The tube cement of Phragmatopoma californica: a solid foam
1 Department of Bioengineering, University of Utah, Salt Lake City, UT
84112
2 Institute for Collaborative Biotechnologies and the Materials Research
Laboratory, University of California, Santa Barbara, CA 93106, USA
3 Marine Science Institute and MCDB Department, University of California,
Santa Barbara, CA 93106, USA
* Author for correspondence (e-mail: waite{at}lifesci.ucsb.edu)
Accepted 11 October 2004
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Summary |
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Key words: polychaeta, sabellariidae, Phragmatopoma californica, bioadhesion, complex coacervation, polyphosphoserine
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Introduction |
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The sabellariids are commonly called sandcastle worms because individual
tubes with their resident worm are honey-combed together into large reef-like
mounds, although the mounds more-closely resemble proletarian apartment
buildings than castles. Their gregariousness is due to a component of the tube
cement that induces larvae to settle, metamorphose and build a new tube on
existing conspecific tubes (Eckelbarger,
1978; Jensen,
1992
). The colonies occur in the inter-tidal zone where there is
sufficient wave action to suspend food and appropriate particles for tube
building and repair. The sandcastle construction, and in particular the cement
bonds, must therefore be robust enough to withstand the siege of a turbulent,
high-energy environment. The cement is an important model for biomimetic
adhesives because of its apparent toughness, because it adheres strongly to a
variety of materials, and because it bonds rapidly to these materials in
seawater.
Two cement precursor proteins, Pc1 and Pc2, have been isolated from the
cement glands of Phragmatopoma californica
(Waite et al., 1992), a
sabellariid that lives off the coast of California. Both precursor proteins
are basic (predicted pI values of 9.7 and 9.95, respectively, from unpublished
gene sequences) and consist of repeated sequence motifs rich in glycine,
lysine, and 3,4-dihydroxyphenyl-L-alanine (DOPA) residues. The DOPA
functional groups may participate in bonding of the cement to mineral surfaces
as well as quinone-tanning of the cement during hardening
(Waite, 1999
). In addition to
the DOPA-containing Pc1 and Pc2, at least one more protein rich in serine was
suggested by amino acid analysis of the whole cement. Ser content of whole
cement approaches 28 mol%, whereas isolated Pc1 and Pc2 contained only 3.7 and
2.5 mol% serine, respectively (Jensen and
Morse, 1988
; Waite et al.,
1992
). The tube cements of other sabellariid polychaetes have been
shown by elemental analysis to contain inorganic elements, namely phosphorus,
magnesium and calcium in relatively large amounts, and in some cases perhaps
traces of manganese, iron, zinc and aluminum
(Gruet et al., 1987
;
Truchet and Vovelle, 1977b
;
Vovelle, 1979
). In all cases,
the phosphorus, calcium and magnesium were perfectly co-localized both in the
external cement and in the secretory granules of the cement glands. Here, we
report further characterization of the structure and composition of the cement
of P. californica, and present a model that accounts for the cement
structure and mechanism of bonding.
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Materials and methods |
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Scanning electron microscopy and energy dispersive spectrometry
For electron microscopy, glass beads containing fresh glue disks either
were mounted on conductive carbon tabs (Ted Pella; Redding, CA, USA) on SEM
posts as individual beads and lyophilized, or mounted as intact sections of
glass tubes, then lyophilized. In the latter case, beads were broken apart
after lyophilization. The samples were sputter-coated with gold using a Denton
Vacuum DESK II coater (Moorestown, NJ, USA), and examined with a Tescan Vega
TS 5130MM thermionic emission scanning electron microscope equipped with an
IXRF Systems energy dispersive spectrometer (Houston, TX, USA).
Laser scanning confocal microscopy
Intact cement disks were pried off glass beads with a forceps and mounted
in seawater between a coverslip and microscope slide then sealed with
fingernail polish. The samples were examined under a 60x planapochromat
objective, numerical aperture 1.4, with an Olympus Fluoview 500 confocal
microscope using ImagePro version 5 to acquire and process images (Melville,
NY, USA). Auto-fluorescence was observed over the entire visible spectrum and
into the infrared. Images were acquired simultaneously in three separate
channels using filter sets designed for Cy3, Cy5 and fluorescein (excitation
488, 543 and 633 nm, emission filters 505-525, 560-600, and >600 nm).
Amino acid analysis
Glass beads (50-100 mg) containing cement disks were hydrolyzed in 100
µl 6 mol l-1 HCl and 10%) phenol in vacuo at 110°C.
