A molecular, morphometric and mechanical comparison of the structural elements of byssus from Mytilus edulis and Mytilus galloprovincialis
Marine Science Institute and Molecular, Cellular and Developmental, Biology Department, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
* Author for correspondence (e-mail: lucas{at}lifesci.ucsb.edu )
Accepted 5 April 2002
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Summary |
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M. edulis threads are typically twice the length and diameter of M. galloprovincialis threads and appear to contain nearly 10 % more collagen. These differences are maintained even when the different thread portions are compared. Despite differences in a number of parameters, most notably that whole M. galloprovincialis threads are stiffer, threads whether whole or separated into proximal and distal portions, have similar mechanical behaviors. It is apparent from this comparison that M. galloprovincialis and M. edulis are seemingly interchangeable models for byssal research.
Key words: byssus, byssal thread, collagen, mussel, Mytilus edulis, Mytilus galloprovincialis, cDNA
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Introduction |
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The byssus is secreted by the foot and can be morphologically separated
into three distinct regions; the plaque, the thread and the stem
(Bairati, 1991). The plaque,
the most distal portion of the byssus, is the direct point of attachment of
the mussel to its surroundings. Interesting in its own right, because of its
underwater adhesive properties, the plaque's contribution to the mechanical
strength of the byssus is predominantly in its bonding ability
(Waite, 1983
). The stem, at
the most proximal end of the byssus, not only serves as an attachment point
for multiple byssal threads but also mediates the direct connection between
the non-living extraorganismal byssal threads and the living tissues of the
mussel. Indeed, the acellular byssus originates from a fusion of retractor
muscles at the base of the foot via the stem, in this way providing
an anchor point as well as allowing for a certain degree of rotation and
tension control.
The thread can be further subdivided into proximal and distal regions. The
delineation of proximal and distal portions of the thread has historically
relied strictly on morphological observations, with the proximal region being
described as corrugated and the distal as smooth
(Brown, 1952). However, such
descriptive partitions may not be nearly so clear-cut. The transition zone
between the two regions is almost certainly not a clean interface, but instead
reflects a graded change in the morphology and mechanical behavior of the
thread. Demarcations based on the mechanical or biochemical properties of the
two regions would seem to be more revealing about the actual discrimination of
proximal from distal thread.
The mechanical properties of excised and separated proximal and distal
thread portions indicate that the thread is a hybrid structure. The proximal
portion is elastic, while the distal portion is stiff with somewhat peculiar
stress-softening and self-healing properties
(Vaccaro and Waite, 2001).
These mechanical generalizations belie the underlying biochemistry of the
disparate segments of the threads. It has been shown that the main structural
components of byssal threads are a series of three collagens with
block-copolymer-like domains (Coyne et al.,
1997
; Qin et al.,
1997
; Qin and Waite,
1998
). Furthermore, two of these collagens are distributed in a
gradient fashion along the length of the thread
(Qin and Waite, 1995
). Byssal
precollagen P (preCol-P) is most abundant in the proximal portion of the
thread with a decreasing distally directed gradient. Complementary to that is
an increasing gradient of preCol-D in a proximal to distal direction. The
third collagen, byssal precollagen NG (preCol-NG), is present along the entire
length of the thread (Qin and Waite,
1998
). Using fiber X-ray diffraction, Mercer
(1952
) demonstrated the
existence of collagen fibers in mussel byssus. However, the fibrillar
arrangement of these molecules in the thread remains unknown, leading to a
number of proposed models (Qin and Waite,
1995
,
1998
;
Vaccaro and Waite, 2001
; Waite
et al., 1998
,
2002
). In addition, it has
been suggested that the fibrillogenesis of byssal collagens may involve an
amino-acid-sequence-dependent self-assembly mechanism.
