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First published online February 20, 2004
Journal of Experimental Biology 207, 1127-1135 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00859
The effect of molluscan glue proteins on gel mechanics
1 Department of Biology, Ithaca College, Ithaca, New York 14850,
USA
2 Department of Materials Science and Engineering, Cornell University,
Ithaca, New York 14850, USA
* Author for correspondence (e-mail: asmith{at}ithaca.edu)
Accepted 29 December 2003
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Key words: mollusc, slug, Arion subfuscus, land snail, Helix aspersa, glue, gel, adhesion, mucus, periwinkle, Littorina irrorata
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).
Most commonly, mucous gels serve as a lubricating and protecting layer. Many
molluscs, however, use similar gels to glue themselves to the substratum when
they are inactive. Limpets use such gels to glue themselves to rocks amid
violent wave surge. Similarly, a thin line of gel along the lip of a marsh
snail's shell can firmly attach it to the top of a wet marsh grass stem,
despite the windswept movements of the grass. These gels, like most mucous
secretions, consist of a dilute network of polymers, usually containing more
than 95% water (Smith, 2002).
One would not expect a dilute gel that is typically a lubricant to form strong
attachments. In order to achieve such strength, it seems likely that the
adhesive gel differs significantly in structure from other forms of mucus.
Smith et al. (1999) and
Smith and Morin (2002) showed
that the adhesive and non-adhesive forms of mucus from these animals are
structurally similar except for the presence of specific proteins in the glue.
These will be referred to as glue proteins. The difference can be striking,
with the glue proteins making up roughly 10–50% of the secretion in the
animals studied. The presence of such quantities of these proteins in the
adhesive mucus suggests that they play a key role in adhesion. Specifically,
we hypothesize that the proteins act on other polymers to stiffen the gel. One
possibility is that the proteins cross-link the other polymers in the gel.
This would have a large impact on the gel's mechanical strength
(Denny, 1983;
Smith, 2002). They may
cross-link the large polymers of the normal, non-adhesive mucus, or similar
large polymers. Alternatively, the proteins may serve as enzymes to catalyze a
cross-linking reaction, or otherwise modify the polymers in the gel. It is
also possible that the proteins themselves form an adhesive bond independent
of the other polymers in the gel. Finally, the presence of these proteins may
only be incidentally related to adhesion.
To determine the role of these proteins, their effect on different gels was measured. We isolated the glue proteins from the adhesive mucus of marsh periwinkles and two other species, terrestrial slugs and land snails. We added these proteins to commercial polymers that can gel or form highly viscous solutions. We predicted that the glue proteins would increase the stiffness and viscosity of gels formed by large polysaccharides. These experiments provide qualitative and quantitative evidence that the identified proteins play a direct role in adhesion.
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Isolation of glue proteins
The glue proteins from the marsh periwinkle L. irrorata (Say) were
tested first. Smith and Morin
(2002) showed that both the
adhesive and non-adhesive mucus from these snails contain MDa-size
carbohydrates. In addition, the adhesive mucus has two proteins of 41 and 36
kDa that appear to be related. These proteins make up roughly half of the
organic material in the adhesive mucus. Samples of the adhesive mucus were
collected as described by Smith and Morin
(2002) from roughly 200
periwinkles kept in a 29 gallon aquarium with recirculating artificial
seawater. Samples were dried and stored at –80°C until use.
The 41 and 36 kDa glue proteins were isolated by gel filtration
chromatography. For each separation, several mg of dried sample were dissolved
in 2 ml of buffer containing 8 mol l–1 urea, 0.5 mol
l–1 NaCl, 0.5% Triton X-100 and 20 mmol l–1
phosphate, pH 7.4. Samples were heated to 80°C, vortexed and sonicated
until they dissolved. They were then loaded onto a 1.6 cm x 60 cm
Sephacryl S-400 column (Pharmacia Biotech, Sweden) with a column buffer of the
same components, but containing urea at a concentration of 6 mol
l–1 instead of 8 mol l–1. In earlier trials,
lower concentrations of urea, Triton and sodium chloride were tried, but these
did not give such consistent results. Fractions from the column were assayed
by the Bradford assay for protein and the orcinol–sulfuric acid assay
for carbohydrates (see Smith and Morin,
2002). This column cleanly separated the MDa-size carbohydrates
from the smaller proteins (Fig.
