A Major Protein Precursor of Zebra Mussel (Dreissena polymorpha) Byssus: Deduced Sequence and Significance

11 Pages·1998·4.4 MB·English

by  K E Anderson

Reference: Biol. Bull. 194: 15(1-160. (April. 1998)
A Major Protein Precursor of Zebra Mussel
{Dreissena polymorpha) Byssus: Deduced
Sequence and Significance
KEVIN ANDERSON AND HERBERT WAITE*
E. J.
Chemistry/Biochemistry- Department and College ofMarine Studies, Newark, Delaware 19716
Abstract. The zebra mus.sel is a nonindigenous invader functioning in tension: marine mussel adhesives, plant
of North American lakes and rivers and one of the few extensins, titin, and trematode eggshell precursors.
—
freshwater bivalve molluscs having a byssus a sclero-
tized organ used by the mussel for opportunistic attach- Introduction
cmeDnNtAtowhhaorsdesucrofmacpeoss.itWeephraotveeinsesqeuqeunecnecdeawfaosot-dsepdecuicfeidc The zebra mussel, Dreissena polymorpha (Pallas), is a
freshwater bivalve indigenous to the river basins of the
cfrDoNmAa sferraigemseonftso.veTrhleapopvienrgalblutdeodcuccaseidonsaelqluyennocneidmeanttcihc—easl tBallalcyk,inBtarlotdicu,ceadndinCtaosopnieanofsetash.e RGerceeanttlLya,keits,waasndacicnidleens-s
tryptic peptides from a major byssal precursor protein than 10 years, its distribution has expanded into the lakes
Dreissenapolymorpha foot protein 1 (Dpfpl ). The calcu- and rivers ofat least a third ofthe North American conti-
lated mass of Dpfpl is 49 kDa; but this is known to be nent (Johnson and Padilla, 1996). The economic impact
extensively hydroxylated and O-glycosylated during mat-
of this expansion has been profound and is due. in large
uration. Purified native Dpfpl analyzed using matrix-as- part, to fouling (Roberts, 1990). Zebra inussels foul by
sisted laser-desorption ionization mass spectrometry with attaching opportunistically and in large numbersto awide
time-of-flight indicates that the protein occurs as at least
variety of surfaces by means of a thread-like structure
two size variants with masses of 48.6 and 54.5 kDa. In known as a byssus (Ackerman et al.. 1992). In this re-
all probability, the sequence variantsreported inthis study spect, they resemble marine mussels (Mytilidae), which
are related to the larger mass variant. Dpfpl has a block
have adopted a similar strategy.
copolymer-like structure defined by two consensus motifs
Zebra mussel byssal threads are fibrous extracellular
that are sharply segregated into domains. The N-terminal structures composed largely of proteins, many of which
side of Dpfpl has 22 tandem repeats of a heptapeptide contain the post-translationally modified amino acid 3,4-
consensus (P-[V/E]-Y-P-[T/S/6]-[K/Q]-X); the C-termi-
dihydroxyphenylalanine (Dopa) (Rzepecki and Waite.