The initial characterization of byssal collagens from Mytilus
edulis suggested a structure/function-type relationship, that is, the
organization and mechanical properties of the underlying structural elements
should reflect the properties of the byssal thread as a whole. Each of these
proteins has a central collagen domain, consisting of between 437 and 521
amino acids, flanked on either side by a unique set of structural motifs
(Waite et al., 1998). The
N-terminal flanking domains of preCol-P consist of a histidine-rich region
followed by an elastic domain, while its C-terminal side contains an acid
patch, a second elastic domain and a terminal histidine-rich region. In a
parallel fashion, preCol-D has both of the histidine-rich domains and the
acidic cluster but the elastic domains are replaced with silk-like regions,
i.e. spider ampullate silk. Four residues of 3,4-dihydroxyphenylalanine (DOPA)
have been detected in the N-terminal flanking domain of preCol-D. DOPA is a
prime candidate for forming cross-links in the byssal threads and is abundant
in a number of other byssal-related proteins
(Waite, 1999
). The
histidine-rich regions and the acid patch are present in preCol-NG, but the
elastic domains are replaced with plant-cell-wall-like sequence motifs. A
further peculiarity arises within the collagen domains of each molecule.
PreCol-NG and preCol-P each exhibit single sequence breaks in the
triple-helical Gly-X-Y repeat. Models indicate that these breaks
cause structural bends or kinks in the collagen triple helix
(Waite et al., 2002
). PreCol-D
has three such kinks. What role these aberrations play in the functionality of
the individual molecules and the byssus as a whole remains a matter of
speculation.
Mytilus edulis has served as the primary model organism for
byssus-related studies, with a paucity of comparative studies in M.
galloprovincialis, M. trossulus and M. californianus
(Bell and Gosline, 1996). It is
generally accepted that M. edulis is the ancestral species from which
the other mytilids have evolved, with M. galloprovincialis and M.
trossulus often being included in the `edulis complex'
(Gosling, 1992
). Even though
hybridization and introgression do occur amongst these groups, they are
nonetheless considered to be distinct species
(Beynon and Skibinski, 1996
).
While hybridization zones do occur, most notably, but not exclusively, in
Japan (Inoue et al., 1997
;
Matsumasa et al., 1999
),
California (Rawson et al.,
1999
) and the British Isles
(Rawson et al., 1996
), each
species tends to be geographically separated from the others. M.
galloprovincialis predominantly inhabits the warmer waters of temperate
latitudes, M. edulis is found in the colder waters of temperate
latitudes and M. trossulus is usually found in the colder waters of
the northern latitudes. Where hybridization zones do occur, M.
galloprovincialis is frequently found in more exposed areas than M.
edulis (Skibinski et al.,
1983
).
Despite genetic and physiological differences among the species
(Hilbish et al., 1994), it has
been suggested that the byssal threads from M. galloprovincialis are
morphologically and mechanically similar to those of the closely related
M. edulis. However, these studies have relied on comparisons between
data from the literature that are often inconsistent
(Smeathers and Vincent, 1979
;
Price, 1981
;
Bell and Gosline, 1996
). No
thorough comparison between the byssal threads of these two mussel species
exists. Ultimately, similarities or differences in the mechanical and
morphometric properties of byssal threads must be correlated with the
underlying biomolecular structural components of the system, in this case the
three byssal collagens.
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Materials and methods |
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RNA extraction and cDNA library construction
RNA was extracted using the Rneasy Plant Mini Kit from Qiagen (Valencia,
CA, USA). In general, two freshly dissected feet from Mytilus
galloprovincialis were used per extraction, and the manufacturer's
protocols were followed after initial tissue disruption under liquid nitrogen
in a mortar and pestle. Purified RNA was ultimately reverse-transcribed and
packaged into a lambda ZAP Express cDNA library (Stratagene, La Jolla, CA,
USA) following standard protocols. This library served as a readily available
source of cDNA.
PCR and cloning of the byssal collagens
A series of PCR primers was constructed for each byssal collagen on the
basis of available gene sequences from Mytilus edulis; the M.
galloprovincialis cDNA library served as template in each reaction. A
3' vector-specific primer was used to obtain the sequence of the
3' untranslated regions.
The initial set of forward primers for preCol-D was as follows: MeDF1-ATCAACATGGTCTACAAACTC, MeDF2-CAGGACATGCCGGTAAACACGGAAC and MeDF3-GTGGTATGGGTAGACGAG. Reverse primers were designed as follows: MeDR1-GTTCCGTGTTTACCGGCATGTCCTG, MeDR2-CTCGTGGTCCTGCTGGTCCTTGT and MeDR3-AACACTTGCAGATTTTATTGATA.