The acid hydrolysates were flash evaporated to dryness with three changes of
de-ionized water and two with 100% methanol using a Buchler vacuum evaporation
unit set at 60°C. Amino acid analysis following acid hydrolysis was
performed as described previously (Waite,
1991). Briefly, a Beckman System 6300 Autoanalyzer equipped with a
Na HPLC ion exchange column (Beckman Coulter #338076; Fullerton, CA, USA) was
used. The elution program was 85 min long to allow full separation of DOPA
from Leu. Amino acid detection based on derivatization by ninhydrin was
monitored at 570 and 440 nm, and absorbances were processed by HP ChemStation
for LC [Hewlett-Packard Rev A.06.03(509); Wilmington, DE, USA] in external
standard mode using a standard amino acid mixture (Sigma # A9 531; St Louis,
MO, USA).
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Results |
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To verify that the foam-like structure was not an artifact of the SEM processing and fracturing procedure, we took advantage of the observation that the cement disks auto-fluoresced across the visible spectrum, strongest in the red, to examine hydrated and unprocessed cement disks by laser scanning confocal microscopy. Optical sections through intact cement disks (Fig. 2) revealed a cellular structure with closed cells and a cell-size distribution similar to the cellular structure observed by SEM. The continuous phase was auto-fluorescent and the discontinuous phase was non-fluorescent. In total, four separate cement disks were optically sectioned and found to have similar cellular structures.
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The elemental composition of the cement was examined by energy dispersive spectrometry (EDS). Cemented beads were washed in excess de-ionized water or 0.5 mol l-1 NaEDTA (pH 8.0). In Fig. 3A, an EDS spectrum from a region of a cement disk washed in water was overlaid onto an EDS spectrum acquired from an adjacent bare region of the same glass bead that had approximately the same orientation to the EDS detector. The major elements consistently observed in relatively greater proportion in the cement than in the glass, in the order of energy, were C, N (not labeled), O, Mg, P and Ca. Convincing peaks above background for other elements, including Fe and Al, were not consistently observed. Eight cement disks had qualitatively similar elemental compositions relative to an adjacent bare region of the silica bead. Overlaid EDS spectra from cement disks and beads washed with 0.5 mol l-1 NaEDTA demonstrated that the relative amount of phosphorus was undiminished by washing with EDTA (Fig. 3B), suggesting that the phosphorus was part of the covalent structure of the cement rather than in a mineral form that would have been dissolved by EDTA. Carbon, N and O were also undiminished by washing with EDTA, as expected for covalent elements of the protein cement. Magnesium and Ca, conversely, were almost entirely extracted and exchanged with Na. Similar spectra were observed with five separate cement disks washed with EDTA. This suggests that the interior of the cement disk is accessible to the external solution. No other effects of EDTA on the cement disks were observed by light microscopy.
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Spatial mapping of the EDS results show that P, Mg and Ca are indeed localized within the cement disks (Fig. 4). After washing with EDTA, P remains in the cement disk while Mg and Ca are exchanged for Na (Fig. 5). The Mg and Ca are most likely complexed to the cement, as opposed to being trapped in the cells of the cement, because they were not removed by thorough washing with de-ionized water.
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During amino acid analysis of cement that was acid hydrolyzed off the glass beads, an acidic amino acid eluted at 3.2 min, about 7 min before the first standard amino acid (4-hydroxyproline). Two lines of evidence suggested that the early non-standard amino acid was O-phosphoserine (pSer). First, an O-phosphoserine standard eluted at the same time (3.2 min). Second, timed hydrolyses of the cement revealed a decrease in the 3.2 min peak and a proportionate rise in the Ser peak over time, reflecting the hydrolytic conversion of pSer to Ser (Fig. 6). Together, pSer and Ser accounted for close to 30 mol% of the cement amino acid residues. After a 1 h hydrolysis, 85% of the Ser was in the form of pSer (Fig. 7). Extrapolating to zero hydrolysis time, we estimate that 95% or more of the Ser is phosphorylated in the cement. Therefore, the bulk of the P observed in the cement by EDS was in the form of O-phosphoserine.
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Discussion |
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Based on sound engineering principles, foamed adhesives offer several
benefits for tubeworms. First, the foam-like structure would increase the
cement's elasticity and toughness - the amount of energy a material can absorb
before failing (Gibson and Ashby,
1997). The more flexible cement junctions would absorb and
dissipate the energy of the impinging surf in the inter-tidal environment to
minimize damage to the tube, like foam packing material. A related benefit is
the demonstrated crack-stopping behavior of foams. Second, the cellular
structure of the cement would save material and metabolic energy. If the
cohesive strength of the solid cement were much greater than the adhesive
strength of the cement to the substrate, then the extra material in a solid
cement would be wasted. Matching the adhesive and cohesive strengths of the
cement is more economical. Third, a foam structure could minimize the
abruptness of the elastic modulus mismatch between the rigid particles and the
flexible cement. A gradient in the cell size from the interface with the
particle to the center of the cement, with the smallest cells being close to
the interface, would create a gradient in the elastic modulus with the highest
cement modulus being at the interface.
