cDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

Abstract

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His 76 , Asp 132 , and Ser 227 , which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys 56 -His 57 ) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.In fertilization, sperm must bind to and penetrate through the extracellular glycoprotein matrix surrounding the egg, which is called the zona pellucida in mammals and the vitelline coat in marine invertebrates. After this process, membrane fusion occurs between the sperm and egg. Upon primary binding of the sperm to the vitelline coat, it undergoes an acrosome reaction, which is an exocytosis of the acrosomal vesicle located on the tip of the sperm head. A lytic agent called lysin is exposed on the surface of the sperm head and partially released into the surrounding seawater during the acrosome reaction. In mammals, it has been believed that a trypsin-like enzyme called acrosin (EC 3.4.21.10) is a zona lysin (1, 2). However, recent studies using acrosin-gene knockout mice have revealed that acrosin is not essential for mouse fertilization, although a significant delay (about 30 min) in sperm penetration through the zona pellucida was observed (3, 4). From these results it is currently thought that sperm proteases other than acrosin may participate in sperm penetration through the mammalian egg coat and that acrosin may be involved in the dispersal of the acrosomal matrix (5).Ascidians (Urochordata) occupy a phylogenetic position between vertebrates and "true" invertebrates. Although all ascidians are hermaphrodites, several ascidians including Halocynthia roretzi are strictly self-sterile. The vitelline-coat lysin system is thought...

Rendezvin: An Essential Gene Encoding Independent, Differentially Secreted Egg Proteins That Organize the Fertilization Envelope Proteome after Self-Association

“…Although CUB domains and LDLrA repeats are common throughout the animal kingdom, where they function in protein-protein binding (Bork and Beckmann, 1993;Sugiyama et al, 2000;Topfer-Petersen et al, 2000;Kodama et al, 2001;Guo et al, 2004;Song et al, 2006), our results provide the first direct evidence that these two modules interact in a specific, high-affinity manner. How might the CUBs of RDZ 120 organize a matrix enriched with LDLrAs ?…”

“…They are present in many extracellular matrix proteins, receptors, and fertilization-related proteins such as the sea urchin vitelline post protein p160 (Haley and Wessel, 2004), the egg bindin receptor 1 (Kamei and Glabe, 2003), the embryonic matrix protein hyalin (Song et al, 2006), ascidian sperm acrosin (Kodama et al, 2001), and mammalian spermadhesins (Topfer-Petersen et al, 1998). Among the fertilization-specific proteins, two sperm-derived proteins have been shown to directly bind the extracellular matrices of their respective eggs, consistent with the involvement of CUBs in gamete binding in vivo (Dostalova et al, 1995;Calvete et al, 1996;Topfer-Petersen et al, 2000;Kodama et al, 2001). Crystal structures of the CUB domain suggest this 10 ␤-strand motif sandwiches a hydrophobic core between two sheets of five ␤-strands Varela et al, 1997).…”

Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica)

“…In mice, ZP2 binds to proacrosin-null sperm considerably less effectively than wild-type sperm, and the binding of proacrosin to ZP2 is mediated by a strong ionic interaction between polysulfate groups on ZP2 and basic residues on an internal proacrosin peptide (Howes et al 2001), resulting to conclude that the ZP2-proacrosin interaction is important for the retention of acrosome reacted sperm on the ZP surface. In case of ascidian sperm, paired basic amino acid residues of acrosin are reported to play a key role in the binding of acrosin to the vitelline coat (Kodama et al 2001). Because ascidian acrosin is released from sperm into the surrounding seawater, acrosin is suggested to be also involved in the process of sperm penetration through the vitelline coat (Kodama et al 2001).…”

“…In case of ascidian sperm, paired basic amino acid residues of acrosin are reported to play a key role in the binding of acrosin to the vitelline coat (Kodama et al 2001). Because ascidian acrosin is released from sperm into the surrounding seawater, acrosin is suggested to be also involved in the process of sperm penetration through the vitelline coat (Kodama et al 2001).…”

Allorecognition and Lysin Systems During Ascidian Fertilization

“…Both HrProacrosin (precursor of HrAcrosin) and HrSpermosin possess several candidate regions for protein-protein interaction, that is, two CUB domains in the C-terminus of HrProacrosin and a Pro-rich region in the N-terminus of the light chain of HrSpermosin (Kodama et al 2001(Kodama et al , 2002. Two VC proteins (25-and 30-kDa VC proteins) were identifi ed as binding proteins to these proteases (Akasaka et al 2010 ).…”