Forward primers for preCol-NG were synthesized as: MeNGF1-ATGGTCCATAATTTCCTGACT, MeNGF2-TTACCAGGTGCACCCGGA and MeNGF3-AAGGAGAACTTGGACCAGTCG. The reverse primers were: MeNGR1-TCCGGGTGCACCTGGTAA, MeNGR2-CGACTGGTCCAAGTTCTCCTT and MeNGR3-AGGAACTTGCACTTTTTAT.
PreCol-P primers were as follows: MePF1-ATGGTTCGGTTTTCCCTAGC, MePF2-GAGGATTCGGTGGACCAGGTAC and MePF3-GTGGCCCAGCAGGTCCAAGA. The reverse primers were: MePR1-TTGGTCCAATTAATCCGATGA, MePR2-GAATAACACCTGGTGCTCCT and MePR3-ACGAAGACTGCAGATTTTAATA.
PCR was performed using standard conditions; buffer, dNTPs and Taq polymerase were from Qiagen. Reaction conditions consisted of a 30s denaturation step at 95°C followed by a 1 min annealing at 50°C with a 2 min elongation at 72°C. This cycle was repeated 35 times. PCR products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Bands of the expected size were excised and gel-eluted using the Qiagen gel purification kit and ligated into Promega's (Madison, WI, USA) pGEMT Easy cloning vector. JM109 cells were transformed and plated, and positive clones were selected after blue/white screening. Positive clones were grown overnight in LB medium, and plasmids were purified using the plasmid purification kit from Qiagen. PreCol inserts were sequenced using vector-specific primers for M13 and SP6 primer sites at the Advanced Instrumentation center of the University of California, Santa Barbara.
RT-PCR and 5'-RACE
RNA was purified from mussel feet as previously described and used to
obtain 5' untranslated sequence information. The GeneRacer kit
(Invitrogen, Carlsbad, CA, USA) was used to obtain sequence information from
full-length transcripts only. In each case, previously synthesized
gene-specific primers against Mytilus edulis sequences were
sufficient for use in 5'-rapid amplification of cDNA ends (RACE)
reactions when coupled with linker-specific primers. Reaction products were
gel-eluted, cloned and sequenced as previously described for standard PCR
reactions.
Byssal collagen sequence comparisons
Previously published Mytilus edulis byssal collagen sequences were
obtained from the GenBank database. The following is the list of accession
numbers for M. edulis preCol sequences; AF029249, preCol-D
(Qin et al., 1997); AF043944,
preCol-NG (Qin and Waite,
1998
); and AF015539, preCol-P
(Coyne et al., 1997
). cDNA
sequences were aligned and translated using Sequencher 3.0 (Gene Codes Corp.,
Ann Arbor, MI, USA). Comparisons between species-specific byssal collagens
were performed using an online version of Clustal W from the European
Bioinfomatics Institute (Thompson et al.,
1994
).
Byssus collection
To ensure that full-length byssal threads were collected from each species,
individual mussels suspended from a Plexiglas plate were killed by inserting a
scalpel between the two halves of the shell and severing the adductor muscles.
The stem was then removed, and single threads were isolated at their
attachment point to the stem. Unless indicated otherwise, all measurements,
both physical and mechanical, encompass the entire length of the thread from
the stem to the point at which the thread joins the top of the plaque but did
not include the plaque.
Morphometric characteristics of byssal threads
Thread dimensions were measured using a stereomicroscope (M3Z, Wild,
Switzerland) equipped with a graticule. After total thread length had been
determined, each thread was separated into its proximal and distal portions as
determined by the point at which its corrugated appearance became smooth. The
diameter of each portion was then determined at its widest point since some
degree of variability in diameter is evident along the length of each
segment.
Determination of collagen content and gradient in byssal threads
The percentage content of collagen in the proximal and distal portions of
the byssal thread was estimated by quantifying the amount of hydroxyproline.
Individual portions of either proximal or distal thread sections were placed
in an ampule with 0.1 ml of 6 mol l-1 HCl and 0.01 ml of
redistilled phenol. The threads were hydrolysed in vacuo for 24 h at
110°C. Samples were then flash-evaporated at 60°C. Amino acids were
quantitated with a Beckman System 6300 analyzer using the modified elution
program described previously by Waite
(1995).