1). As the only significant proteins present in the protein peak
were the 41 and 36 kDa glue proteins
(Smith and Morin, 2002),
further purification steps were not taken. The protein peak was pooled and
dialyzed exhaustively against phosphate-buffered saline (PBS; 20 mmol
l–1 phosphate, 130 mmol l–1 NaCl, pH 7.4)
or, in some cases, distilled water. There was no detectable difference in
results between samples dialyzed in either way. The dialysis bags were then
placed on high molecular mass polyethylene glycol chips to concentrate the
protein by osmosis. Discontinuous sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) using the methods of Laemmli
(1970) and Hames
(1990) confirmed that the
samples contained the 41 and 36 kDa proteins and little else. This procedure
yielded 1–2 ml of these proteins at a concentration of roughly 1 mg
ml–1. Attempts to increase the amount isolated were often
complicated by the difficulty in dissolving more than a few mg of dried glue
per ml of buffer and because of possible aggregation at the top of the column.
One might expect this given that the polymers function by forming gels at low
concentrations.
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Qualitative effects of L. irrorata glue proteins
The effect of L. irrorata glue proteins on gel-forming polymers
was assessed qualitatively. After dialysis, solutions of glue protein were
added to dried gel-forming polymers, mixed, allowed to set in microcentrifuge
tubes, then removed for observation and comparison to controls. Controls were
the same as the treatments, except with bovine serum albumen (BSA) instead of
glue proteins. To ensure that the only difference between treatments and
controls was the type of protein added, the BSA was dissolved in the bathing
solution from the last dialysis bath used on the glue protein sample. Thus, it
should have had the same concentration of ions, the same pH, and the same
amounts of trace contaminants. This is particularly important since traces of
urea and detergent may interfere with gelling. In addition to BSA, other
control proteins were tested, including gelatin, ovalbumin and non-fat dried
milk. The presence or absence of BSA, or any of these control proteins, had no
clear effect on gels. Nevertheless, every trial using glue proteins was
compared with a paired control using the same concentration of BSA.
The gel-forming polymers that were tested included agar, pectin (apple and
citrus), polygalacturonic acid and guar gum. Phillips and Williams
(2000) provide detailed
information on these polymers. All of these polymers are roughly MDa-size
polysaccharides except for agar, which has subunits of roughly 100–150
kDa. Agar, guar gum and pectins are composed of different subunits, while
pectins are similar to polygalacturonic acid, except that they are partially
methylated. Unlike the other polymers, guar gum is neutral and its mechanics
depend solely on intermolecular tangling. The others are capable of gelling by
different mechanisms.
Several different concentrations of gel-forming polymer were tested for each gel. The concentrations that worked best, and were thus used in later trials, were 2% for guar gum, polygalacturonic acid or pectin, and 0.5 or 0.6% for agar. Dried samples were weighed out on an electronic balance that was accurate to the nearest 0.01 mg. The dissolved proteins were added with a digital pipettor. Care was taken to ensure that the concentration of gel-forming polymers varied by less than 1% among samples (i.e. 2% gels may have actually been 1.98–2.02%). For the pectin samples, calcium chloride (to 50 mmol l–1) was added to assist gelling. Similarly, hydrochloric acid (to 20 mmol l–1) was added to polygalacturonic acid samples to help dissolve the polymer. Agar was heated to 100°C for 2 min before being allowed to set.
Samples were placed on a flat surface and compared visually. They were categorized as being either primarily liquid, gelled slightly into loose clumps, or firmly gelled, retaining the shape of their container. Statistical tests were not performed on these qualitative experiments. Instead, it was concluded that the glue proteins had an effect on a gel if they produced a repeatable change between the aforementioned states in each of at least four trials.
