Probing the Adhesive Footprints of Mytilus californianus Byssus

California mussels Mytilus californianus owe their tenacity to a holdfast known as the byssus, a fibrous extracellular structure that ends distally in flattened adhesive plaques. The “footprints” of freshly secreted plaques deposited onto glass coverslips were shown by matrix-assisted laser desorption ionization time of flight mass spectrometry to consist chiefly of proteins ranging in mass from 5200 to 6700 Da. These proteins, variants of a family known as mcfp3 (M. californianus foot protein 3), were purified from acetic acid/urea extracts of plaques and foot tissue. Mcfp3 appears to sort into fast and slow electrophoretic variants. Both are rich in Gly and Asn and exhibit post-translational hydroxylation of Tyr and Arg to Dopa and 4-hydroxyarginine, respectively, with the fast variant containing more than twice as much Lys + Arg. Both the slow and fast variants were partially sequenced from the N terminus, and the complete sequences of 12 variants were deduced from cDNA using degenerate oligonucleotides, PCR, and rapid amplification of cDNA ends. Mcfp3s are highly polar molecules and contain up to 28 mol % Dopa, which remains intact and may be crucial for adhesion to metal and mineral surfaces. California mussels Mytilus californianus owe their tenacity to a holdfast known as the byssus, a fibrous extracellular structure that ends distally in flattened adhesive plaques. The “footprints” of freshly secreted plaques deposited onto glass coverslips were shown by matrix-assisted laser desorption ionization time of flight mass spectrometry to consist chiefly of proteins ranging in mass from 5200 to 6700 Da. These proteins, variants of a family known as mcfp3 (M. californianus foot protein 3), were purified from acetic acid/urea extracts of plaques and foot tissue. Mcfp3 appears to sort into fast and slow electrophoretic variants. Both are rich in Gly and Asn and exhibit post-translational hydroxylation of Tyr and Arg to Dopa and 4-hydroxyarginine, respectively, with the fast variant containing more than twice as much Lys + Arg. Both the slow and fast variants were partially sequenced from the N terminus, and the complete sequences of 12 variants were deduced from cDNA using degenerate oligonucleotides, PCR, and rapid amplification of cDNA ends. Mcfp3s are highly polar molecules and contain up to 28 mol % Dopa, which remains intact and may be crucial for adhesion to metal and mineral surfaces. The fabrication of strong and durable adhesive bonds between macromolecules and metal or mineral surfaces in a wet environment is one of the most persistent challenges for modern adhesive technology (1Comyn J. Developments in Adhesives.in: Kinloch A.J. Applied Science Publishing, Barking, UK1981: 279-313Google Scholar). Ironically, this is “business as usual” for the marine mussels who inhabit rocky wind-swept seashores. Mussels (Mytilus) thrive despite persistent surf and tides thanks in part to a robust holdfast structure called the byssus, which consists of a bundle of threads each of which is tipped distally by an adhesive plaque that bonds to mineral and metal surfaces. The adhesive strategies of mussels and other sessile marine invertebrates are increasingly envisioned as paradigms for designing tough water-resistant adhesives (2Deming T.J. Curr. Opin. Chem. Biol. 1999; 3: 100-105Crossref PubMed Scopus (215) Google Scholar, 3Dalsin and Messersmith (1990) (2005) Materials Today 8, 53-57Google Scholar). How is water a problem for adhesion? There are many complicating factors, but one of the most fundamental involves the dielectric constant of water. Most known cases of practical and biological adhesion are mediated by noncovalent interactions between the adhesive and the adherend surface. The interaction energy of a charge-charge interaction, for example, is governed by Coulomb's law as E=-(Q1Q2)/[4πεr](Eq. 1) where Q1 and Q2 are the charges on two interacting molecules, ϵ is the dielectric constant of the medium, and r is the interatomic distance (4Israelachvili J. Intermolecular and Surface Forces. Academic Press, London, UK1985: 22Google Scholar). The dielectric constant of water (ϵ = 80) is much higher than that of vacuum (ϵ = 1) or nonpolar solvents (ϵ = 2-3), and this, consequently, greatly diminishes the magnitude of interaction energies that are possible in water. Ligand-receptor adhesion typically gets around this limitation by conformation-dependent protein-ligand interactions, in which ligand binding usually occurs within hydrophobic protein pockets having a much lower dielectric constant than the surrounding bulk water (5Chen B.N. Piletsky S. Turner A.P.F. Combinatorial Chem. High Throughput Screening. 