Percentage collagen content was determined by assuming that all proline
residues in the Y position of the Gly-X-Y motif are
converted to hydroxyproline. Qin and Waite
(1995) and Qin et al.
(1997
) demonstrated this
tendency in both preCol-D and preCol-P from Mytilus edulis. From
this, a sequence-determined percentage hydroxyproline value is calculated for
each preCol. A mean sequence-derived hydroxyproline content was calculated for
the proximal portion by averaging the hydroxyproline content of preCol-NG and
preCol-P. A similar value was derived for the distal region by averaging the
hydroxyproline content of preCol-NG and preCol-D. The percentage
hydroxyproline content for each thread portion, determined by acid hydrolysis,
was then divided by the sequence-determined mean for each thread portion. The
resulting value approximates the byssal collagen content of each thread
portion. This does not account for the contribution of hydroxyproline from
foot protein-1 (FP-1) to acidhydrolysed samples. The FP-1 of M.
edulis contains 10% hydroxyproline
(Taylor et al., 1994
). Given
the identical consensus decapeptide repeats in FP-1 from both species
(Inoue and Odo, 1994
), the
proportion of hydroxyproline is not likely to differ greatly in M.
galloprovincialis.
Following the procedure established by Mascolo and Waite
(1986) to determine whether a
collagen gradient occurs along the length of a byssal thread based upon
changes in hydroxyproline, proline and glycine content, Mytilus
galloprovincialis threads were sequentially cut into 0.25 cm segments
starting at the stem. The final segment before the plaque was often shorter
than 0.25 cm. Five threads with a mean length of 2.0±0.15 cm (mean
± S.E.M.) were analyzed. Each segment was acid-hydrolysed, and the
amino acids were quantified as described previously. For comparison, M.
edulis threads were also used, but their mean length was 3.0±0.20
cm (mean ± S.E.M., N=5).
Biomechanical properties of byssal threads
Young threads, 2-3 days post-deposition, were utilized for mechanical
studies. Mechanical properties were measured for whole threads and for the
separated distal and proximal portions. Newly collected threads were allowed
to rest in filtered sterilized sea water for up to 24 h before testing. In an
effort to avoid grip slippage during extension, the ends of each thread
portion were sandwiched between double-sided tape. Dehydration during testing
was prevented by enclosing one end and approximately three-quarters of the
thread in a polyethylene bag with filtered sterilized sea water. Both ends
were then clamped into the grips of a Bionix 200 tensile tester (MTS Systems,
Cary, NC, USA) equipped with a 10 N load cell. Biomechanical variables were
measured using a crosshead speed of 5 mm min-1 and an initial gauge
length of between 5 and 10 mm for distal and whole threads and between 3 and 6
mm for proximal thread portions. Threads were stretched to their breaking
point. Cyclical testing of threads was also performed as described by Vaccaro
and Waite (2001). Young's
modulus for whole threads and the distal region was measured as the slope of
the linear portion of the stress/strain curve at low strain (strain <10%).
For the proximal portion of the thread, Young's modulus was measured at the
steepest portion of the stress/strain curve. Stress and strain are defined as
the load per cross-sectional area (in N m-2=Pa) and the change in
length per initial length, respectively. No correction for the cross-sectional
area was applied. The yield point offsets were measured using the `zero-slope'
method, i.e. at the first point at which the slope of the stress/strain curve
is asymptotic (Ferry, 1980
).
The strain energy was determined by integrating the area under the curve.
Statistical significance was established by single-factor analysis of variance
(
=0.05).
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Results |
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Despite the high degree of identity for preCol-D between Mytilus galloprovincialis and M. edulis, a number of potentially important differences exist (Fig. 1). Of note is an additional histidine in the N-terminal flanking domain and two additional histidines in the C-terminal histidine-rich domain from M. galloprovincialis. Furthermore, the M. galloprovincialis sequence has fewer glycine clusters in the silk-like domains with the pattern XGG (where X=L, A, F or V), 40 versus 29.