To provide further support that any effects were due to the presence of the glue proteins, tests were performed to see if the effect depended on their concentration and their structure. First, different concentrations of glue proteins were tested on 0.6% agar or 2% polygalacturonic acid. The glue protein concentrations typically ranged from 0.1 to 3 mg ml–1, with only a few higher concentrations due to the difficulty of getting sufficient material. The concentration was manipulated by diluting concentrated samples with the bathing solution from the last dialysis step. As described above, the same concentration of BSA was used as a control. Overall, 15 trials were performed with agar, and 21 with polygalacturonic acid.
To test the effect of the structure of the glue proteins, some trials were
performed with 1% dithiothreitol (DTT) added to 0.6% agar and 1.3 mg
ml–1 of glue protein or BSA. Reduction of disulfide bonds
changes the structure of the glue proteins, based on samples run on SDS-PAGE
(Smith and Morin, 2002). Thus,
if the proteins' structure is important for their effect, then DTT should
weaken or eliminate that effect. This test was repeated with two different
samples.
Identification of glue proteins in other species
Since both limpets and marsh periwinkles have specific proteins associated
with adhesion, it is likely that this is a common method of controlling gel
mechanics among molluscs. Thus, two other molluscs capable of strong adhesion
were studied: the slug Arion subfuscus (Draparnaud) and the land
snail Helix aspersa Müller. The dorsal surface of the slug
produces copious amounts of highly sticky, orange mucus when disturbed. This
mucus is secreted quickly, often as a liquid, and stiffens within seconds into
a sticky, rubbery mass. In contrast, the land snail H. aspersa
secretes a mucus that dries to a hard film for a long-term adhesion
(Barnhart, 1983). Because of
the size of this snail and its predictable behavior, it is easier to collect
sufficient quantities of sample for quantitative testing.
Adhesive samples were collected from roughly 100 land snails and 30 slugs kept in terraria. Land snails survived well for over a year with periodic feedings of lettuce, chicken feed and crushed oyster shells. The slugs survived for weeks in humid containers with grass and chicken feed. Adhesive mucus was collected from the slugs by rubbing their dorsal surface with a metal spatula. Non-adhesive trail mucus was collected by allowing the slugs to crawl over a glass surface and scraping the glass behind them, being careful to avoid contamination from the adhesive mucus produced by their backs. Despite this, contamination was a continual problem. It was difficult to collect pure trail mucus without inducing the slug to attempt to stick to the glass, or to produce mucus from its back that slid off onto the glass. Such contamination was not a problem with land snails. Dried adhesive mucus was collected from land snails that were firmly attached to the terrarium wall. Snails were detached and the glue was scraped off the glass with a razor blade and carefully removed from the shells with forceps. After this, trail mucus was collected by allowing the snails to crawl in a damp plastic tub for roughly 1 h, draining the excess water, then rubbing the mucus off the surface of the tub with a rubber spatula.
Trail and adhesive samples were then compared by SDS-PAGE. Equal amounts of trail mucus and adhesive mucus were dissolved in sample buffer (0.125 mol l–1 Tris-Cl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 1.6 mol l–1 urea). Polyacrylamide gel concentrations of 10% and 15% were used. Coomassie Blue-stained gels were photographed using a digital camera and analyzed using Kodak 1D software (Rochester, NY, USA) to identify the molecular mass and net staining intensity of the bands above background. To identify proteins correlated with adhesion, six samples of trail and adhesive mucus from slugs were compared, and four samples of trail and adhesive mucus from land snails.
Qualitative effects of H. aspersa and A. subfuscus glue proteins
Proteins characteristic of the adhesive mucus of H. aspersa and
A. subfuscus were isolated by gel filtration, dialyzed and
concentrated as described previously. The effect of these proteins on the
degree of gelation of gel-forming polymers was compared visually to the effect
of other fractions from the column. For H. aspersa, the effects of
the glue proteins and of the MDa-sized polymers from early fractions were
tested at 1 mg ml–1 on 0.6% agar. These experiments were
performed twice using material from different column runs. For A.
subfuscus, four separate experiments were performed, using material
from different column runs. For each column run, fractions were pooled into
five groups and each was adjusted to 0.3 mg ml–1 and tested
on 2% citrus pectin (one set of experiments) or adjusted to roughly 0.1 mg
ml–1 and tested on 0.6% agar (three sets of experiments).