2002; 5: 409-427Crossref PubMed Scopus (28) Google Scholar). The extent to which mussels can exploit this approach in byssal adhesion seems limited given how polar the mineral and metal surfaces to which they stick are. The biochemistry of mussel byssus has been investigated in primarily two species, Mytilus edulis and Mytilus galloprovincialis, both relatively sheltered species (6Waite J.H. Integr. Comp. Biol. 2002; 42: 1172-1180Crossref PubMed Scopus (310) Google Scholar). Not surprisingly, all of the proteins characterized so far in the two species show a very high degree of sequence homology. The California mussel, Mytilus californianus, in contrast to its congeners, dominates the most wave-swept and exposed habitats along the Pacific coast from Baja California to Vancouver Island. Its mean dislodgement tenacity was determined to be 250 N/mussel, forty times greater than that of M. edulis (7Witman J.D. Suchanek T.H. Mar. Ecol. Prog. Ser. 1985; 16: 259-268Crossref Google Scholar). Despite this superiority, almost nothing is known about the comparative biochemistry of adhesion in M. californianus. At least six different proteins have been characterized from freshly secreted adhesive plaques of M. edulis. These are mefp1, 2, 3, 4, and 5 and various preCols (6Waite J.H. Integr. Comp. Biol. 2002; 42: 1172-1180Crossref PubMed Scopus (310) Google Scholar). Extensive loss of solubility and epitopes caused by rapid protein processing has greatly impeded the precise immunochemical localization of each of these in the byssus (8Anderson K.E. Waite J.H. J. Exp. Biol. 2000; 203: 3065-3076Crossref PubMed Google Scholar). Our hypothesis in the present study is that one of these proteins is strategically localized near the interface for adhesion. We have interrogated the footprints of M. californianus byssal adhesive plaques by direct laser desorption ionization mass spectrometry and identified several new Dopa-rich mcfp3 proteins. The polar and highly modified nature of these proteins suggests that adhesion may be governed by novel interactions in addition to the usual noncovalent types. Protein Purification from Mussels—Mussels were collected locally from rocks and jetties around Campus Point and Goleta Pier in Santa Barbara, CA and immediately transferred to shallow holding tanks with circulating raw seawater at 15 °C. About 60 mussels were tethered to each 18 × 25-cm plate made of acrylic or glass (thickness, 1 cm), from which byssal plaques were harvested daily using a clean single-edge razor blade, briefly rinsed with 200 volumes of MilliQ water, and stored at -80 °C. About 1000 accumulated plaques were thawed and extracted in a small volume (5 ml/200 plaques) of 5% acetic acid (v/v) containing 8 m urea and 0.1 mm tri(carboxyethyl)-phosphine by homogenization on ice using a small hand-held tissue grinder (Kontes, Vineland, NJ). The homogenate was centrifuged for 30 min at 20,000 × g and 4 °C. The supernatant was collected (10 ml), and half was dialyzed against 4 liters of MilliQ water using membrane tubing with a 1,000-dalton cut-off (Spectrum Industries). Dialysis resulted in a turbidity, which was clarified by centrifugation (30 min at 20,000 × g and 4 °C), and sedimented material was redissolved in 5% acetic acid with 8 m urea, whereas the supernatant was freeze-dried at -80 °C. The acetic acid/urea plaque extracts were subjected to reverse phase HPLC 2The abbreviations used are: HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; NBT, nitro blue tetrazolium; RACE, rapid amplification of cDNA ends; CB, Coomassie Blue. using a 260 × 7-mm RP-300 Aquapore (Applied Biosciences Inc., Foster City, CA) column eluted with a linear gradient of aqueous acetonitrile (9Papov 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). Eluant was monitored continuously at 280 nm, and collected 1-ml fractions were assayed by amino acid analysis and electrophoresis following freeze-drying. Electrophoresis—Routine electrophoresis was done on polyacrylamide gels (7% acrylamide and 0.2% N,N′-methylenebisacrylamide) containing 5% acetic acid with 8 m urea (10Waite J.H. Benedict C.V. Methods Enzymol. 