nal side has 16 repeats of a tridecapeptide motif (K-P-
1993). Peptidyl Dopa is thus aconvenient markerofbyssal
G-P-Y-D-Y-D-G-P-Y-D-K). Both consensus repeats are
precursor proteins and is thought to play an important role
unique, with some limited homology to other proteins
in adhesion and the maturational cross-linking of byssal
threads (Waite. 1990). Three polymorphic Dopa-con-
taining protein families have previously been isolated and
R*eTcoeivwehdo2mDceocrermebsepornd1e9n9c7e; aschcoeupltdedbe10adFderbersusaerdy. 1E9-9m8a.il:hwaite@ paitially characterized from zebra mussel foot tissue, the
udel.edu site of byssal protein synthesis and storage. The largest
Abbreviations: DIG. digoxigenin; Dopa. 3. 4-dihydroxyphenylala- of these proteins. Dreissena polymorpha foot protein 1
nine; Dpfpl, Dreissena polymorplia foot protein 1; MALDI-TOF, ma- (Dpfpl ). has an apparent molecular weight of76 kDa and
trix-assisted laser desorption-ionization mass spectrometry with time- Dopa at levels up to 6.6 mole 9c (Rzepecki and Waite,
ooff-fcliDghNtA; Meenfdpsl:.RMTx-tPihCiRs,edrieilviesrsfeoottrparnostceriinp:taRsAeC-pEo,lyrmaepriadseamcphlaiifnicarteiaoc-n 1993). Like many byssal precursors from marine mussels,
tion; SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electro- Dpfpl features Dopa residues in repeating consensus mo-
phoresis. tifs. Despite this similarity, Dpfpl is markedly different
150
ZEBRA MUSSEL BYSSAL ADHESIVE PROTEIN 151
from the marine proteins in two respects. First, members internal peptide (Dpl.A(-) TTRTCRTAIGGICCRT-
ofthe Dpfpl family have acidic isoelectric points ranging CRTA). Each 50-//1 reaction contained 0.25 niM of each
from 5.3 to 6—.5; marine byssal precursors, in contrast, are dNTP, 100 pmoles of each primer, and 2.5 units of
highly basic many with pis exceeding the effective re- Taq2QQQ) polymerase (Stratagene, La Jolla. CA). in a
solving range of available ampholytes. Second, dreissenid buffercontaining 10 mMTris-Cl, 1.5 mMMgCU, 75 mM
byssal precursors, including Dpfpl. are glycosylated with KCland 15 mA/(NH4):S04. Samples were initially dena-
A'-acetylgalactosamine O-linked to serine and threonine tured at 95°C for 4 min 30 s followed by 30 cycles of
residues; there is, however, no evidence for glycosylation amplification as follows: 95°C for 30 s, 50°C for 30 s,
in byssal proteins from any marine taxa. It is not known and 72°C for 2 min. A final extension for 5 min at 72°C
whether these differences reflect two generally valid solu- was carried out to ensure addition of3' A overhangs. The
tions to the problem of adhesion underwater or represent resulting amplification product was ligated into the pCRII
genuine differences in the requirements for adhesive bond vector(Invitrogen, San Diego,CA) accordingtomanufac-
formation in freshwater and marine systems. turer's instructions. The insert from the newly constructed
Ourefforts to determine the complete primary sequence plasmid, pDPl.NA, was sequenced on both strands using
of Dpfpl by traditional peptide mapping have been vector-specific and degenerate oligonucleotide primers.
thwarted by the repetitive structure and protease-resistance 5' RACE was performed to obtain cDNA sequence
oflarge regionsofthe protein (Rzepecki andWaite, 1993). data upstream of the region coding for the N-terminus
In this study, we report on the complete primary sequence (Frohman et al.. 1988) and to independently establish the
of Dpfpl deduced using molecular techniques. cDNA se- cDNA sequence of the N-terminus. All reactions were
quence data reveal that Dpfpl is a tandemly repetitive performed using reagents contained in the 5' RACE Sys-
protein composed of two motifs: a novel heptapeptide se- tem V2.0 (Life Technologies, Bethesda, MD) according
quence and a tridecapeptide consensus sequence. Unusu- to manufacturer's instructions. Briefly, 1 pg of D. poly-
ally, these motifs are segregated to distinct regions of the morpha foot tissue total RNA was reverse-transcribed
protein, a fact which almost certainly has important conse- using a gene-specific primer (Dpl.GSPl(-) TATTTT-
quences to the self-assembly of the zebra mussel byssus. GTAGGAGTGGG). The purified first-strand cDNA was
tailed with dCTP. and PCR was performed using the sup-
(GGCCACGCGTCGACT-
Materials and Methods plied abridged anchor primer
AGTACGGGIIGGGIIGGGIIG) and Dp1.GSP1(-). Each
RNA extractions 50-//1 reaction contained 0.25 mM of each dNTP and
All tissues used in these experiments were excised, 20 pmoles of each primer in 1x PCR buffer (Life TemchM-
irmnediately frozen in liquid nitrogen, and ground in a nologies, Bethesda, MD) supplemented with 2
mortar chilled to -80°C. Tissue was homogenized in a MgCL. Samples were denatured at 95°C for 4 min 30 s
hand-held glass homogenizer (Kontes, Vineland, NJ), and and equilibrated to 72°C. Two-and-one-half units of
total RNA was extracted according to the methods of TaqlQQQ polymerase were added and amplification for
25 cycles was performed under the following conditions:
Chomczynski and Sacchi (1987).