Quantitative effects of H. aspersa glue proteins
Quantitative measurements of the effect of glue proteins from H.
aspersa were made using a dynamic rheometer (Ares, TA Instruments, New
Castle, DE, USA). The storage and loss moduli of the gels were determined,
thus giving a measure of the elastic and viscous contributions to the gels'
mechanics (Denny, 1983). The
elastic contribution will often be referred to as the stiffness. To be an
effective glue, either a high stiffness, a high viscosity or both are
necessary to prevent flow within the adhesive
(Wake, 1982). Thus, these are
useful measures for characterizing gel mechanics. It is important to note,
however, that the overall adhesive strength also depends upon interfacial
adhesion and the ability to dissipate energy by deformation
(Gay, 2002).
Samples were tested in shear between 25 mm parallel plates at room temperature. They were tested at a strain of 5% and a frequency of 10 rad s–1. Based on repeated measurements of materials with a constant modulus, the precision of the rheometer was within 0.5–1 Pa.
Agar (0.5%) and citrus pectin (2%) were tested since they responded well and were easy to manipulate. Powdered polymers were mixed with either glue proteins or with BSA in dialysis solution. The glue proteins were tested at concentrations ranging from 0.1 to 2 mg ml–1. Agar samples were loaded on the rheometer while still liquid and analyzed for 10 min. Since the modulus increased for several minutes, then leveled off as the agar gelled, the peak values for each trial were recorded. Pectin samples were fully gelled when loaded and 5–10 measurements were taken per sample. These were consistent and showed no increasing trend, so the average values were recorded. Values for agar were compared using non-parametric statistics, as the variation did not follow a normal distribution.
To determine the importance of charge, other gel-forming polymers were also tested quantitatively. In these experiments, the glue proteins were used at a concentration of 1 mg ml–1. The effect of the glue proteins on 2% citrus pectin and 2% apple pectin was compared. Similarly, the effects on 0.6% agar and 0.2% agarose were compared and the effects on 2% carboxymethylcellulose (high viscosity grade) and 2% methylcellulose (high viscosity grade) were compared. In each pair, the gel-forming polymers were structurally similar except that the former was more negatively charged than the latter.
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The effect of the glue proteins was concentration-dependent. For both agar and polygalacturonic acid, the firmest gels were formed with roughly 1–2 mg ml–1 of glue proteins. With polygalacturonic acid, all 13 trials with 1–3 mg ml–1 of glue proteins showed a clear effect. At lower concentrations (0.1–0.6 mg ml–1), only two of five trials showed a clear effect. At higher concentrations (near 5 mg ml–1) there was no clear effect in three trials.
Changing the structure of the glue proteins by breaking their disulfide bonds with DTT correspondingly weakened their effect. Instead of forming a firm gel with a distinct shape, the treated samples formed loose clumps of gel that were only slightly stiffer than the control. In contrast, treatment with DTT had no effect on control gels, which were uniformly loose and barely gelled.
Identification of glue proteins in other species
As was true of periwinkles and limpets, there were specific proteins
correlated with adhesion in the mucus of the slug A. subfuscus
(Fig. 3A). There were six major
protein bands ranging from 10 to 200 kDa that were found in both the trail and
adhesive mucus. Two proteins of roughly 15 kDa and61 kDa, however, were
consistently associated with adhesion. They were, respectively, 1.8 and 2.2
times as concentrated in the adhesive mucus (paired Student's t-test,
N=6, P=0.02 and 0.04, respectively). This difference would
probably have been greater but for contamination of trail mucus samples with
adhesive mucus during collection. In contrast, none of the other proteins
differed significantly between the two types of mucus. As was true with
periwinkles, the glue proteins made up a substantial fraction of the mucus. In
the adhesive mucus, the 15 kDa protein accounted for 47% of the protein on
SDS-PAGE, based on staining intensity, while the 61 kDa protein accounted for
8%. Both of these proteins were associated with fainter bands that traveled
slightly farther in the gel and whose staining intensity seemed to correspond
with the larger proteins.