Several key differences are also found in the collagen flanking domains of preCol-NG (Fig. 2). Perhaps of greatest importance is an extra poly-A run in the N-terminal plant-cell-wall-type domain of Mytilus galloprovincialis. It is presumed that this stretch of amino acids would serve to stiffen the molecule. Also, a single additional histidine is found in its C-terminal histidine-rich domain. Individual preCol-D and preCol-NG chains have cysteine residues whose location is maintained between the species. How these couple to form disulfide bonds is unknown, but such cross-links may prove crucial in mature byssal threads.
When comparing the byssal preCol-P protein sequences (Fig. 3), differences in histidine content are evident, with an additional three residues occurring in the N-terminal flanking regions and another three in the C-terminal flanking domains in Mytilus galloprovincialis. Also evident are fewer glycine clusters with the pattern ZGG (where Z=I, F, V and A) in M. galloprovincialis, 31 versus 39 for M. edulis.
While these differences may prove to have functional significance, e.g. the presence of more histidine residues may give the byssal collagens from Mytilus galloprovincialis a greater potential to form metal-chelating cross-links, it is the similarities that may underscore the true benefit of constructing a material out of these three collagen molecules. For each molecule, the acid patch region remains unchanged except for a single isoleucine/valine difference in preCol-P. A similar absence of differences is found when comparing the collagen domains of the molecules. PreCol-NG and preCol-P have only a few substitution differences, and preCol-D from M. galloprovincialis has a 12-amino-acid insertion, although the integrity of the collagen sequence is maintained. Furthermore, each collagen domain has exactly the same breaks in the Gly-X-Y repeat sequence, suggesting that the molecules from both species will have similar kinked structures and properties.
Morphometric characteristics of byssal threads
Table 1 shows a comparison
of the physical dimensions of byssal threads from Mytilus edulis and
M. galloprovincialis. For each species, 25 individual threads were
measured. Of particular note, the overall thread length in M. edulis
is approximately double the length of M. galloprovincialis threads.
Furthermore, the elastic proximal region makes up a higher percentage of the
thread length in M. edulis. A similar result is seen when diameter is
compared. Both the proximal and distal portions of M. edulis threads
are approximately double the diameter of the corresponding diameters for
M. galloprovincialis threads. Furthermore, the diameter of the
proximal portion is 36% and 20% larger than that of the distal portion in
M. galloprovincialis and M. edulis, respectively.
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Determination of collagen content and gradient in byssal threads
The byssal collagen content of the respective proximal and distal portions
of threads was calculated on the basis of the hydroxyproline content of
acid-hydrolysed threads without correction for the hydroxyproline content of
FP-1. Representative amino acid compositional analyses are shown in
Table 2. The calculated values
for byssal collagen content of the distal and proximal portions for both
species are shown in Table 1.
Mytilus edulis appears to have a larger concentration of byssal
collagens in both its proximal and distal byssal thread portions compared with
the same thread portions from M. galloprovincialis. In both cases,
the distal thread has approximately 20% more collagen than the proximal
region.
|
The manner in which this apparent difference in the proportion of collagen varies along the length of a thread was investigated by plotting the percentage composition of glycine, proline and hydroxyproline relative to location in the thread from the proximal to the distal end. Fig. 4 shows a comparison of the gradients for Mytilus edulis and M. galloprovincialis threads. As demonstrated by these plots, the collagen content of M. edulis threads appears to increase in a gradual fashion, whereas the gradient is much more abrupt in M. galloprovincialis. The inflection point in the M. galloprovincialis gradient suggests that the transition zone between the proximal and distal regions occurs abruptly over a length scale of a few millimeters, whereas the gradual gradient in M. edulis may extend over several centimeters.
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Biomechanical properties of byssal threads
By testing whole threads as well as their corresponding portions under
tension, several mechanical properties were measured. Representative
stress/strain curves are shown in Fig.