95°C for 30 s, 42°C for 30 s, and 72°C for 30 s. A final
5-min extension was performed at 72°C. A second round
Reverse transcriptase (RTj-polymerase chain reaction of PCR was performed using AAP and a nested gene-
(PCR) and 5' rapid amplification ofcDNA ends specific primer (Dpl.GSP2(-) TTGTTGTATAGTTCG-
(RACE)
GAATTTTAG). The reaction volume and component
mRNA was purified from total RNA using the Oligo- concentrations were as outlined in the previous reaction.
tex mRNA spin column kit (Qiagen, Chatsworth, CA). Samples were initially denatured at 95°C for 4 min 30 s
After purification, 1 ytg mRNA was reverse transcribed followed by 30 cycles of amplification as follows: 95°C
using 20 pmoles of a primer specific to polyA tracts for 30 s, 42°C for 30 s, and 72°C for 60 s. A final exten-
(polyT-LD AGAGAGATTTTTTTTTTTTTTTTTVN) with sion for 5 min at 72°C was carried out to ensure addition
200 units of MM-LV reverse transcriptase (Superscript of 3' A overhangs. The resulting amplification products
II, Gibco-BRL) for 2 h at 37°C in buffer supplied by the were cloned into the pGEM-T vector(Promega, Madison,
manufacturer. The reaction was quenched with 1 ml of 1 WI) according to manufacturer's instructions. The insert
X TE, pH 7.5. One percent (v/v) of the resulting first- from the newly constructed plasmid, pDP1.5'UTA, was
strand cDNA was amplified with the polymerase chain sequenced on both strands using gene-specific primers.
reaction (PCR) using degenerate oligonucleotide primers
Probe synthesis and cDNA library screening
based on the previously determined (Rzepecki andWaite,
1993) amino acid sequence of the N-terminus of Dpfpl Two probes were created in this experiment to screen
(Dpl.N(-h) GGIACITAYGAYTGGACNGA) and an a D. polymorpha foot tissue cDNA library (Eddington,
152 K. E. ANDERSON AND J. H. WAITE
1996). Adigoxigenin (DIG)-labeledantisense RNA probe RNA dot blots
(probe #1) was generated from Ddel-digested pDPl.NA
using T7 polymerase and the DIG-RNA labeling kit Ten micrograms of total RNA separately extracted
from D. polymorpha foot, adductor mussel, mantle, and
s(tBrouecthiroinns.geAr-MDaInGn-hleaibme)leadccdoorudbilneg-sttoramnadneudfacDtNurAer'psroibne- gill tissue were diluted in an equal volume of RNA dilu-
(probe #2) spanning the 5' untranslated region of Dpfpl tion buffer (water: 20x SSC; formaldehyde; 5:3;2) and
and the first 172 nt coding for the mature protein was spotted onto a positively charged nylon membrane (MSI,
generated using the PCR DIG probe synthesis kit (Boeh- Westboro, MA). The membrane was hybridized to either
probe #1 as described above or to an actin-specific dou-
ringer-Mannheim) according to manufacturer's instruc- DNA
tions. pDPl.S'UTA was used as a template for this reac- ble-stranded DIG-labeled probe (Patwary et al..