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In addition to the proteins that were small enough to appear on SDS-PAGE, a substantial amount of protein eluted from the gel filtration column with an apparent molecular mass in the MDa range. This fraction was variable in magnitude but roughly equal to half of the organic material in the adhesive mucus. This high molecular mass fraction was primarily proteinaceous with some associated carbohydrates. It should be noted, however, that the carbohydrates seemed to be lost more readily through the procedure and were less readily detected due to the specificity of the staining reaction. Thus, the carbohydrate content may have been underestimated.
The difference between adhesive and trail mucus was even more striking in the land snail H. aspersa (Fig. 3B). The trail mucus had few proteins in the size range that could be visualized on SDS-PAGE. In contrast, the adhesive mucus had prominent protein bands at molecular weights of approximately 82, 97 and 175 kDa. As with periwinkles and slugs, the results from the gel filtration column showed that the adhesive also had an MDa-sized component. The amount of this large molecular mass fraction varied considerably, but was often roughly similar to the amount of the glue proteins. It appeared to be primarily proteinaceous, though there were also carbohydrates, as with the slug adhesive. It should be noted that in all three species tested, the MDa-sized polymers constitute roughly half of the organic material of the adhesive mucus. The glue proteins make up the rest in H. aspersa and L. irrorata, and roughly half of the rest in A. subfuscus.
Qualitative effects of H. aspersa and A. subfuscus glue proteins
As with L. irrorata glue proteins, the glue proteins from A.
subfuscus and H. aspersa stiffened gels. The 15 kDa protein from
A. subfuscus triggered citrus pectin to gel, while other proteins
that were equally common to both the trail and adhesive mucus did not appear
to have this effect. The purity of the fractions was not sufficient to be
certain of this, however. Gel filtration resulted in early fractions enriched
in proteins larger than 500 kDa, middle fractions enriched in proteins ranging
from 40 to 500 kDa, and later fractions that contained primarily the 15 kDa
glue protein. The 15 kDa protein, however, was present to a lesser extent in
all earlier fractions as well. In fact, while most of this protein eluted as
expected based on its mass, SDS-PAGE analysis of the pooled fractions showed
that a significant peak of this protein also eluted in the void volume with
the MDa-sized polymers. This suggests that it may bind to the larger polymers,
even under the dissociating conditions used here. In any case, the extent to
which pooled column fractions with the same overall protein concentration
triggered gel formation was roughly proportional to the amount of 15 kDa
protein in the fraction. When the overall protein concentration of the pooled
fractions was roughly 0.1 mg ml–1, only the last fractions
containing primarily the 15 kDa protein triggered gelling. In another set of
tests with all the fractions concentrated to 0.3 mg ml–1, the
last fractions again triggered strong gelling. In addition, the fraction
containing the MDa-sized polymers, which contained roughly half as much of the
15 kDa protein as the last fractions, also triggered strong gelling. The other
fractions, which had less than a third as much of the 15 kDa protein, only
caused the formation of softer, looser lumps of gel.
For H. aspersa, the primary protein peak contained the 82, 97 and 175 kDa glue proteins and little else. These triggered gel formation in agar, while fractions containing the MDa-sized polymers at the same concentration had no qualitative effect.
Quantitative effects of H. aspersa glue proteins
Quantitative measurements confirmed the qualitative results. The land snail
glue proteins had a strong effect on gels, and this effect was concentration
dependent (Fig. 4). There was a
clear linear increase in pectin stiffness as the glue protein concentration
increased from 0.1 to 1.3 mg ml–1. In this range, the slope
was significantly different from zero (linear regression;
P=2x10–5, r2=0.75). At
higher concentrations, the effect dropped off. Note that at 1.3 mg
ml–1 the glue proteins constituted 6% of the total organic
material.
