Reference: Biol. Bull. 205: 8-15. (August 2003) © 2003 Marine Biological Laboratory 250 Million Years of Bindin Evolution KIRK S. ZIGLER^'2'* AND H. A. LESSIOS' ' Smithsonian Tropical Research Institute, Balboa, Panamá; and ^ Department of Biology, Duke University, Durham, North Carolina Abstract. Bindin plays a central role in sperm-egg attach- level, often within species, sometimes within genera, but ment and fusion in sea urchins (echinoids). Previous studies rarely across an entire class. There are good reasons for this determined the DNA sequence of bindin in two orders of the focus: such studies are likely to uncover mutational changes class Echinoidea, representing 10% of all echinoid species. that are important in mate recognition and in speciation. We report sequences of mature bindin from five additional However, comparisons across broad taxonomic levels can genera, representing four new orders, including the distantly offer insights into the evolution of such molecules. They can related sand dollars, heart urchins, and pencil urchins. The reveal which features of these molecules are conserved (and six orders in which bindin is now known include 70% of all are thus essential for basic functions) and which features are echinoids, and indicate that bindin was present in the com- free to vary. For the parts that do vary, such comparisons mon ancestor of all extant sea urchins more than 250 million can determine common features of evolution. Most of all, years ago. Over this span of evolutionary time there has the comparisons can address the question of the universality been (1) remarkable conservation in the core region of of a particular molecule by asking how far back in evolution bindin, particularly in a stretch of 29 amino acids that has one needs to search to find the point at which a completely not changed at all; (2) conservation of a motif of basic different molecule has taken over the essential functions amino acids at the cleavage site between preprobindin and involved in gamete binding and fusion. mature bindin; (3) more than a twofold change in length of Echinoids (sea urchins, heart urchins, and sand dollars), mature bindin; and (4) emergence of high variation in the with their readily obtainable gametes, have long been model sequences outside the core, including the insertion of gly- organisms for fertilization studies. Because fertilization is cine-rich repeats in the bindins of some orders, but not external, the molecules involved in gamete recognition and others. fusion are associated exclusively with the gametes. Bio- chemical studies in sea urchins identified the first "gamete Introduction recognition protein," bindin (Vacquier and Moy, 1977). Various studies have shown that molecules involved in Bindin is the major insoluble component of the sperm reproduction (and particularly in gamete interactions) acrosomal vesicle and has been implicated in three molec- evolve rapidly, often under the influence of positive selec- ular interactions (Hofmann and Glabe, 1994). First, after the tion (reviewed in Swanson and Vacquier, 2002). Among acrosomal reaction, bindin self-associates, coating the acro- these proteins there are examples of both high (Metz and somal process. Second, it functions in sperm-egg attach- Palumbi, 1996) and low (Metz et ai, 1998b) levels of ment by binding to carbohydrates in the vitelline layer on intraspecific variation. In some cases a single molecule the egg surface. Third, it is involved in the fusion of sperm displays domains that are highly conserved and other do- and egg membranes (Ulrich et al, 1998, 1999). mains that are highly variable (Vacquier et al, 1995). Vari- Bindin is translated as a larger precursor, from which the ation in such proteins is usually studied at a low taxonomic N-terminal preprobindin portion is subsequently cleaved to produce mature bindin (Gao et al, 1986). The mature bindin molecule contains an amino acid core of about 55 residues Received 25 February 2003; accepted 3 June 2003. * To whom correspondence should be addressed. Current address: Fri- that is highly conserved among all bindins characterized to day Harbor Laboratories, University of Washington, 620 University Road, date (Vacquier et al, 1995). An 18 amino acid section of Friday Harbor, WA 98250. E-mail: [email protected] this conserved core (B18) has been shown to fuse lipid EVOLUTION OF BINDIN vesicles in vitro, suggesting that this region functions in differences in molecular size, amino acid sequence, and sperm-egg membrane fusion (Ulrich et ai, 1998, 1999). number of repeats. Thus far, bindin is known only from echinoids; no homol- ogous molecules have been identified in any other organism Materials and Methods (Vacquier, 1998). Samples To date, the nucleotide sequence of bindin has been determined in six genera of sea urchins. In Echinometra The pencil urchins (order Cidaroida) were represented in (Metz and Palumbi, 1996), Strongylocentrotus (Gao et al, our study by Eucidaris tribuloides, collected on the Atlantic 1986; Minor et al, 