Zp domain proteins in the abalone egg coat include a paralog of verl under positive selection that binds lysin and 18-kda sperm proteins

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ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins
Jan E. Aagaard,*,1 Victor D. Vacquier,2 Michael J. MacCoss,1 and Willie J. Swanson1
1 2

Department of Genome Sciences, University of Washington Scripps Institute of Oceanography, University of California San Diego *Corresponding author: E-mail:jaagaard@u.washington.edu. Associate editor: Scott Edwards

Abstract
Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitellineenvelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common toVERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putativelysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm–egg fusion. This work identifies an egg coat paralog of VERL under positive selection andthe candidate sperm proteins with which it may interact during abalone fertilization. Key words: reproductive proteins, gamete recognition, reproductive isolation.

Research article

Introduction
A major focus of reproductive biology is identifying how sperm and egg proteins interact during fertilization. Understanding these molecular interactions is of broad biological importance, butcognate sperm and egg molecules have been identified in just a few cases (Swanson and Vacquier 1997; Kamei and Glabe 2003; Harada et al. 2008). One of the best characterized involves fertilization proteins from abalone (Haliotis spp.). Abalone sperm contain two principle acrosomal proteins, lysin (Lewis et al. 1982) and an 18-kDa protein (Swanson and Vacquier 1995a), which are released upon binding ofsperm to the elevated abalone egg envelope (the vitelline envelope [VE]). Lysin creates a hole in the VE, allowing sperm to cross this fertilization barrier by a nonenzymatic process involving binding to a large VE glycoprotein (the vitelline envelope receptor for lysin [VERL]) (Swanson and Vacquier 1997). VERL contains a tandemly repeated array of an ;150 amino acid motif that is believed to bethe lysin-binding sequence (Swanson and Vacquier 1997; Galindo et al. 2002). Though 18 kDa is ineffective at dissolving the VE, this fusagenic sperm protein coats the sperm acrosomal process and localizes to a second egg coat, structurally very similar to the VE, that overlies the plasma membrane at the egg surface (Mozingo et al. 1995). The 18-kDa sperm protein may bind a receptor

at the eggplasma membrane, mediating sperm–egg fusion (Swanson and Vacquier 1995a). The similar structure and physical characteristics of lysin and 18 kDa support paralogy between these two sperm proteins, although they have evolved different functions in fertilization (Kresge et al. 2001a). Crystallography shows that these paralogs have the same alpha-helical bundle structure but strikingly different...
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