*Department of Biological Sciences, Louisiana State University at Baton Rouge;
and
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego
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Abstract |
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Introduction |
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Proteins mediating the interaction between sperm and egg of free-spawning marine invertebrates are characterized by extensive interspecific divergence. In primitive marine gastropods, including abalone (Haliotis) and top snails (Tegula), the protein lysin performs a critical role in fertilization by dissolving a hole in a tough glycoproteinaceous envelope which surrounds the egg. Interspecific comparisons of lysin cDNA among closely related species of these gastropods reveal extensive divergence and rapid accumulation of nonsynonymous substitutions (Lee, Ota, and Vacquier 1995
; Hellberg and Vacquier 1999
). In abalone, a second major acrosomal protein also evolves extremely rapidly (Swanson and Vacquier 1995
; Metz, Robles-Sikisaka, and Vacquier 1998
).
Bindin, a sperm-egg attachment protein from sea urchins, likewise evolves rapidly (but see Metz, Gómez-Gutiérrez, and Vacquier 1998
). Positive selection, however, is more localized within bindin than within gastropod acrosomal proteins (Metz and Palumbi 1996
). Repetitive sequence elements play a substantial role in the interspecific divergence of bindin (Minor et al. 1991
; Biermann 1998
). To date, internal repeats have not been reported for gastropod acrosomal proteins.
Here, we report rapid interspecific divergence of cDNA encoding the major acrosomal protein (TMAP) from the sperm of five species of teguline gastropods: Tegula aureotincta, Tegula brunnea, Tegula montereyi, Tegula regina, and Norrisia norrisii. Previous work has established that T. brunnea and T. montereyi are sister taxa and that T. regina forms a monophyletic clade with this pair (Hellberg 1998
). Tegula aureotincta and N. norrisii are relatively distant from this trio, and their relationships have not yet been resolved. A molecular clock that can be used to estimate divergence times (and, therefore, rates of substitution) between teguline species has also been calibrated (Hellberg and Vacquier 1999
). We find that, like other acrosomal proteins from marine gastropods, TMAP exhibits high rates of nonsynonymous nucleotide substitution and positive selection between closely related species. In addition, a region containing the propeptide's cleavage site has been duplicated in one species (T. aureotincta), resulting in two mature peptides, one of which incorporates a portion of the presumed ancestral propeptide into the mature peptide. Such alternative processing may have given rise to the highly divergent N-termini seen in TMAP for two other species (T. regina and N. norrisii).
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Materials and Methods |
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mRNA Isolation, cDNA Synthesis, and Sequencing
Total RNA was isolated from testes (either fresh or preserved in 70% ethanol) by homogenization in 4 M guanidinium isothiocyanate, 25 mM sodium acetate (pH 6), 0.5% ß-mercaptoethanol (Chomczynski and Sacchi 1987
). The resulting homogenate was layered over 5.7 M CsCl (in 25 mM sodium acetate (pH 6) with 0.5% ß-mercaptoethanol) and centrifuged for 18 h in a Beckman SW41 rotor at 26,000 rpm (20°C). cDNA was produced by reverse transcription using oligo-dT (for T. brunnea and T. montereyi) or primer TB3END (for the others, see below). A T. brunnea testis cDNA library (Lambda ZAP II, Stratagene) was constructed following the manufacturer's instructions.