1984; 107: 397-413Crossref PubMed Scopus (79) Google Scholar). This system is ideal for basic Dopa-containing proteins because it can be processed for protein or Dopa staining with equal facility. The proteins were stained with Serva Blue R (Serva Fine Chemicals, Westbury, NY), whereas Dopa was stained with either the Arnow reagent (10Waite J.H. Benedict C.V. Methods Enzymol. 1984; 107: 397-413Crossref PubMed Scopus (79) Google Scholar) or nitro blue tetrazolium redox cycling (11Paz M. Flückinger R. Boak A. Kagan H.M. Gallop P.M. J. Biol. Chem. 1991; 266: 689-692Abstract Full Text PDF PubMed Google Scholar). Amino Acid Analysis and Sequencing of Peptides—The peptides and proteins were hydrolyzed in 6 n HCl with 5% phenol in vacuo at 110 °C for 12-48 h to correct for the losses of certain amino acids (12Tsugita A. Uchida T. Mewes H.W. Ataka T. J. Biochem. (Tokyo). 1987; 102: 1593-1597Crossref PubMed Scopus (101) Google Scholar). Recovery of tryptophan required hydrolysis in 4 n methanesulfonic acid (13Simpson R.J. Neuberger M.R. Liu T.-Y. J. Biol. Chem. 1976; 251: 1936-1940Abstract Full Text PDF PubMed Google Scholar) or in 6 n HCl with 30% phenol in vacuo for 20 and 40 min at 165 °C (14Muramoto K. Kamiya H. Anal. Biochem. 1990; 189: 223-230Crossref PubMed Scopus (63) Google Scholar). Routine amino acid analysis was done by ion exchange and a ninhydrin-based detection system (Beckman System 6300 Auto Analyzer) using a previously described step elution program (15Waite J.H. Anal. Biochem. 1991; 192: 429-433Crossref PubMed Scopus (27) Google Scholar). Hydroxyarginine eluted at 80 min and was quantified using the molar color yield of arginine. Because of its coelution with ammonia, tryptophan was separately quantitated on System 6300 using a 40-min program of NaD (5% sodium chloride and 1.9% sodium citrate at pH 6) at a column temperature of 70 °C. The amino acid sequence of protein and peptides was derived by automated Edman degradation using a Porton Instruments 2090 Microsequencer (Beckman-Coulter, Fullerton, CA). Phenylthiohydantoin derivatives of amino acids were chromatographically separated according to a gradient program specified earlier (15Waite J.H. Anal. Biochem. 1991; 192: 429-433Crossref PubMed Scopus (27) Google Scholar). The elution time for phenylthiohydantoin-4-hydroxyarginine (8.2 min) was determined in a previous study by Papov et al. (9Papov 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). Adhesive Plaque Footprints—To obtain adhesive plaque footprints, the mussels were tethered over glass coverslips, and when one or more plaques had been deposited onto the substrates, the connecting threads were severed, and the coverslips were removed. The coverslips were thoroughly rinsed with two changes of MilliQ water. The underside of the glass coverslip was dried so that the locations of the adhering plaques could be marked with a black marker pen. The plaques were then removed with a clean single-edge razor. The footprints were imaged by stereo light and scanning electron microscopy. MALDI-TOF Mass Spectrometry—MALDI-TOF experiments were performed using a Voyager DE (linear delayed extraction) mass spectrometer (Applied Biosystems, Foster City, CA). The MALDI matrix was prepared by dissolving sinapinic acid (10 mg/ml) in 70% acetonitrile. The Mcfp3 protein or peptides derived thereof were dissolved in this matrix solution to give a final concentration between 1 and 10 pmol/μl. About 1μl of this solution was applied to the target plate and allowed to evaporate. The sample spots were using an laser Inc., with a of and a of 8 and at a of 5 MALDI ionization and and for the Mcfp3 protein for that were using either or The was about which was to mass of the caused by the different hydroxylation of the Mcfp3 of byssal plaques were for proteins by