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The results were similar but more variable with agar. At low concentrations
of glue proteins (
0.5 mg ml–1), there was no significant
difference from controls (Wilcoxon two-sample test; P>0.1,
N=8). There was a clear effect in some samples, but it was not
consistent. At concentrations of 0.75–2 mg ml–1, the
glue proteins increased the storage modulus of agar by a factor of 4.2
(P<0.001, N=13). The storage modulus was greatest with
glue protein concentrations of 1–1.85 mg ml–1
(17–27% of the total organic material) and weakened at 2 mg
ml–1 where the mean stiffness was only slightly higher than
that of the control. The loss modulus (viscosity) followed the same pattern as
the storage modulus in this and all other quantitative experiments, though the
values were typically much lower and in the range where the rheometer was less
accurate.
The glue proteins worked best with negatively charged polymers. While the H. aspersa glue proteins clearly stiffened agar, they had no significant effect on agarose (Fig. 5A). The primary difference between agarose and agar is that agarose is neutral, while agar also contains sulfated and acetylated sugars. Similarly, the glue proteins caused a 27-fold increase in the stiffness of citrus pectin, but no change in the stiffness of apple pectin (Fig. 5B). The glue proteins did, however increase the loss modulus of apple pectin from 1.0±0.2 Pa to 1.8±0.1 Pa (t-test, P=0.0005). The primary structural difference between these two forms of pectin is that apple pectin is more methylated, blocking a larger fraction of its negative charge. Finally, methylcellulose, which is neutral, had lower moduli with the glue proteins, though it went into solution more rapidly. With negatively charged carboxymethylcellulose, the glue proteins had no effect on the measured stiffness (Fig. 5C). In six of the seven comparisons with carboxymethylcellulose, however, the samples with glue proteins appeared lumpier, somewhat stiffer and more gelled than the controls. Thus, the lack of a change in modulus may be due to measurement error resulting from sample inhomogeneity. For example, the presence of clumps could break up the sample and lower the measured modulus. In addition, the glue proteins caused substantial bubble formation during sample mixing and these bubbles were trapped within the high viscosity cellulosic samples. The presence of trapped bubbles would presumably lower the measured moduli. The carboxymethylcellulose did become sticky with the glue proteins, adhering to the spatula used to spread them on the test plates. This was the only material that responded in this way.
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In addition to the effect on gel mechanics, the glue proteins from all species created a characteristic increase in the ability of a solution to wet any surface. Solutions containing the glue proteins were easily recognizable by their ability to wet plastic and acrylic (Plexiglass) surfaces fully, forming a thin film that stayed spread on the surface, while solutions containing other proteins beaded up.
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The mechanism of action of molluscan glue proteins
The experiments described in this paper effectively rule out several other
possible interpretations of the difference in structure between adhesive and
trail mucus. It seems unlikely that the proteins serve as enzymes that act on
the other polymers in the mucus. If they were, one would not expect them to
affect unrelated polymers such as agar and pectin. Furthermore, the changes
often occur almost instantly, which would not be expected for enzymes. Given
these observations and the fact that the glue proteins make up as much as half
of the secretion, it seems likely that they play a direct structural role. It
is unlikely, though, that the proteins act independently of the other polymers
in the gel. Even at a concentration of 5 mg ml–1 (0.5%), a
solution of glue proteins has roughly the same viscosity as water. It is only
in concert with other gel-forming polymers that an effect is seen. It is still
uncertain whether the glue proteins cross-link the trail mucus polymers and
thus literally convert the non-adhesive mucus into a glue, or whether they
crosslink large polymers in the adhesive mucus that may differ from the trail
polymers in an as yet undetermined way. Because the glue proteins are
non-specific, they may work with any large polymer in mucus, but some
modifications of the large polymer may improve the adhesiveness.
