Short sequence-paper Characterization of two zebra¢sh cDNA clones encoding egg envelope proteins ZP2 and ZP3 Hai Wang, Zhiyuan Gong * Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 2 March 1999; received in revised form 14 April 1999; accepted 26 April 1999 Abstract Two zebrafish cDNA clones encoding homologs of mammalian zona pellucida proteins ZP2 and ZP3 were isolated from a whole adult cDNA library. The ZP2 clone encodes a protein of 428 amino acids. Unlike other teleost ZP2s that contain an N-terminal repetitive domain enriched with prolines and glutamines, the zebrafish ZP2 has no such repetitive domain. In the C-terminal non-repetitive domain, the zebrafish ZP2 shares 55^76% sequence identity with other teleost ZP2s. The ZP3 cDNA clone encodes a protein of 431 amino acids, which shares 61% sequence identity with a carp ZP3. Similar to mammalian ZP proteins, both zebrafish ZP2 and ZP3 contain several potential phosphorylation sites. However, unlike mammalian ZP proteins, both zebrafish ZP proteins contain almost no glycosylation site, which has been proposed to be important for interaction with sperm; thus, the ZP proteins may behave differently in mammals and teleosts. Northern blot analysis indicated that both zebrafish ZP2 and ZP3 mRNAs were expressed exclusively in the ovary and hence the ovary is likely the only site for ZP2 and ZP3 biosynthesis. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Glycosylation ; Phosphorylation ; Ovary ; Teleost ; Zona pellucida Fish eggs, like mammalian eggs, also contain a layer of non-cellular membrane, or zona pellucida. The mammalian zona pellucida consists mainly of three glycoproteins: ZP1, ZP2 and ZP3. While ZP2 and ZP3 function as sperm receptors, ZP1 provides structural integrity to egg membrane and may also be involved in sperm binding [1,2]. ZP3 is also an inducer of acrosome reaction in spermatozoa during fertilization [3]. In the present study, through parti- ally sequencing randomly selected clones from a whole adult cDNA library [4], we have isolated two full length clones encoding zebra¢sh ZP2 and ZP3 and found that they share the highest sequence iden- tities with the previously reported carp ZP2 and ZP3 cDNA clones, respectively [5,6]. The zebra¢sh ZP2 cDNA clone (A71) contains an insert of 1349 bp, including a complete coding region (1284 bp) for 428 amino acids (Fig. 1A). Based on available sequence data, all known ¢sh ZP2s possess an N-terminal repetitive domain rich in proline and glutamine residues. Both the number and pattern of the repeats are variable among di¡erent ¢sh species (Fig. 2). Alignments of ZP2s from carp [6], gold¢sh [6], winter £ounder [7], medaka [8] and Atlantic sal- mon (GenBank accession No. AJ000665) show that the zebra¢sh ZP2 does not have the repetitive do- main. It has been proposed that the repetitive do- main is involved in egg-hardening upon fertilization 0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4781(99)00066-4 * Corresponding author. Fax: +65 7792486; E-mail : dbsgzy@nus.edu.sg Biochimica et Biophysica Acta 1446 (1999) 156^160 www.elsevier.com/locate/bba