming in on the m/z range of 8000 to 13,000. The larger of the new proteins, mcfp-6, has a [M+H]+ of 11.6 kDa that falls between the two mcfp-3 dimers [2M+H]+ centered at 10.6 and 13 kDa, respectively (Fig. 1). The dense cluster of peaks centered at m/z 12,000 accumulated gradually after the peak at 11,600 formed, possibly suggesting matrix adducts. Another smaller protein, mcfp-5, was detected below these at 8.9 kDa (Fig. 1). Footprint Protein Characterization from Plaques—To more fully characterize the new footprint proteins, it was first necessary to isolate them from the plaques and, for more material, from the phenol gland in the mussel foot. Collected plaques were extracted sequentially with acid-urea and acid-GdnCl, respectively, according to details outlined under "Materials and Methods." After precipitation by ammonium sulfate (20% w/v), the acid-urea extract was dialyzed against dilute acetic acid, concentrated, and fractionated by gel filtration chromatography (Fig. 2B). Pure mcfp-6 fractions were identified by their mass on MALDI and pooled for biochemical characterization. In contrast, acid-GdnCl plaque extracts were enriched in mcfp-5, which could easily be purified by passage through C8 HPLC, eluting at 20 min (Fig. 2A). The composition of footprint proteins isolated from plaques was determined by amino acid analysis following hydrolysis (Table 1). Accordingly, nearly three-quarters of all residues in mcfp-5 are represented by glycine (24%), lysine (15%), and Dopa (30 mol %). In contrast, mcfp-6 has much less Dopa (<5 mol %), but elevated levels of glycine (14%), aspartate (14%), and tyrosine (20%) are noteworthy; cystine is present at 3.4% in mcfp-6. Both mcfp-5 and -6 contain some phospho-O-serine estimated between 3 and 5 mol %. N-terminal sequences (Figs. 3 and 5) for mcfp-5 and -6 are distinct, but Dopa occurs in both. The mobilities and staining properties with redox cycling on polyacrylamide gels of plaque-derived mcfp-5 and -6 are illustrated in Fig. 2. Strong redox cycling with NBT is typical for Dopa/Topa-containing proteins (10Paz M. Flückinger R. Boak A. Kagan H.M. Gallop P.M. J. Biol. Chem. 1991; 266: 689-692Abstract Full Text PDF PubMed Google Scholar, 11Waite J.H. Methods Enzymol. 1995; 258: 1-19Crossref PubMed Scopus (66) Google Scholar).TABLE 1Amino acid composition of footprint proteins isolated from plaques and foot tissue of M. californianus given in mol % (Res/100 Res)ResidueMcfp-5Mcfp-6FootPlaquePredictedaCompositions predicted from the mcfp-5α and mcfp-6β sequences, respectively.FootPlaquePredictedaCompositions predicted from the mcfp-5α and mcfp-6β sequences, respectively.mol %mol %mol %mol %mol %mol %pSerbDetermined independently after modification or timed hydrolysis.4.64.803.52.80CM-CysbDetermined independently after modification or timed hydrolysis.0002.93.40Asx3.53.64.213.513.414.2Thr1.21.51.43.13.02.0Ser1.21.28.33.64.37.1Glx4.00.601.72.30Pro2.73.62.84.44.93.0Gly21.219.620.814.413.714.2Ala2.92.72.82.72.93.0Cys/20003.52.95.5Val0001.11.61.0Met000000Ile0000.61.01.0Leu1.30.81.42.32.22.0Dopa30.430.403.73.20Tyr0.50.227.820.019.220.2Phe0003.13.14.0His3.14.85.6000Hylys2.600000Lys18.819.820.89.29.89.2Arg3.03.14.26.66.58.1Total100100100100100100a Compositions predicted from the mcfp-5α and mcfp-6β sequences, respectively.b Determined independently after modification or timed hydrolysis. Open table in a new tab FIGURE 3Purification of footprint proteins from the mussel foot. A, elution profile for mcfp-5 subjected to C8 reverse phase HPLC. B, gel filtration chromatography on Shodex-803 of acetic acid-extracted mussel feet for mcfp-6 purification, followed by further C8 reverse phase HPLC for more pure mcfp-6 preparation (C). (Inserted sequences denote the N termini by Edman analysis.) Bracketed peaks denote pooled fractions. D, polyacrylamide gel electrophoresis of key steps in protein purification from mussel foot. Lanes 1–3 are SDS-PAGE and stained with CBR-250. Lane 1 contains the low molecular weight markers (5 μg each): phosphorylase B (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme (14,400). Lanes 2 and 3 contain purified mcfp-6 and -5, respectively. Lanes 4–10 are acid-urea PAGE. Lane 4 contains crude mcfp-5 obtained by GdnCl extraction. Lanes 5 and 6 contain the pooled fractions following C8 reverse phase HPLC (3-A) and are stained with CBR-250 and NBT, respectively. Lane 7 contains the crude extract of mcfp-6 following dialysis against 5% acetic acid. Lane 8 illustrates the mcfp-6 containing fractions pooled following chromatography with Shodex-803 column. Lanes 9 and 10 contain mcfp-6 following C8 reverse phase HPLC and are stained with CBR-250 and NBT, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5A, complete deduced sequences of mcfp-5 and -6. There are two variants of mcfp-5 that differ by a single amino acid and five variants of mcfp-6 identified as α, β, γ, δ, and ϵ. Italicized initial sequence denotes the signal peptide, and underlined portions indicate N-terminal sequence by Edman degradation. Shaded areas denote variable positions. Deduced sequences have the following GenBank™ accession numbers DQ444853 (mcfp-5), DQ351537 (mcfp-6α), DQ351538 (mcfp-6β), and DQ351539 (mcfp-6γ). Homology (B) sequence searches resulted in 76% identity between mcfp-5 and mefp-5 and 31% identity between mcfp-6β and snake venom phospholipase A2 (BLAST query).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Footprint Protein Isolation from Feet—With mass, composition, N terminus, and electrophoretic mobility established for plaque-derived mcfp-5 and -6, protein counterparts from foot tissue could be prepared from considerably more complex extractions. mcfp-6 was readily extracted with dilute acetic acid from phenol glands. Most co-extracted contaminants were precipitated by adding perchloric acid to 1.5% (v/v). Subsequent protein precipitation with ammonium sulfate led to the removal of remaining contaminants including mcfp-1 and -2. High purity mcfp-6 was thus easily prepared by gel filtration chromatography of the protein soluble in ammonium sulfate (Fig. 3B). Reversed phase HPLC confirmed the homogeneity of mcfp-6 (Fig. 3, C and D). In contrast to mcfp-6, isolation of mcfp-5 from the phenol gland required unusually aggressive conditions including sequential extraction with acetic acid, acid-urea, and acid-GdnCl. The sequential treatment removed most unwanted proteins, which simplified the chromat