Regarding composition, the cement appears to consist of a set of
polyelectrolytes with opposite net charges at physiological pH. The previously
identified precursor proteins Pc1 and Pc2 are positively charged. They contain
relatively high percentages of basic residues (approximately 15 mol% each),
few acidic residues and, accordingly, their isoelectric points are predicted
to be 9.75 and 9.95, respectively (Waite
et al., 1992). They also contain almost 10 mol% DOPA residues
each. The other major cement protein (or proteins) whose soluble precursor has
yet to be isolated is negatively charged. It contains a high proportion of
acidic pSer. All in all, the cement contains about 19% basic residues and
about 30% acidic residues, predominantly pSer. It also contains significant
quantities of complexed divalent cations. The Ca2+ and
Mg2+ of P. californica cement are not likely to be in a
phosphate mineral form, as has been suggested of P. koreni cement
(Truchet and Vovelle, 1977a
),
because EDTA washed away the vast majority of Ca2+ and
Mg2+ while not significantly diminishing the amount of phosphorus.
If the phosphorus occurred in mineral form, it too would have been dissolved
when Ca2+ and Mg2+ were sequestered by EDTA. In
Sabellaria alveolatea (Gruet et
al., 1987
) and in Pectinaria koreni
(Truchet and Vovelle, 1977a
),
and therefore almost certainly in P. californica, high concentrations
of Ca2+ and Mg2+ are present within secretory granules
of the cement glands.
The mechanisms by which polychaetes produce the solid cellular foam
structure in their cement is of considerable biological and technological
interest. It is not likely to involve the blowing of a molten polymer as in
the manufacture of StyrofoamTM and polyurethane foams
(Shutov, 1986). Such processes
require a gaseous blowing agent and, practiced underwater, would produce a
material with undesirable buoyancy. Rather, the foam-like structure is
probably formed from two interspersed liquid phases. A possible mechanism that
would account for phase separation during cement formation is suggested by
earlier cytological observations, by the composition of the tube cement, and
by classic studies of protein colloid chemistry. We consider each of these in
turn. Electron micrographs of thin sectioned cement glands of P.
koreni (Truchet and Vovelle,
1977a
) and S. alveolatea
(Vovelle, 1965
) revealed
polymorphic secretory granules originating in distinct cell types. Some of the
secretory granules, still deep within the cement glands, clearly had the
`bubbly' appearance of the deposited cement. This suggests that the process
that leads to phase separation in the cement may begin during the maturation
of the secretory granules. A common feature of secretory granule formation is
the condensation of a polyanionic matrix through crosslinking by multivalent
cations. Two well-studied examples are the condensation in mast cells of
negatively charged heparin glycosaminoglycan by histamine, a divalent cation
(Nanavati and Fernandez, 1993
;
Verdugo et al., 1987
); and the
condensation in goblet cells of the negatively charged mucin glycoprotein by
Ca2+ (Verdugo,
1990
). It follows that condensation of the cement into dense
secretory granules may occur by interaction of the anionic polyphosphoserine
protein with polycationic Pc1, Pc2, Ca2+ and Mg2+.
Bungenberg de Jong (Bungenberg de Jong,
1949a
,b
)
described numerous examples of the spontaneous separation of an aqueous
solution of two more oppositely charged polyelectrolytes into two immiscible
aqueous phases - a dilute equilibrium phase and a denser solute-rich phase -
in a process referred to as complex coacervation. Complex coacervate systems
have the following characteristics. (1) Both phases are predominantly water.
The coacervate phase is an isotropic liquid containing amorphous associative
particles that move freely relative to one another. (2) Coacervation occurs
when the charges of the polyelectrolytes are balanced. Coacervation is
therefore pH dependent, occurring to the maximum extent at the pH where the
solution is electrically neutral; ionic strength dependent, since shielding of
charges can change the charge balance of the system; and dependent on the
ratio of polyelectrolyte concentrations. (3) Complex coacervation usually
involves at least one flexible random coil polymer. The flexibility may
mediate associative interactions between the colloidal aggregates of the
liquid coacervate phase. (4) Coacervates occur in diverse morphologies,
including, under certain conditions, foam structures with transient
water-filled vacuoles (Bungenberg de Jong,
1949b
).