cDNA was initially amplified by PCR using the T. brunnea testis cDNA library as template and degenerate primers based on amino acid sequences of TMAP peptides. A primer based on internal amino acid sequence ENRMKN (5'-GARAARAAYMGNATGAARAA-3') was paired with the T7 vector primer. DNA sequence obtained from the resulting amplicon was used to design a primer specific to the 3' untranslated region of TMAP cDNA (TB3END: 5'-TGAACTGCAGGTTATTTATTTCA-3'), which was paired with the T3 vector primer to complete the T. brunnea cDNA. This TB3END primer, in combination with a degenerate primer based on the amino acid sequence EAKIDYDY (5'-GARGCNAARATHGAYTAYGAYTA-3'), was used to amplify the 3' end of T. aureotincta TMAP cDNA. A reverse primer (5'-TARTCRTARTCDATYTTNGCYTC-3') based on the same amino acid sequence was primed with oligo-dC22 to amplify the 5' end from dG-tailed T. aureotincta cDNA. Finally, TMAP from the remaining three species was amplified using TB3END and a forward primer based on signal sequence shared between T. brunnea and T. aureotincta (TMAPSIGSEQ: 5'-TGATGTTGGTGTCGATCATATGG-3').
PCR reactions contained each primer at 0.5 µM (except that concentrations of degenerate primers were increased in direct proportion to their degeneracy), Taq polymerase at 10 U/ml, TaqExtender (Stratagene) at 10 U/ml, 1 x TaqExtender buffer, 0.2 mM of each dNTP, and 12 µl of template DNA in a total volume of 50 µl. Thermal profiles consisted of 35 cycles of 40 s at 94°C, 2 min at 46°C, and 1.5 min at 72°C.
PCR products were either sequenced directly (T. aureotincta, T. brunnea) using amplification primers or blunt-end cloned (T. montereyi, T. regina, N. norrisii) into pBlueScript, which was then used to transfect DH5-competent Escherichia coli cells. Both strands were sequenced using ABI Prism FS or BigDye chemistry. The five new sequences presented here have been assigned GenBank accession numbers AF190895AF190899.
Southern Blotting
Southern blotting was used to determine gene copy number for T. brunnea TMAP. Genomic DNA digested with BglII, ClaI, EcoRI, EcoRV, HindIII, or XbaI was separated on a 0.6% TBE agarose gel and blotted onto Hybond N filters using the manufacturer's instructions. After UV cross-linking, the filter was probed with a radiolabeled T. brunnea TMAPSIGSEQ/TB3END PCR product.
Analysis of Protein and cDNA Sequences
Searches for proteins with sequences similar to the TMAPs were performed using BLASTp. Molecular weights and isoelectric points were calculated using MacVector. MacVector was also used to identify repeated sequence elements.
cDNA sequences were aligned by eye. Proportions of nonsynonymous (Dn) and synonymous (Ds) substitutions per site were calculated by method one of Ina (1995)
using FENS (de Koning et al. 1998
). Indels were dropped in pairwise fashion. The N-termini of T. regina and N. norrisii could not be aligned with any certainty and were excluded from the analysis. t-tests determined whether nonsynonymous substitutions were statistically more frequent than synonymous ones.
The scaled 2 method was used to assess codon usage bias (Shields et al. 1988
). Nucleotide biases were calculated following Irwin, Kocher, and Wilson (1991)
. Because the purpose of these tests was to determine whether nucleotide or codon biases could have produced high Dn values, nonalignable sites (the N-termini of T. regina, N. norrisii, and the larger form of T. aureotincta) were excluded from these analyses.
Divergence times between pairs of teguline species were estimated using a molecular clock based on a 639-bp fragment of mitochondrial cytochrome oxidase I (mtCOI). This clock was previously calibrated at one silent transversion per million years using a pair of Tegula species (T. verrucosa and T. viridula) isolated by the rise of the Isthmus of Panama (Hellberg and Vacquier 1999
). Although species presently separated by the Isthmus may have diverged long before the Isthmus' rise (Knowlton and Weight 1998
), this particular pair belongs to a subgenus that arose 4 MYA and likely split 3 MYA (Coates and Obando 1996
). Times of divergence were estimated conservatively using silent transversions (Irwin, Kocher, and Wilson 1991
) instead of Kimura (1980)
two-parameter distances, because the latter consistently gave more recent estimates of divergence (Hellberg and Vacquier 1999
).
