tailieunhanh - Báo cáo khoa học: Spermosin, a trypsin-like protease from ascidian sperm cDNA cloning, protein structures and functional analysis

We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi[Sawadaet al. (1984),J. Biol. , 2900±2904; Sawadaet al. (1984), Dev. , 246±249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. | Eur. J. Biochem. 269 657-663 2002 FEBS 2002 Spermosin a trypsin-like protease from ascidian sperm cDNA cloning protein structures and functional analysis Eri Kodama1 Tadashi Baba2 Nobuhisa Kohno2 Sayaka Satoh2 Hideyoshi Yokosawa1 and Hitoshi Sawada1 1 Department of Biochemistry Graduate School of Pharmaceutical Sciences Hokkaido University Japan 2Institute of Applied Biochemistry University of Tsukuba Tsukuba Science City Japan We have previously reported that two trypsin-like enzymes acrosin and spermosin play key roles in sperm penetration through the vitelline coat of the ascidian Urochordata Halocynthia roretzi Sawada et al. 1984 J. Biol. Chem. 259 2900-2904 Sawada et al. 1984 Dev. Biol. 105 246-249 . Here we show the amino-acid sequence of the ascidian preprospermosin which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides and an open reading frame encoding 388 amino acids which corresponds to a molecular mass of 41 896 Da. By sequence alignment it was suggested that His178 Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classihed as a novel trypsin family member. The mRNA of preprospermosin is specihcally expressed in ascidian gonads but not in other tissues. Purihed spermosin consists of 33- and 40-kDa bands as determined by SDS PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain residues 130-388 and a long light chain designated L1 residues 23-129 whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 residues 97-129 . The L1 chain contains a proline-rich region designated L1 AL2 which is lacking in L2. Investigation with the glutathione-S-trans-ferase GST -spermosin-light-chain fusion proteins includ-ingGST-L1 GST-L2 andGST-L1 AL2 revealedthatthe proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus we propose that sperm spermosin is a