1991; Biermann, 1998; Debenham et al, coast of Panama; the order Diadematoida by Diadema an- 2000), and Heliocidaris (Zigler et al, 2003), there are many tillarum, also from the Atlantic coast of Panamá. The sand sequence rearrangements among individuals and species, dollars (order Clypeasteroida) were represented by Encope and indications of positive selection in regions on either side stokesii from the Pacific coast of Panamá; the heart urchins of the core. In Arbacia (Glabe and Clark, 1991; Metz et al, (order Spatangoida) by Moira clotho collected at the Perlas 1998a) and Tripneustes (Zigler and Lessios, 2003), there are Islands in the Bay of Panamá. Heliocidaris erythrogramma fewer sequence rearrangements and no evidence for positive (order Echinoida) was collected near Sydney, Australia. selection. In Lytechinus, only one sequence has been pub- lished (Minor et al, 1991), so the mode of evolution of the DNA isolation and sequencing molecule remains unknown. The five genera in which bindin was previously se- We injected various individuals of each species with 0.5 quenced belong to two echinoid orders, the Echinoida and M KCl until we encountered one that produced sperm. The testes of this ripe male were removed and used either the Arbacioida. These two orders contain only 10% of all directly for mRNA extraction, or after preservation in either extant echinoid species (Kier, 1977; Smith, 1984; Little- RNALater (Ambion Inc.) or in liquid nitrogen. The methods wood and Smith, 1995). The molecular structure of bindin for mRNA isolation, reverse transcription reactions, initial in the other 13 orders of the class Echinoida has not been polymerase chain reactions, 3' and 5' rapid amplification of studied. The only evidence that bindin is present outside the cDNA ends (RACE) reactions, and DNA sequencing were Echinoida and Arbacioida comes from Moy and Vacquier as described in Zigler and Lessios (2003), with the follow- (1979), who reported that an antibody to bindin of Strongy- ing modifications. (1) A fragment of the core region of locentrotus purpuratus reacted with sperm from one species bindin was amplified from the reverse transcriptase reac- of the order Phymosomatoida and two species of the order tion product or from genomic DNA, using primers Clypeasteroida. As Vacquier (1998) has pointed out, mole- MB1130+ (5'-TGCTSGGTGCSACSAAGATTGA-3') and cules that mediate fertilization•in contrast to those central either core200- (5'-TCYTCYTCYTCYTGCATIGC-3') or to other basic life processes•often differ between taxa. For corel57- (5'-CIGGRTCICCHATRTTIGC-3'). These prim- example, in the molluscan class Bivalvia, completely dif- ers correspond to amino acids VLGATKID, ANIGDP, and ferent proteins are involved in gamete recognition of oysters AMQEEEE, respectively (Vacquier et al, 1995). (2) When (Brandriff et al, 1978) and of mussels (Takagi et al, 1994). complete 5' mature bindin sequences were not obtained It is, therefore, not safe to assume without empirical evi- during the first round of 5' RACE, new primers were dence that bindin is present in all orders of echinoids, or that designed at the 5' end of the obtained sequence; then a it has the same general structure as in the taxa in which it second round of RACE amplification was conducted. (3) A has already been characterized. 5 ' preprobindin primer was designed based on a comparison As a first step in determining which orders of echinoids of preprobindin sequences of Moira clotho (this study) to possess bindin and, if they do, how its structure varies, we preprobindin sequences oi Arbacia (Glabe and Clark, 1991), cloned and sequenced mature bindin from five genera of sea Strongylocentrotus (Gao et al, 1986; Minor et al, 1991), and urchins, four of which belong to orders in which bindin was Lytechinus (Minor et al, 1991). This primer, prolSO (5'- previously unknown. We combined our data with those of AAGMGIKCL^GYSCIMGIAAGGG-3'), which corresponds previous studies of bindin in genera belonging to the orders to the conserved amino acids KR(A/S)S(A/P)RKG of the Echinoida and Arbacioida. The final data set includes bindin preprobindin, was used in combination with exact primers from 10 genera of sea urchins, pencil urchins, sand dollars, from the bindin core to amplify mature bindin sequences 5 ' and heart urchins, and the results indicate that the molecule of the core from Eucidaris tribuloides testis cDNA. (4) was present in the common ancestor of all extant echinoids Bindin sequences obtained from RACE were subsequently that diverged from each other over 250 million years ago. confirmed by amplification, cloning, and sequencing of full The core sequence has remained remarkably unchanged mature bindin sequences from testis cDNA. over this period of time, whereas the areas flanking the core Sequencing of both DNA strands was performed on have undergone substantial modification, resulting in great an ABI 377 automated sequencer, and sequences were 10 K. S. ZIGLER AND H. A. LESSIOS edited using Sequencher 4.1 (Gene Codes Corp.).