Secondary structure may reveal homologies between distantly related proteins even when DNA sequence comparisons cannot. Such was the case for lysin and an 18-kDa protein in abalone (Swanson and Vacquier 1995
; Metz, Robles-Sikisaka, and Vacquier 1998
). TMAP secondary structure was analyzed using tools available from PredictProtein (http://dodo.cpmc.columbia.edu/pp/submit_adv.html).
Secondary structure was inferred using PHDsec (Rost and Sander 1993, 1994
). PHDsec employs neural networks trained on observed position-specific replacements to make predictions for secondary structure at individual sites in a target protein of unknown secondary structure. These initial predictions are refined by observed replacements in aligned input reference proteins. Each of the five teguline TMAPs was used in turn as a target protein, with the remaining four serving as reference proteins. PROSITE (Bairoch, Bucher, and Hofmann 1997
) and ProDom (Corpet, Gouzy, and Kahn 1998
) were used to search for functional motifs and putative domains, respectively.
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Results |
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Gas phase sequencing of purified T. brunnea TMAP yielded 14 amino-terminal residues, beginning with Gly1 (fig. 2 ). A CNBr fragment yielded an additional 45 contiguous residues. In T. aureotincta, TMAP resolved as two bands (fig. 1 ), both of which were subjected to gas phase sequencing. We also obtained an additional 28 internal residues of T. aureotincta sequence from a CNBr fragment (fig. 2 ).
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In T. aureotincta, the sequence M-43 to R-1 represents a prepro sequence element of 43 residues. However, direct sequencing indicated that the N-terminus of the longer (and more abundant) of the two TMAPs appears within these 43 residues. This suggests that the two forms of T. aureotincta TMAP are alternatively processed mature forms of the same translation product, with the N-terminus of the longer form being G-9 and the N-terminus of the shorter form being K1. The terminus of the longer form is adjacent to a furin cleavage site (R-X-R/K-R), typical of proteins with prepro regions. All species have additional furin sites occurring at R-23. Adjacent to the N-terminal K-1 of the shorter form is the sequence R-4-R-E-R-1, which must be cleaved by another protease. The predicted starts of the propeptides of the four other species all align with position -42 of T. aureotincta and were either 26 (T. brunnea) or 24 residues long. The propeptides of the three Tegula species which mitochondrial sequences suggest are monophyletic (T. brunnea, T. montereyi, and T. regina; Hellberg 1998
) are nearly identical, differing by only a twoamino acid insertion and an I
F replacement in T. brunnea. The presumed N-termini of mature TMAPs of T. regina and N. norrisii (-10 to +6 in fig. 2
) are highly divergent (only 1 residue in 11 shared) and do not obviously align with any region of the other species.
The longer prepro peptide of T. aureotincta contains three imperfect repeats which align with the prepro region of the other four species (fig. 3 ). The identity between T. aureotincta and the other species is greatest for the first T. aureotincta repeat (positions -31 to -20). The two other elements apparently arose by duplication of this 12amino acid segment.
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The alignable portions of the mature proteins from the five species vary in amino acid identity from 36% to 72% (table 1 ). Both cysteine residues do not vary among the five species, nor do most positions occupied by aromatic residues (Y and F). There were no significant matches to GenBank or to any recognized functional motifs or domains. The TMAP signal sequence matches the lysin signal sequence at only 2 of 18 alignable residues (one of them being the start methionine) in T. brunnea, the only species for which complete signal sequences are available for both molecules. In contrast, the signal sequences of the distant homologs lysin and 18-kDa of Haliotis rufescens match at 8 of 16 alignable residues.
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Rates of Nucleotide Divergence
Using the cytochrome oxidase I silent-transversion molecular clock, estimated times of divergence for the five species range from 4 to 20 Myr (table 2
). Nonsynonymous substitution rates for the mature protein based on these times of divergence are high: between 26.3 and 60.5 per site per billion years (table 2 ). Synonymous substitution rates are similarly high for most comparisons but are less than one third the nonsynonymous rate for the contrast between the sister species T. brunnea and T. montereyi (Table 2
). The Kimura two-parameter clock (not shown) estimated shorter times of divergence than did the silent-transversion clock and, hence, higher TMAP substitution rates than those presented here.