MALDI-TOF mass The as involves the glass coverslips were collected plaque The coverslips were of and by thoroughly with MilliQ water, which the plaques were using a clean single-edge razor. The glass with the was dried and onto a MALDI sample plate with of matrix sinapinic were applied to the and to laser to matrix acid can be for in analysis with a lower and for and and respectively, for were used as for possible on MALDI by different as glass and the sample were on each surface. and was extracted from a foot of M. californianus using a was one freshly mussel foot in liquid with a and cDNA was from using reverse with an from The of the reverse was used in The was with the degenerate and which to the sequence of two variants of and respectively, and an amplification from The was performed in of and 5 of each 5 of each 1 of and of for on a of 30 at 30 at and 20 at with a final of 5 The were subjected to into a and into 10 for and The the sequence of Mcfp3 with the The and which the of two variants of respectively, and were used to the of Mcfp3 by a amplification of cDNA ends using a from and were the as described Plaque mass spectrometry was used to plaque footprints plaque from glass coverslips so that to could be by light or scanning electron and MALDI mass small proteins ranging from 5 to in the footprints which are of magnitude more than other proteins in the of of the proteins exhibit example, the at is a of separated from one by other were at and with more and is at the higher plaques were extracted with acetic acid 8 m urea, and the was subjected to MALDI mass and that was because many plaque proteins with The proteins in the of that were with of the footprints many proteins, which when stained in for protein and Dopa strong staining 2, plaque the stained for protein the most in the half of the whereas in the stained for Dopa are very strong in the lower half as the more proteins were by of how they were The of proteins from plaque extracts was at about g of collected 4 of protein was that than of of the plaques was Protein and the proteins, from both foot tissue and plaques as were using previously for (9Papov 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). of M. californianus two or fast proteins could be by and a redox cycling 2, the was crucial for detection because of by of these were purified by HPLC, partially and by MALDI-TOF The most of has an at had the sequence to but was with at Protein had a N and was at Dopa 20 mol % in the of these proteins with greater than could be in the foot acid of foot fast and slow variant plaque and variants in in a new proteins on phase HPLC resulted in elution of by a with fractions were subjected to and analysis for amino acid and the fast and slow proteins on HPLC to the fast and slow electrophoretic and The proteins in the the fast and proteins in both mass and sequence and more Dopa than fractions the Amino acid analysis of fast fractions 30 and proteins rich in Gly mol Lys mol Arg + mol (10 mol Dopa + Tyr mol and and mol the at in the gradient elution from the phase HPLC the and fast plaque fractions in having by Gly and Asn of these is that they are from the by against MilliQ water and Lys and Arg and a lower of Tyr to of slow fractions to be from the fast as MALDI mass exhibit both and different mass for example, protein to the and of the and proteins in the fast a of about 1 in the in 30 and in and in the in and in At the slow fractions protein that were and present in the The most were from and in other proteins, the variants with higher Dopa eluted with lower Dopa when the in the lower Dopa and Sequencing of on the N were used to a of by reverse sequences were using sequences from the with complete sequences have been determined and are in is to the of these variants with the MALDI of the footprints and purified proteins. with the in hydroxylation of Arg and Tyr in the in M. edulis and M. (9Papov 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, J. Waite J.H. Mar. 2000; PubMed Scopus Google Scholar, Waite J.H. Mar. Biol. 1999; Scopus (63) Google the for each mcfp3 variant are to from hydroxylation to hydroxylation of the target of the for each variant on the sequence as as the with to and Dopa, The