1996). Hybridization with actin specific probe was per-
tion, and a primer specific to the 5' untranslated region
of Dpfpl (Dpl.5' UT(-f-) ATACTTCAGAGCATCAA- formed at 37°C with a stringency wash using 0.5X SSC/
CCAA) and Dpl.GSPl(-) were used as primers. Both 0.1% (w/v) SDS at 68°C.
probes were individually incoiporated at a concentration
of lOOng/ml into standard hybridization buffer -I- 50% Northern hybridizations
fomiamide (5x SSC. 1% Blocking buffer (Boehringer- mRNA
Three micrograms offoot tissue were subjected
Mannheim). 0.17c (w/v) sarcosyl, 0.02% (w/v) SDS. 50%
to formaldehyde/agarose gel electrophoresis according to
(fpormoibaemi#d1e) (ovr/v)4)2.°HCybr(ipdriozbaeti#o2n)s. wSetrreincgaernrciyedwoautshaets60f°oCr Sambrookelal. ( 1989). RNA was transferred onto a posi-
tively charged nylon membrane and hybridized overnight
both probes were conducted with O.lx SSC/0.2% (w/v)
with probe #1.
SDS at 68°C.
One million plaques generated from a \ZAP-Express
cDNA library (Stratagene. La Jolla, CA) were doubly Mass analysis ofnative Dpfpl
screened with probes #1 and #2. No plaques positive for
Native Dpfpl was purified from the foot ofadult zebra
probe#2 were detected, suggestingthat afull-lengthclone
mussels according to Rzepecki and Waite (1993). The
poofsiDtpifvpelfowraspronbote p#r1eswenetreincotrheids,lieblruatrey.d FinortSyMplbauqfufeesr mass of the native protein was determined by matrix-
(100 mM NaCI. 50 mM Tris-CI pH 7.5, 8 mM MgSOj. wasistihstteidme-loafs-efrlidgehsto(rpMtAiLonD-Iio-nTiOzaFt)iounsimnagsasPesrpSeecpttriovmeetBriy-
0.1% gelatin), and tested for insert size by PCR using osystems Voyager model in the positive ion mode and
vector-specific primers Hanking the cDNA insert. After delayed extraction. A 20-^M solution of Dpfpl in 0.1%
secondary screening. cDNA from the plaque bearing the
acetic acid was mixed with three volumes of a saturated
largest insert was rescued as a phagemid using the ExAs- sinapinic acid solution (40% acetonitrile/0.1% TEA); 2 pi
sist interference-resistant helperphage kit (Stratagene, La of the resulting mixture (lOpmoles Dpfpl) was placed
Jolla. CA) and sequenced using the nested deletion tech- on a sample plate and allowed to air dry. The sample was
nique (see below). inserted into a vacuum chamber (I x 10"' torr) and the
spectra generated from 256 pulses ofa 337-nm laserwere
Nested deletions averaged. The acceleration voltage was 25,000 with a
90% grid voltage and a guidewire setting of 0.1%.
Nested deletions were performed using the double-
stranded nested deletion kit (Pharmacia Biotech. Piscata-
way. NJ). In each case. 5 ^g of template was doubly Results
digested with EcoRI and Pstl. and the restriction enzymes RNA dot blots and Northern hybridizations
were heat inactivated. Digested clones were precipitated
M
in ethanol and resuspended in a buffer containing 1.5 The tissue specificity of Dpfpl is demonstrated in Fig-
potassium acetate, 37.5 mM Tris-acetate pH 7.6, 15 mM ure 1. RNA dot blots show that Dpfpl mRNA transcripts
magnesium acetate. 750 ^M /9-mercaptoethanol. and were detected only in total RNA extracts from foot tissue
15 /7g/ml bovine serum albumin (BSA). A 2-/yg sample and not in extracts from gill, adductor muscle, or mantle
of each digest was used for digestion with Exonuclease tissue. Identical dot blots hybridized to an actin-specific
III. The reactions were earned out at 23°C and aliquots probe were positive for all tissue types although the
taken every 5 min. All clones yielding deletions larger strength of the signal varied considerably between tissue
than the size of the empty vector were ligated, trans- types (data not shown). These results are consistent with
formed into XL1-Blue MRP' cells (Stratagene, La Jolla, data obtained from othermarine byssal precursorproteins
CA), purified, and sequenced using a vector-specific ilnoue et al.. 1995. 1996a; Coyne t'/ «/.. 1997; Qin t'? «/..