Based on the above considerations, we propose that phase separation of the sabellariid cement may occur as a result of a complex coacervation process during secretory granule maturation (Fig. 8). The complex coacervation occurs between Pc1 and 2 (polycations), the pSer-rich protein (polyanion) and Ca2+/Mg2+. These components are mixed in the cement glands in a ratio that is electrically neutral and thus phase separate (Fig. 8A,B). Water (E) is expelled from the coacervating ions as they condense and desolvate. Initially, when the coacervate droplets are small, the excluded water will diffuse directly into the E phase (Fig. 8B). As the size and viscosity of the coacervate droplets increases, the excluded water will become increasingly entrapped in the coacervate phase as discontinuous vacuoles (Fig. 8C).
|
Bungenberg de Jong (1949a)
referred to the process as `vacuolation', and this could well give rise to the
cellular foam structure of the cement. Our model suggests, as a parsimonious
explanation for the origin of the sabellariid cement, that the ordinary
secretory process may have been adapted to the production of a foam cement.
This adaptation may have occurred through natural selection of the type of
polyanionic secretory matrix in the cement gland. For example, a polyphosphate
matrix may coacervate more readily than the more common polycarboxylate
matrices because at the pH of the secretory vesicle (pH 5) the polyphosphate
(pKa1 2) would be more charged than the polycarboxylate (pKa 4).
Likewise, a polysulfate matrix such as the heparin glycosaminoglycan matrix of
mast cells would likely be too bulky and stiff to efficiently form
coacervates. Additional parameters that may have been adjusted by natural
selection to maximize coacervation and optimize cell dimensions and size
distributions include the charge density of the polyions, the flexibility of
the polyions, and of the ratio of polyions to divalent cations.
During secretion, the vacuolated cement droplets apparently coalesce
(Fig. 8D) into a single
cohesive cement disk with a diameter of about 200 µm. Secretion is
accompanied by a jump in pH from 5 in the secretory granule to 8.2 in
seawater that could trigger two events in the coacervate. First, deprotonation
of histidine residues (pKa
6.5) and the consequent loss of the
positive charge would free up their phosphate counterions to interact with
Ca2+/Mg2+ cations. Second, the nature of the
interactions between Ca2+/Mg2+ and phosphate groups
would change from coulombic interactions between solvated ions to bonds more
akin to ionic bonds in an insoluble salt, the effect of which would be to
harden spontaneously and solidify the cement
(Fig. 8D).
Another environmental change accompanying cement secretion is exposure to a
high concentration of monovalent Na+ cations (>0.5 mol
l-1). The large excess of monovalent Na+ could displace
some of the multivalent cations crosslinking the polyphosphoserine matrix,
leading to the rapid absorption of water
(Verdugo et al., 1987). This
sudden thirst may contribute to removing water at the interface with the
substrate to facilitate the underwater adhesion of the cement proteins to the
substrate, particularly the DOPA residues of Pc1 and 2
(Waite, 2002
). The swelling
due to the absorption of water would also increase the volume and more
importantly the adhesive area of the cement disk. With regard to adhesion, the
polyphosphoserine protein may also play a direct role in adhesion of the
cement to calcareous substrates. Several phosphoproteins have been shown, or
suggested, to bind strongly to calcareous minerals. These include the saliva
protein statherin, which binds to hydroxyapatite
(Long et al., 2001
);
osteopontin, a matrix protein of bones
(Boskey et al., 1993
) and
calcareous kidney stones (Kohri et al.,
1992
); phosphoryn, the extremely acidic phosphoprotein of dentin
(Ritchie and Wang, 1996
), and
mefp-5 from the adhesive pad of mussel byssal threads
(Waite and Qin, 2001
).
The presence of DOPA residues in Pc1 and Pc2 suggest that the cement is, at
least in part, hardened by diDOPA crosslinks. Quinone-tanning occurs in a wide
range of DOPA-containing structural proteins
(Waite, 1995). The mechanism
of oxidative crosslinking between DOPA residues has been most extensively
studied in the DOPA-containing proteins of the mussel byssal thread
(Burzio and Waite, 2000
;
McDowell et al., 1999
). Since
the phenolate form of DOPA is more redox active than the protonated form, DOPA
crosslinking would be accelerated by the shift to the elevated pH of seawater.
That the bulk of the quinone tanning probably occurs after deposition of the
cement onto the particle is suggested by the observation that freshly
deposited cement is white and creamy but turns brown within a few hours.
Solidification through covalent cross-linking after secretion of the complex
coacervate would prevent separation of the two dispersed aqueous phases,
locking in the final solid foam structure of the cement, while providing its
ultimate cohesive strength.
In conclusion, the robust underwater adhesion to diverse substrates, the composition, and the foam-like structure of its cement, hint at a sophisticated materials engineering practice of P. californica. There remain many questions, particularly regarding the details of the molecular structure of the cement, the cytological location of individual cement proteins, and changes in the structure during and after the secretion process. In pursuing these questions, it is likely that there is much that Phragmatopoma californica will teach us about designing adhesives.
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Acknowledgments |
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