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Bias in nucleotide usage at silent sites or in codon usage could result in underestimates of Ds, leading to inaccurate conclusions of positive selection (Ticher and Graur 1989
). The percentage of G+C shows little bias over the full coding region (45.2%48.0%) or at third positions of codons (46.7%53.6%; table 3
). The percentage of C at third positions varies from 21.1% to 28.1%. Nucleotide usage falls toward the low end of the theoretical range (from 0 = no bias to 1 = maximum bias; Irwin, Kocher, and Wilson 1991
). Codon usage biases are also low (table 3
): all values are lower than that of chymotrypsin from the abalone H. rufescens (Lee 1994
). These data suggest that neither nucleotide nor codon usage bias can account for the significant excess of Dn relative to Ds seen for four of the TMAP interspecific comparisons.
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Discussion |
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Four comparisons of the mature TMAP reveal a significant excess of nonsynonymous substitutions relative to synonymous substitutions (table 1
). Several observations suggest that this significant excess of Dn relative to Ds results from selection for amino acid change in TMAP. First, relatively high values of Dn are restricted to the mature protein (table 1
). Dn/Ds values for the immediately adjacent propeptide are below unity (although not significantly so). Signal sequences from T. aureotincta and T. brunnea (not shown) evolve still more slowly than the propeptide. Similar relative rates of nonsynonymous change (conserved signal, moderate propeptide, rapid mature protein) have been reported for interlocus divergence of three mating pheromones from the ciliate Euplotes raikovi, another instance of reproductive protein radiation marked by extensive amino acid replacements (Miceli et al. 1991
). Second, the relatively high value of Dn is not likely due to either nucleotide or codon bias (table 3 ; Lee 1994
), although the species with the highest codon bias (N. norrisii) is involved in three of the four significant Dn/Ds comparisons.
Finally, the Dn/Ds value of >1 is not likely to be due to the comparison of nonorthologous loci, at least for the species pair yielding the highest ratio (T. brunnea/T. montereyi). Southern analysis of T. brunnea shows TMAP to be a single-copy gene in this species (fig. 4
), and the probability of duplication and subsequent extinction of one copy of the gene during the brief time (2 Myr) separating T. brunnea and T. montereyi seems low. TMAP PCR products were directly sequenced in T. aureotincta, suggesting that TMAP occurs as a single mRNA and is probably a single-copy gene in this species as well. Results for T. regina and N. norrisii (which were cloned) are less clear; ultimately, Southern analysis of all of these species will be needed to ascertain copy number.
Six of 10 interspecific TMAP comparisons did not show Dn/Ds > 1. Comparisons of gastropod acrosomal proteins generally do not exceed unity when Ds > 0.2 (see Hellberg and Vacquier 1999
), regardless of whether species co-occur (Lee, Ota, and Vacquier 1995
). Here, Ds > 0.2 for 9 of the 10 pairwise comparisons (the T. brunnea/T. montereyi pair, with the highest Dn/Ds value, being the sole exception). Thus, relatively low Dn/Ds values probably result from the downward bias of estimators of Dn/Ds when divergence is great (Ina 1995
).
The function of TMAP remains unknown; purified TMAP did not dissolve vitelline envelopes. Previous assays of lysin activity (Hellberg and Vacquier 1999
) used whole acrosomal extracts enriched for lysin, so the possibility remains that TMAP serves some role with lysin in dissolving vitelline envelopes. However, observed dissolution activity in those experiments varied directly with the proportion of lysin in the preparation, suggesting little role for TMAP. The localization of TMAP within the acrosome strongly suggests some role in fertilization.
