Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

Abstract

Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins.

Fig. 1.

ZP domain genes identified from a pink abalone (H. corrugata) ovary EST library. RACE was used to extend ESTs predicted to have homology to ZP domain proteins, identifying nine ovary-expressed genes in addition to VERL. Phylogenetic analysis of the ZP domain from these genes (≈768 nucleotides) employing likelihood was performed under the general time reversible model with four rate categories by using PAUP (38). Terminal branches in the tree are labeled with a proposed gene name specifying whether the protein is found within solubilized VE material (VERL, VEZP2 to VEZP9) or not (ZPA), and bootstrap support based on 100 bootstrap replicates is shown for nodes with ≥50% support. Protein domains previously characterized for VERL (48) or computationally predicted via SMART (www.embl-heidelberg.de) for recently identified ZP genes are shown, including signal peptides (SP), internal repeats, and ZP, as well as transmembrane domains (TMD).

Fig. 2.

Vitelline envelope proteins from abalone eggs. (A) Abalone eggs are surrounded by a raised VE separated from the egg (EC, egg cytoplasm) by a perivitelline space (PS). (B) Five micrograms of total solubilized protein from purified VEs of both green (H. fulgens) and pink (H. corrugata) abalone were run on 2.5–15% SDS/PAGE gels and silver stained. The high molecular mass bands (boxed) identified as VERL (9) were confirmed by MS/MS of gel slices. Approximate molecular masses of size standards (M) are shown to the right. Aliquots of the same total VE proteins from both species were used for peptide identification in MS/MS experiments.