5. Table 3 shows
Young's modulus, the strain at breaking, the strength at breaking and the
strain energy for proximal and distal portions from Mytilus edulis
and M. galloprovincialis. For the distal region, the yield strength
and strain at yield were also determined. Many of the biomechanical properties
do not differ statistically between the species, as determined by
single-factor analysis of variance. However, Young's modulus is higher for the
proximal portion of M. edulis compared with M.
galloprovincialis, while the strain at breaking is lower. This is
indicative of a stronger, stiffer proximal region in M. edulis. For
the distal region, only the yield strain is statistically different, with
M. edulis having a higher strain, which is also indicative of its
increased toughness. Cyclic testing of thread portions was performed as in
Vaccaro and Waite (2001) with
threads repeatedly loaded to 50% of their initial length to determine whether
there was a difference in the hysteresis or recovery of initial modulus in
threads from the two species. No significant differences were observed (data
not shown). The mechanical properties of whole byssal threads from each
species are very similar, with M. galloprovincialis having slightly,
but significantly, lower strain energy.
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Discussion |
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It has been proposed that metal ions, possibly Zn2+,
Cu2+ and Fe2+, play a significant role in the structural
integrity of the thread. An exact comparison between the metal composition of
byssal threads from M. edulis and M galloprovincialis has
not been made. However, Vaccaro and Waite
(2001) demonstrated using
metal chelators, such as EDTA, that such ions are essential for the normal
recovery of threads under cyclic tension. No cross-links have been isolated
from byssal threads; however, McDowell et al.
(1999
) have detected
5,5'-di-DOPA in the byssal plaques of Mytilus edulis using
rotational echo double-resonance nuclear magnetic resonance. Waite
(1990
) has suggested that
similar di-DOPA-or other quinone-based cross-links might also be present in
byssal threads. Such cross-links may exist between byssal collagen molecules
because DOPA has been detected in preCol-D and is possibly a component of the
other preCol variants. It has been surmised that, at least in the case of the
byssal collagens, metal chelate complexes represent a significant
cross-linking alternative. Such complexes involving histidine, DOPA or even
cysteine residues would differ from di-DOPA in their reversible, sacrificial
breakage under tension (Thompson et al.,
2002
).
The sequence of the preCols makes them strong prospects for polyvalent metal interactions given the histidine-rich regions found in both the N and C termini of all three preCols, the DOPA residues in preCol-D and the cysteine residues in preCol-D and preCol-NG. If the sheer number of histidines is any indication of cross-linking potential, then Mytilus galloprovincialis would be expected to produce stronger, stiffer threads by virtue of having more histidines in the flanking domains of all its preCols. Comparisons of the mechanical properties of threads from M. edulis and M. galloprovincialis partially support this conclusion. When the performance of intact threads is considered, M. galloprovincialis threads have a higher Young's modulus that is indicative of a more cross-linked or crystalline structure. This value is somewhat offset by the higher strain energy of M. edulis threads; however, because of variance in the values, the difference in strain energy is not as significant. Overall, mechanically, the threads appear to be fairly similar with only slight differences between the two species. Something more must be going on.
Mytilus galloprovincialis threads are shorter and slimmer than
those of M. edulis which, in conjunction with having a lower
concentration of collagen, may mitigate any increased cross-linking advantage.
Perhaps what is achieved in both cases is a set of mechanical properties
influenced by evolutionary pressures whereby M. galloprovincialis has
found the need to construct a thread with a lower potential for extension.
Recent mathematical modeling of marine organisms (such as mussels) with
flexible attachments suggests that `going with the flow' does not necessarily
reduce dislodgment forces under all conditions
(Denny et al., 1998). In
particular, the strategy of making extensible byssal threads under high-flow
regimes to reduce forces due to drag and lift could, in some instances,
increase inertial forces on the mussel because of momentum. As the degree of
byssal extension will have a direct bearing on the consequent momentum and
inertial energy of the systems, thread length represents perhaps the simplest
way for species to adapt their holdfast to flow.
Although sequence comparisons tend myopically to focus on differences, it
may be the similarities in this case that prove to be most significant. The
flaws in the collagen triple helix are maintained between the species.
Modeling of the collagen domains has revealed that these flaws correspond to
kinks in the collagen structure, thus requiring precise lateral packing for
fibers to form or allowing space for requisite matrix proteins
(Waite et al., 2002).