are by of amino + + + + + + + + + + + + + 1 in a new The 12 sequences are into within and and to the of and fast HPLC fractions and to the in and 4 when hydroxylation is The to with of 15 possible with 8 of Tyr Arg and by a Gly to Arg at and in an to Asn at has on at has has and has and have 4 or 5 Arg and 10 Tyr whereas to have a Arg and Tyr This is to the mass in of to a of at least for hydroxylation and were the two most variants in footprints, for example, a mass of to and to is for and These are with of in footprints and of the and purified proteins. Dopa in or is very to in this, that Dopa in intact for one of two 1) it is by the interface or it is by present in the plaque the extracted 20 plaques with of these were from the glass and in seawater for h the other were extracted immediately the interface to seawater to the of the fast or slow protein with an is by 2, whereas is of and exposed plaque footprints by MALDI-TOF a are by a of footprints Our that the California mussel two Dopa-rich variants called for adhesion of the byssal plaques to These are the fast and slow variants. previous study using M. in that MALDI-TOF mass spectrometry could be used to the protein on the underside of adhesive plaques J. Waite J.H. Mar. 2000; PubMed Scopus Google two limited of these the laser is known to up to 1 into K. M. Anal. Chem. Scopus Google of which the plaque is and matrix by the plaque of proteins to the surface. was possible to the proteins were from the interface or from within the plaque MALDI-TOF of M. californianus plaque footprints on glass show that a of is present on the and that they are by the MALDI of footprints are are more than The most to be variant mass of is in footprints as as in extracted purified protein fractions The in the and to the and to the at is with a mass of and an of for the The that purified with than 10 and against its The at is to contain two and with of and At is variant but all to the slow MALDI-TOF analysis of HPLC fractions in suggests the of in fractions and to is in fractions and Mcfp3s from other mussels in their small and in having by and but sequence were at The persistent in the sequences involves near the this to a in a 1990; PubMed Scopus Google Scholar). Arg and its in are at than half the of in the from M. edulis (9Papov 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 which suggests that may have a on tenacity of in species of Mytilus from Papov et al. (9Papov 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 sequence from and Waite Waite J.H. Mar. Biol. 1999; Scopus (63) Google sequence from et al. K. S. S. Waite J.H. J. Biochem. PubMed Scopus Google sequences 4 and 5 from the present in a new the most of is the high of Dopa, which could approach 28 mol % in variants This is in one other plaque J.H. PubMed Scopus Google Scholar) in adhesion is because of its in The of in plaque is for two from in the slow is and and in from plaques despite the having been in seawater for several The of Dopa is given the of the redox to = pH J. Chem. Scopus Google Scholar) and the high pH and redox of seawater Because it is from in footprints following of the the of to from into the There are several possible that the sequence around Dopa may lower the that a may be present in the plaque or that Dopa may be by of a as for At this all to be possible Dopa at the interface and from the structure of the of Dopa, one is that it is for Chem. 1984; Scopus Google Scholar, J.H. J. Biol. 1990; PubMed Scopus Google Scholar) a analysis of Dopa on et al. S. J. M. Messersmith P.M. PubMed Scopus Google Scholar) have shown that Dopa is by a of metal on are to be by J. Scopus Google Scholar). these interactions to byssal adhesion in then it is of fundamental for and water-resistant adhesive bonds as between Dopa and from noncovalent charge-charge and interactions in that they are on the dielectric constant of the M. R. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the other can K. S. S. Waite J.H. J. Biochem. PubMed Scopus Google Scholar). these two to water and a new of adhesives that be both and than is We for a cDNA from M. californianus foot tissue. and with protein The is for with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.