primer. 1997) and support the hypothesis that Dpfpl plays a role
ZEBRA MUSSEL BYSSAL ADHEMSIVE PROTEIN 153
A G
F
Figure 1. Total RNA dot blots ofDreissenapolymorpha foot (F). adductor muscle (A), mantle (M).
and gill (G) tissue hybridized to a digoxigenin-labeled RNA probe specific to Dpfpl. The probe was
hybridized to 1 /j.g of total RNA from each tissue.
as abyssal structural protein. Northern blots offoot tissue sequence data was determined from at least two indepen-
mRNA
indicatedthatDpfpl transcripts range in size from dently amplified samples. The combined transcript is
1200b to 1500 b. suggesting the presence ofsize variants 1481 bp in length and contains an open reading frame of
(Fig. 2). 1332 bpcoding fora protein of443 amino acids. Included
in the transcript is a start codon at nucleotide position 36
Dpfpl cDNA sequence and two overlapping canonical polyadenylation signals
(Kozak. 1986) at nucleotide positions 1464 and 1468.
In Figure 3 the aligned nucleotide sequence data ob-
The calculated molecular weight of the deduced primary
tained from 5' RACE. RT-PCR with degenerate oligonu-
cleotide primers, and from the largest cDNA clone iso- sequence is 49 kDa. with a predicted isoelectric point
of 5.29.
lated are presented. Each sequence differs slightly from
The first 19 amino acids code for a putative signal
the other, and therefore the consensus sequence generated
peptide that conforms to the rule of von Heijne (1985).
from this alignment does not represent any single Dpfpl
Computer-based modeling of signal peptide cleavage
sequence. It is likely that differences in the data sets
(Nielsen et al., 1997) correctly predicts cleavage of the
reflect the existence of Dpfpl variants rather than errors
introduced during amplification, because each set ofPCR signal peptide preceding the previously determined N-
terminal glycine residue of the mature protein (Rzepecki
and Waite, 1993). The N-terminus of Dpfpl, as coded
for by sequences generated using 5' RACE, differs from
< the previously reported N-terminal sequence (Rzepecki
2.4 kb
and Waite, 1993) in that it substitutes serine residues
for threonine at position #2, tyrosine at position #3, and
aspartic acid at position #10. None of the three indepen-
dently generated 5' RACE clones exactly coded for the
previously reported N-terminus of Dpfpl. N-terminal se-
quence data generated with degenerate oligonucleotide
primers more closely resemble the previously reported N-
terminal sequence but also substitute serine for aspartic
acid at position #10. It is not possible to determine from
these data if the N-terminal sequence deduced from
cDNAs generated via degenerate oligonucleotide primers
<
1.0 reflects a genuinely different N-terminus or is simply an
artifact forced by the primers used during amplification.
The N-terminal 38 amino acids of the mature protein
are relatively enriched in threonine and serine residues
Figure2. NorthernblotofDreissenapolymorphafoottissuemRNA and quickly give way to a tandemly repeating heptapep-
hybridizedtoadigoxigenin-labeled RNA probe specific to Dpfpl; 3fjg tide. This generally basic motif (P-[V/E|-Y-P-[T/S|-[K/
offoot tissue mRNA was used. Q]-X) isrepeated 22times inthe N-terminal halfofDpfpl
154 K. E. ANDERSON AND J. H. WAITE
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ZEBRA MUSSEL BYSSAL ADHESIVE PROTEIN 155
B
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156 K. E. ANDERSON AND J. H. WAITE
with some variation, particularly at position #7 of the Ma.'is analysis ofnative Dpfpl
consensus sequence; however, proline residues at posi-
tions #1 and #4 and tyrosine residues at position #3 are MALDI-TOF analysis of native Dpfpl indicates that
highly conserved (Fig. 4). RT-PCR data differ from the purified protein is represented by two major mass
cDNA clone data in this region ofthe transcript by omis- variants. The lighter of the two variants has a mass [M
ssuilotninogfftrhormeoanitnrean1s7ve5rsainodn batynaucGle1o9ti0dEe spuobssittiitounti6o6n1.re- +-I- HH^*]Y^ =— 5448..56kkDDaa.. Nwhoerpeeaasksinwetrhee dheetaevciteerdvianritahnte,6[0M-
80 kDa range.