The non-specific action of the glue proteins is intriguing. They appear to
be able to cross-link a variety of carbohydrates. They appear to work best
with large, negatively charged polymers. This would make sense given that the
mechanics of molluscan mucus depend heavily on giant carbohydrate-rich
molecules with substantial negative charge due to the presence of sulfated
sugars or uronic acids (Denny,
1983). These findings suggest that the glue proteins have charged
regions that mediate ionic cross-links. Consistent with this, Smith et al.
(1999) and Smith and Morin
(2002) found that the glue
proteins of limpets and periwinkles have a large number of charged amino
acids. Overall, the proteins are acidic, but they may have regions with
substantial positive charge, since they contain 15–17% basic amino acid
residues. The fact that the glue proteins affected both agar and
polygalacturonic acid was interesting. Both of these are capable of gelling,
but while polygalacturonic acid normally forms electrovalent cross-links, agar
depends on association between helical regions
(Williams and Phillips, 2000).
Perhaps the glue proteins provide additional crosslinks to stabilize the ones
that typically form. It is worth noting that the effect on agar was not as
large as the effect on more highly charged citrus pectin or polygalacturonic
acid.
Several other characteristics of the glue proteins may give insight into
their nature. The fact that they increased the ability of solutions to wet
surfaces makes sense since an adhesive must be able to bond to an interface in
addition to stiffening the bulk adhesive. Since the proteins also triggered
substantial foaming, one or more of them may have some surfactant properties.
This may also explain why they caused the neutral polysaccharides to go into
solution more rapidly. A structure involving separate non-polar and charged
regions would be intriguing. Several other researchers have had success
developing gels from synthetic block copolymers with this type of structure
(Petka et al., 1998;
Nowak et al., 2002).
Another aspect to consider is the possibility that different glue proteins
within a secretion play different roles. In the species tested, there was
typically one glue protein that was present in large quantities, along with
one or two others. It is possible that the others are minor variants of the
major glue protein. There is some evidence from amino acid composition and
isoelectric points that the two glue proteins in periwinkles are related, and
some evidence that the two glue proteins in limpets may be related as well
(Smith et al., 1999;
Smith and Morin, 2002). In
some cases, the glue protein forms a dark band in SDS-PAGE with one or two
fainter bands below it. These may be different size variants, possibly
differing in degree of glycosylation. As yet, though, there is no indication
of whether or not the different proteins have different roles. In this study,
no attempt was made to separate the different glue proteins from each other.
Hence, for example, we know that together the 41 and 36 kDa proteins from
periwinkle glue affect gels, but whether each can work on its own is
unknown.
While the effect of the glue proteins depended on their concentration, the
effect weakened at concentrations above approximately 1.5 mg
ml–1. There are several possible explanations for this. As
noted in the Materials and methods, it is difficult to achieve much higher
concentrations in the initial extract. This may be partly due to a tendency to
aggregate, as demonstrated for limpet glue proteins
(Smith et al., 1999). Thus,
while it may be possible to concentrate the proteins beyond 1.5 mg
ml–1 after chromatography, they may begin to interact with
each other and cease functioning normally. The weakened effect at higher
concentrations may also have been an artifact of the experimental system;
there may have been problems because of inhomogeneous mixing and the use of
commercial gel-forming polymers rather than the native ones.
It should be noted that this type of concentration dependence is not unusual for an adhesive. In commercial adhesives consisting of two components, there is often an optimum ratio of the components. Given this, it is worth noting the ratio of glue proteins to large polymers in the adhesive mucus of the molluscs that have been studied. The glue proteins from the land snail, slug and periwinkle constitute roughly 25–50% by mass of the total organic material. In limpets, the glue proteins make up a somewhat smaller percentage of the whole. With the gel-forming polymers in this study, the glue proteins worked best when they constituted roughly 6–27% of the total material. This suggests that the glue proteins should make up a substantial fraction of the material, but not more than half.