Imperfections in invertebrate collagens are not uncommon. Flaws in the triple
helix of collagens have been found in the tubeworm Riftia pachyptila
(Mann et al., 1992
), the
cnidarian Hydra vulgaris (Fowler
et al., 2000
) and the marine worm Arenicola marina
(Sicot et al., 1997
). In each
case, the imperfections have been either modeled or clearly demonstrated by
rotary shadowing electron microscopy as kinks or bends in the triple helix.
The function of such bends remains unknown, but clearly they are
well-tolerated and presumably designed to achieve some sort of structural
integrity. The exact nature of the structural packing of byssal collagens
remains elusive, but it is almost certainly key to understanding the
mechanical properties displayed by byssal threads.
In all three sets of byssal collagens, the acidic domains remain virtually
unchanged between species, except for a single isoleucine/valine difference in
preCol-P. This lack of variability indicates a high degree of evolutionary
pressure to maintain this sequence among species. It has been suggested that
the acid patch in spider silks may coordinate the self-assembly of these
fibers (Hayashi et al., 1999;
Xu and Lewis, 1990
). While
only conjecture, a similar role is conceivable in mussel byssus. Furthermore,
the XGG (where X=L, A, F or V) repeat pattern found in the
silk-fibroin domains of preCol-D has been implicated in forming the extended
flexible secondary structure of flagelliform silks and may provide both silk
and byssus with their abilities to stretch and recoil
(Hayashi and Lewis, 2001
).
Suresh (2001) reviews the
benefits of constructing graded materials and discusses how many biomaterials
may be capable of serving as models for the construction of synthetic
materials. Byssal threads clearly fall into the category of graded materials.
Through the manipulation of gradients at the macro, micro and molecular
levels, mytilids have constructed a material, the byssus, that adheres under
water, can resist periodic exposure to the sun and air, yet is able to
maintain its ability to stretch and `self-heal' in the face of repeated
deformation by waves (Waite et al.,
2002
). At the macroscopic level, the plaque, the distal and
proximal thread, the stem and the retractor muscle form a graded series of
differing materials that are carefully transitioned to produce a functional
thread. At the microscopic level, collagen fibers are aligned and interspersed
with matrix proteins in such a way that their interactions mediate stresses
along the thread and allow for some degree of reorganization or
`self-healing'. Finally, at the molecular level, individual preCol molecules
of at least three types, each with possible multiple variants, are carefully
titrated along the length of the thread to achieve the desired degree of
inter- and intramolecular cross-linking. A comprehensive understanding of the
structure may prove to be an ideal model for the synthesis of materials with
similar `self-healing' properties.
Mytilus edulis and M. galloprovincialis represent only
two of the many mytilid species. It is clear from this comparison that the
species are largely interchangeable as a model for byssal studies, especially
with regard to conventional biomechanical variables. Comparison of these
mechanical properties with those of the byssus of other mytilids is intriguing
but must be undertaken with some caution. Values in the literature for M.
californianus, M. trossulus, M. edulis and M. galloprovincialis
are often inconsistent (Bell and Gosline,
1996; Price, 1981
;
Smeathers and Vincent, 1975). Similarly, comparisons with the values reported
here do not correspond precisely with those reported in the literature. While
the basis for these inconsistencies is speculative, sample size, mussel
health, reproductive stage, thread age, equipment limitations and the precise
separation of threads into distal and proximal portions are all possible
factors contributing to disparities. Even with that caveat, M.
californianus threads (Young's modulus 868±181.2 MPa) are 2-3
times stiffer than those of M. edulis and M.
galloprovincialis irrespective of the values with which they are compared
(Bell and Gosline, 1996
).
Furthermore, mechanical values for mussels in the `edulis complex' are much
more similar to each other than they are to more distantly related
mytilids.
With these mechanical considerations, it would be both telling and
interesting to know how the byssal collagens of other mytilids compare with
those of these two species. Mytilus californianus, in particular, is
notorious for producing very long, thick, tough threads
(Bell and Gosline, 1996).
Information about whether such remarkable threads are built upon similar
underlying structural components with similar distributions and assemblages
would undoubtedly lead to further refinements to the functional and structural
models of these unique extraorganismic fibers.
The sequence data reported here have been submitted to GenBank under accession numbers AF448526 (MgpreCol-D), AF448524 (MgpreCol-NG) and AF448525 (MgpreCol-P).
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References |
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