Strong interspecific positive selection has previously been reported for lysins from Haliotis (Lee, Ota, and Vacquier 1995
) and Tegula (Hellberg and Vacquier 1999
) and for an 18-kDa acrosomal protein from Haliotis (Swanson and Vacquier 1995
). Loci encoding these other gastropod fertilization proteins are either orthologous (the two lysins) or paralogous (the two Haliotis proteins; see Metz, Robles-Sikisaka, and Vacquier 1998
) to each other. Comparisons of these to cDNA and predicted secondary structure of TMAP do not suggest any obvious relationship. Thus, TMAP and lysin appear to be two historically independent, male-specific sex proteins, both experiencing strong diversifying selection between species.
One possible explanation for positive selection on fertilization proteins is to avoid heterospecific fertilization. The high Dn/Ds value for the T. brunnea/T. montereyi comparison is striking in this light, as these are co-occurring sister species with significant overlap in microhabitat (Riedman, Hines, and Pearse 1981
) and spawning season (Watanabe 1982
). However, in gastropod sperm proteins, Dn/Ds ratios are generally highest for closely related species (Lee, Ota, and Vacquier 1995
; Swanson and Vacquier 1995
), and closely related species tend to be sympatric among these taxa (Hellberg 1998
), so this single observation can be regarded as merely consistent with a role for reinforcement.
Propeptide Repeats and Alternative Processing
The propeptide of T. aureotincta is 20 residues longer than those of T. brunnea and T. montereyi (fig. 2
). Tegula aureotincta has two ~12-residue repeats showing 60% amino acid identity and 77% nucleotide identity to each other (fig. 3
). The first of the three T. aureotincta repeats is more similar to presumed homologous sites in the other four species analyzed. The two repeats must have originated by duplication of the first 12-residue region (fig. 3
).
Most interestingly, the duplication includes at its C-terminal end a dibasic repeat (RR or RK), the usual recognition sequence for the endolytic cleavage which separates propeptide regions from mature peptides (Bond and Butler 1987
). Direct sequencing of two gel-purified acrosomal proteins from T. aureotincta confirmed that two different mature proteins, one corresponding to each of the duplicated cleavage sites (fig. 2
), are expressed.
The net result of the duplication of cleavage sites is that some amino acid residues previously restricted to the propeptide are, under one alternative processing, incorporated into the mature peptide. As with intron capture (Golding, Tsao, and Pearlman 1994
), and unlike exon shuffling or duplications of regions already encoding mature peptides, such propeptide capture should have the effect of introducing truly novel sequence into a mature protein. The nonalignable N-terminal residues of T. regina and N. norrisii (fig. 2
) may have been introduced initially in such a fashion, with subsequent deletions and substitutions leaving no trace of the duplication.
Incorporation of residues that alter protein structure might be expected to have negative selective consequences. Such consequences, however, may be limited for TMAP. The N-terminal differences would not alter the distance between the two conserved cysteines; thus, forms both with and without the N-terminal residues would be expected to have similar folds. Furthermore, gastropod acrosomal proteins often show interspecific length variation of several residues at their amino- and carboxy-termini (Lee, Ota, and Vacquier 1995
; Swanson and Vacquier 1995
). In addition, overcoming the potential selective barrier of replacing an ancestral T. aureotincta 90-residue TMAP with one 10 residues larger may have been facilitated by the fact that both mature proteins would initially be produced (Smith, Patton, and Nadal-Ginard 1989
). Alternative processing may thus provide another genetic mechanism, along with positive selection on point mutations, for promoting diversification of reproductive proteins.
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Acknowledgements |
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Footnotes |
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1 Abbreviation: TMAP, Tegula major acrosomal protein.
2 Keywords: positive selection
fertilization
Tegula,
sperm
prepro duplication
alternative processing
3 Address for correspondence and reprints: Michael E. Hellberg, Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803. E-mail: mhellbe{at}lsu.edu
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