The C-temiinal halfof Dpfpl is dominated by the pre-
viously reported 13 amino acid consensus sequence: K-P-
Discussion
G-P-Y-D-Y-D-G-P-Y-D-K (Rzepecki and Waite, 1993).
This acidic sequence is found tandemly repeated 16 times Theprimary structure ofDpfpl, deduced from overlap-
with only slight variations from the consensus (Fig. 4). ping cDNAs, represents the first complete sequence for
The deduced amino acid composition of the composite a dreissenid byssal protein and an important advance in
Dpfpl sequence, without signal peptide sequence agrees understanding the attachment strategy of the zebra mus-
well with that of native Dpfpl (Table I), suggesting that sel. Two observations suggest that the composite se-
the composite sequence described above is representative quencegenerated from thesedatasets is likelytoresemble
of Dpfpl mRNAs present in zebra mussel foot tissue. full-length transcripts for Dpfpl. First, the size of the
Examination ofcodon usage for Dpfpl (Table II) reveals composite sequence (1481 bases) closely matches the size
a significant degree of codon bias in amino acids that ofthe largest Dpfpl transcript as determined by Northern
occur in conservedpositions ofthe above-mentioned con- blots of zebra mussel foot tissue mRNA hybridized to a
sensus sequences (e.g.. P, Y, D. K. T. G). Dpfpl-specific probe. Second, the deduced amino acid
N-
ZEBRA MUSSEL BYSSAL ADHESIVE PROTEIN 157
Table I
Amino acidcomposition ofdeducedand native Dpfpl
Amino acid
—
158 K. E. ANDERSON AND J. H. WAITE
positions (#2 and #8, Fig. 3) (Rzepecki and Waite, 1993). kDa plaque-specific protein of M. gallopmvincialis. is
The nucleotide sequences presented in Figure 3 suggest largely composed of larger, epidermal growth factor-like
the existence ofat least two ofthese variants. Differences repeats (Inoue et al.. 1995).
between these variants in regions of cDNA overlap are The N-terminal half of Dpfp1 is dominated by a hepta-
limited to the deletion of a single codon in the RT-PCR peptide motifthat is repeated 22 times with some variation,
data and a single transversion resulting in an amino acid particularly at position #7 ofthe consensus sequence. Vari-
substitution in one of the heptapeptide sequences. ability notwithstanding, the spacing ofproline and tyrosine
An examination of the codon usage data (Table II) residues is well conserved, suggesting that these amino
indicates that compositionally dominant amino acids are acids play an important functional role in the motif. No
predominantly coded for by halfof the potentially avail- tryptic peptides exactly matching the deduced primary se-
able codons for these residues. This is especially true quencecouldbemappedtothispartoftheprotein;however,
of proline, tyrosine, aspartic acid, lysine, threonine, and a fragment of one tiyptic peptide (tryptic peptide #13 in
glycine residues, which together account for almost 75% fig. 6 of Rzepecki and Waite. 1993) containing the subse-
of the amino acid composition of Dpfpl. The pattern of quence S-P-L-Y-G-W ... is found to bridge two of the
codon bias in compositionally dominant residues has also heptapeptide repeats. Although thetyrosine in this sequence
been noted in other marine byssal precursor proteins isefficientlyconverted to Dopa. theaminoacidcomposition
notably Mefpl (Filpula et ciL. 1990), Mgfpl (Inoue and of residual undigested Dpfpl suggests that, as a whole,
Odo. 1994). Mcfpl (Inoue et ciL. 19—96b), and. to a lesser this region contains relatively little Dopa (Rzepecki. pers.
extent, Mgfp2 (Inoue et al., 1995) and may reflect a
comni.).
