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First published online January 12, 2004
Journal of Experimental Biology 207, 621-632 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00785

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Purification and cDNA cloning of the ovigerous-hair stripping substance (OHSS) contained in the hatch water of an estuarine crab Sesarma haematocheir

Oleg Gusev¹, Hideki Ikeda¹, Tetsushi Okochi¹, Jae Min Lee², Masatsugu Hatakeyama², Chiyoko Kobayashi³, Kiyokazu Agata³, Hidenori Yamada⁴ and Masayuki Saigusa^1,*

¹ Laboratory of Animal Behavior and Evolution, Graduate School of Natural Science and Technology, Okayama University, Tsushima 3-1-1, Okayama 700-8530, Japan
² Developmental Mechanisms Laboratory, Developmental Biology Department, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba 305-8634, Japan
³ Laboratory for Evolutionary Regeneration Biology, Center for Developmental Biology, RIKEN Kobe, Minatojima-minamimachi 2-2-3, Chuo-ku, Kobe 650-0047, Japan
⁴ Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima 3-1-1, Okayama 700-8530, Japan

View larger version (59K): [in a new window]   Fig. 1. Embryo attachment system and its stripping from the ovigerous hairs after hatching, in the estuarine crab Sesarma haematocheir. (A) Diagram of the incubation chamber of the female (space between the thorax T and the abdomen Ab). (B) The abdominal appendage of the female. Ovigerous hairs (oh) are arranged in whorls on the ovigerous seta (os). Embryos are attached to the ovigerous hairs by the stalk (funiculus), but not to the fine hairs arranged on the plumose seta (ps). (C) Embryos attached to an ovigerous hair (living specimen). (D) The egg attachment system remained on the ovigerous hairs just after hatching. (E) Ovigerous hairs stripped several hours after hatching. (F) The ovigerous seta with its attached embryos was divided into five segments, one of which is shown here. (G) A segment of the ovigerous seta from which the embryos were gently pulled with a forceps after detaching from the female and subdividing. (H) A segment of the ovigerous seta from which the embryos were gently pulled with a forceps after immersion in the hatch water for 1.5 h. wl, walking leg; go, gonopore; an, anus; em, embryo cluster; sg, segment of the ovigerous seta. Scale bars: A, 5 mm; B,D,E, 2 mm; C, 0.5 mm; F–H, 1 mm.

View larger version (27K): [in a new window]   Fig. 2. Final step of purification of S. haematocheir OHSS. (A) RP-HPLC analysis of the proteins. The active fractions eluted by molecular-sieve chromatography (gel filtration) were concentrated, and the proteins were eluted with a linear gradient of 8%–52% acetonitrile containing 0.1% HCl over 80 min (flow rate at 0.6 ml min–1). Numbers (1–6) indicate peaks of protein. (B) OHSS biological assay of fractions eluted by FPLC (gel filtration) and RP-HPLC. Bioassay carried out for 1.5 h with two ovigerous setal segments per fraction (see Saigusa and Iwasaki, 1999). C, control assay in distilled water. Values are means ± S.D. (C) Protein analysis by SDS-PAGE. The polyacrylamide gel was stained with Coomassie Brilliant Blue. The marker proteins were ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa; Amersham). Two bands (25 and 22 kDa) appeared in fractions 3–5, while a single band (25 kDa) appeared in fraction 6.

View larger version (61K): [in a new window]   Fig. 3. Nucleotide sequence and deduced amino acid sequence of S. haematocheir OHSS. Nucleotides are numbered to the left from the first base at the 5' end, and amino acids are numbered to the right from the initiating methionine. The putative signal peptide sequence is shown in bold. The boxed amino acids indicate determined sequences. The putative cleavage site accompanying activation is shown by a solid triangle. The catalytic triad of the serine protease domain is enclosed by a circle. A putative polyadenylation signal is doubly underlined. Putative N-glycosylation sites are indicated by asterisks. Locations of three introns (Int-1, 240 bp; Int-2, 316 bp; and Int-3, 842 bp) are marked by upward-pointing arrows. Positions of nucleotide primers used for cloning and expression analysis of OHSS gene are indicated by red arrows (see text for details).

View larger version (52K): [in a new window]   Fig. 4. Multiple alignment of the deduced amino acid sequence of the serine protease domain of S. haematocheir OHSS with those of known serine proteases of six species. Regions of high homology across all species are highlighted in black (100–90%); regions with less homology are highlighted in gray (>80%) and purple (>60%). The numbering of amino acids corresponds to the original sequences in each animal. Information for comparison: chymotrypsin of Penaeus vanameii (database accession number S29239); trypsin of Paralithodes camtschaticus (AF461036); proclotting enzyme (procl.) of Tachypleus tridentatus (P21902); defensin of Pacifastacus leniusculus (AJ007668.1); human hepsin (P05981); human matriptase (Q9Y5Y6). Residues in the catalytic triad (His-293, Asp-346 and Ser-441) are indicated by an asterisk. Residues in the substrate pocket (Asp-435, Gly-463 and Gly-473) are indicated by diamonds. Six conservative cysteines needed to form three intramolecular disulfide bonds are likely pairings as follows: Cys278–Cys294, Cys411–Cys426, and Cys437–Cys466. The disulfide bond Cys246–Cys366 (the cysteines are boxed) is observed in two-chain serine proteases, but not in trypsin and chymotrypsin.

View larger version (31K): [in a new window]   Fig. 5. RT-PCR analysis of the expression of the OHSS gene. PCR products of 27 cycles of RT-PCR amplification using OHSS cDNA were loaded in 0.8% agarose gel. RNAs were extracted from embryos detached from the female (Emb), the zoea larvae (Zoea), and muscles (FM), hepatopancreas (FH), ovigerous setae (FOs) and brain (FB) of the adult female. Numbers below Emb and Zoea indicate days before (–) and after (+) hatching, and the day of hatching (h). Lane G represents the products of PCR using genomic DNA from embryos as template. Size standards in kb are shown on the left.

View larger version (82K): [in a new window]   Fig. 6. Egg attachment system formed on the maternal ovigerous hair and its stripping after hatching. (A) The egg attachment system formed on an ovigerous hair. This system consists of a single layer with two sublayers (E1a, E1b). The fine structure of the attachment sites (enclosed by the open rectangle) is shown in C. (B) The egg attachment sites arranged at intervals of 130–160 nm on the maternal ovigerous hair. (C) Fine structure of the attachment of layer E1 to the ovigerous hair (left), and schematic drawing of this structure (right). A portion of the attachment site is shown by two small arrows (as). (D) Separation of layer E1 after the embryos have been immersed in hatch water (left), and its schematic drawing (right). Two small arrows (as) show a portion of the attachment sites separated from the investment coat (E1a). oh, maternal ovigerous hair. Scale bars, 100 nm.