Finally, the use of commercial polymers may lead to an underestimate of the effectiveness of the glue proteins. There are many characteristics of gel-forming polymers that could impact their response to a potential cross-linker. For example, they may have structural features or charge distributions that are not ideally suited for interactions with the glue proteins. The glue proteins may also directly or indirectly change the structure of the gel-forming polymers. For instance, they may cause the polymers to aggregate or to take on a less extended conformation. Thus, they could actually weaken the gel. Given this, it will be important to characterize more precisely the features of the mucus gel-forming polymers that are important for adhesion.
Different animals that use glue proteins to modify gel mechanics
The identification of gel-stiffening glue proteins in A. subfuscus
and H. aspersa broadens the potential scope of this adhesive
mechanism. To date, four molluscan species have been tested, all from very
different environments. L. irrorata lives in intertidal salt marshes
attached to blades of marsh grass. Limpets live in the rocky wave-swept
intertidal and are subject to strong wave forces. Both alternate between glued
and active states with a periodicity of several hours
(Smith et al., 1999;
Smith and Morin, 2002). In
contrast, the slug A. subfuscus lives in temperate forests and
gardens and rapidly secretes a sticky, orange mucus when disturbed. This
suggests that it is a defense mechanism. Finally, the land snail H.
aspersa also lives in temperate forests, but secretes a sheet of mucus
around its aperture to form a dried attachment that can last for months. This
sheet is called an epiphragm, and in addition to adhesion, it contributes to
desiccation resistance (Campion,
1961; Barnhart,
1983). Though the adhesive mucus is used in different environments
for different purposes, in all four cases there were similar changes in mucus
structure between non-adhesive mucus and adhesive mucus. In the three species
tested, the glue proteins had a similar effect on gel mechanics. Thus, it is
likely that this is a commonly used mechanism for controlling gel mechanics
among invertebrates.
It is intriguing that there were differences in the size of the glue
proteins and the carbohydrate content of the MDa-sized polymers of the
adhesive. Such differences may play a role in the mechanics. For example, a 15
kDa protein may spread through the gel more rapidly than a 100 kDa protein,
causing the gel to set sooner. Given the differences in environment and
functional needs, it will be interesting to compare the effectiveness of each
glue protein quantitatively. It will also be interesting to look for similar
proteins in other animals. Many marine invertebrates such as molluscs,
interstitial worms and echinoderms secrete adhesive gels
(Smith, 2002). Echinoderms are
particularly interesting because of the strength of their adhesion and their
ability to stay attached for extended periods or let go rapidly. Echinoderms
are also interesting since they have been shown to stiffen their dermis with a
system that may be analogous; holothurians appear to cross-link large collagen
fibers with a proteinaceous `stiffening factor'
(Trotter and Koob, 1995;
Koob et al., 1999;
Trotter et al., 2000).
Summary and future work
This research has clear practical implications. The adhesive gels produced
by molluscs form strong attachments in wet, irregular environments, using a
minimum of organic material. Adhesives used by marine animals are likely to
have unusual and useful characteristics because of the demands of adhesion
underwater (Waite, 2002).
Furthermore, there is substantial interest in the development of gels with
unusual properties, particularly for applications such as drug delivery and
biomedical adhesives (Petka et al.,
1998; Miyata et al.,
1999; Wang et al.,
1999; Peppas et al.,
2000; Nowak et al.,
2002), as well as food science
(Williams and Phillips, 2000).
Despite this potential, there has been little research on the gels produced by
animals. The results described in this paper provide the first direct evidence
of a mechanism by which a dilute gel can become adhesive. Until recently,
almost no work has been done linking changes in biochemical structure to
function in these kind of gels (Davies and
Hawkins, 1998). Now, specific differences in composition have been
shown to control gel mechanics. Further work needs to be done to determine the
structure of these glue proteins and to elucidate how they act on the
gel-forming polymers. Also, in addition to changes in stiffness, other
characteristics of the adhesive mucus should be investigated, such as the
ability to bond effectively to the substrate and the ability to dissipate
fracture energy. Finally, other animals should be investigated, as the
addition of similar proteins to control gel mechanics may be a widespread
phenomenon.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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