need to express byssal structural proteins rapidly in re- Given the frequency oflysine and arginine inthehepta-
sponse to developmental cues and changing environmen-
peptide repeat region, the resistance ofthe repeattocleav-
tal conditions. It is well established that in bacterial sys- age by ti7psin is intriguing. An examination of the
tems, codon bias is positively correlated with the rates of
deduced primary sequence indicates that K-P or R-P se-
gene expression (Robinson et al.. 1984; Varenne et al..
quences cannot be the basis for this resistance. Interest-
1984; Sorensen et al.. 1989), presumably through selec-
ingly, lysine and arginine residues in this domain fre-
tion of codons that recognize the most abundant isoac-
cepting tRNAs for a given amino acid. Precedence for quently occur adjacent to threonine and serine residues.
this hypothesis can also be found among highly expressed That observation, coupled with the detection ofhigh lev-
els of threonine and A'-acetylgalactosamine in partially
genes in multicellular organisms such as Drosophila inel-
anogaster. whose chorion genes, important eggshell com- digested tryptic peptides (Rzepecki and Waite. 1993).
ponents known to be highly expressed during egg devel- leads to the hypothesis that Arg and Lys are protected
ocopdmoenntbi(aKsaf(aAtkoasshiet, a1l.9.94)1.98S7)u,chalasohyepxohtihbeitsissighnaisfiaclasnot afcriodms.tArypssiinmilcalreapvraogteectbiyonadajpapceeanrtsgtloycboesyliamtpeadrtaemdinboy
been advanced to explain observed codon bias in the glycosylated residues in an extensin-like glycoprotein
highly expressed silk fibroin heavy chain ofthe silk moth, from Volvo.x carter! (Ertl et al.. 1992).
Bomby.x mod (Mita et al.. 1994). The N-terminal halfofDpfpl differs significantly from
More than 80% of the deduced primary amino acid the C-terminal domain with its repeated 13 amino acid
sequence of Dpfpl is composed of tandemly repeated motif(Fig. 4). Previous peptidedata(Rzepecki andWaite,
and segregated motifs: one is a heptapeptide; the other, 1993) and the deduced sequence of Dpfpl are consistent
a tridecapeptide consensus motifthat coincides with pep- with the hypothesis that glycosylation is more extensive
tides sequenced previously (Rzepecki and Waite, 1993). in the N-terminal region ofthe protein, whereas hydroxyl-
Theoccurrence oftwo relatively short tandemly repeating ation of tyrosine to Dopa occurs more frequently in the
motifs in Dpfpl is consistent with its proposed role as a remaining C-terminal portion. Additionally, the average
byssal structural protein. However, the absence of data isoelectric point of Dpfpl in the region occupied by the
on the distribution of Dpfpl within the byssus makes it heptapeptide is moderately basic (pi = 8.7), whereas the
difficulttoassignaspecificrole atthistime. Therepetitive C-terminal domain isquite acidic (pi = 4.7). These diver-
nature of Dpfpl is shared by many of the structural pro- gent characteristics suggest that the segregation ofmotifs
teins of marine byssi. Two of three characterized Dopa- plays a significant role in the architectural design of the
containing byssal proteins in Mytilus are known to be zebra mussel byssus. Recently, two byssal structural pro-
composed almost entirely of tandem repeats. Mefpl. a teins from M. edulis have also been shown to be com-
110-kDa protein thought to play a role as a cuticular posed of "block copolymer""-like domains. Both proteins
lacquer in the byssus ofM. edulis. is dominated by non- have a central collagenous core flanked by sequences re-
segregated hexa- and decapeptide repeats (Filpula et al.. sembling eitherelastin (Coyne etal.. 1997) or silk fibroin
1990; Waite et al. 1985; Laursen, 1992). Mgfp2. a 49- (Qin et al.. 1997). The distribution of these proteins can
ZEBRA MUSSEL BYSSAL ADHESIVE PROTEIN 159
A. Heptapeptide comparisons
Protein ConsensusSequence Repeats