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PLoS One
2012 Jan 01;711:e46850. doi: 10.1371/journal.pone.0046850.
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The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.
Morin V
,
Sanchez-Rubio A
,
Aze A
,
Iribarren C
,
Fayet C
,
Desdevises Y
,
Garcia-Huidobro J
,
Imschenetzky M
,
Puchi M
,
Genevière AM
.
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Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.
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23144790
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Figure 1. Nucleotide and peptide sequences of S. granularis SpH protease. A: The sequence of the SpH protease cDNA (1569 nt) is reported with the longest ORF. Two in frame ATG codons are found (underlined) before the suggested first Met (M<$>\kern -8.5pt\scale 120%\raster="rg1"<$>) B: Alignment of putative N-terminal peptide sequences of S. granularis, S. purpuratus and P. lividus SpH-proteases.
Figure 2. Alignment of SpH proteases sequences (ClustalW).The following sequences are compared: S. granularis, S. purpuratus (XP_779916.1), cephalochordate B. floridae (AAQ01138.1), G. gallus (XP_425038.2), X. laevis (NP_001087489.1), O. latipes (Medaka, gi|50251128|), M. musculus (NP_034114.1), H. sapiens L1 (NP_001903.1) and L2 (NP_001324.2, also referred as cathepsin V). The 21 encoded amino acids between the first ATG and the putative first Met of the S. granularis sequence are indicated in italic. The catalytic triad of Cys-144, His-283 and Asn-303 as well as the ERFNIN, GNFD and GCNGG motives are enframed. The theoretical cleavage site of the prodomain is indicated (<$>\raster="rg2"<$>) upstream from the peptide homologous to the N-terminal sequence of T. niger mature SpHp (underlined).
Figure 3. Western blot analysis of native and recombinant SpH proteases.The recombinant mature S. granularis SpHp is immunostained by the antibodies raised against the N-terminal peptide of the previously purified T. niger SpH protease (lane 1); reciprocally, antibodies raised against the recombinant S. granularis mature protease recognized the protein purified by sucrose gradient [8] from T. niger fertilized eggs (lane 2). Both antibodies labelled a common electrophoretic band in chromatin extracts from S. granularis fertilized eggs (lane 3 and 4). Chromatin in lane 2–4 was prepared from eggs collected 5 min post-fertilization.
Figure 4. Phylogenetic tree for several deuterostomian taxa.The tree was constructed via Bayesian inference (BI) and maximum likelihood (ML) on cathepsin amino-acid sequences (shown with relevant family, pred.: mentioned as “predicted†in databases, and accession numbers): A: all cathepsin families; B: only L-like cathepsins. The S. granularis sequence produced in this study is in bold. Numbers are posterior probability (pp, BI)/bootstrap proportions in % (bp, ML). Internal branches are represented for pp>0.5 or bp>50, scale represents the number of substitutions per site.
Figure 5. Timecourse of SpH protease mRNA expression along development.Expression of SpH transcripts along development was evaluated by Northern blot analysis (A) and semi-quantitative RT-PCR (B). PCR was performed as described in Material and Methods using 1 µg of cDNA and 35 amplification cycles (95°C-1 min, 62°C-1 min, 72°C-1 min). The stability of SpH transcripts was estimated (C) by treating embryos with actinomycin D (50 µg/ml) 3 min post-fertilization and comparing the abundance of mRNAs in control and treated embryos by semi-quantitative RT-PCR (RT on DNA-free total RNA, PCR with 0.2 µg cDNA, 27 cycles). Inhibition of RNA synthesis with actinomycin D blocks development beyond hatching as visualized in (1) 20 hours p.f.. In these conditions the abundance of SpHp mRNAs remains invariant in treated embryos when compared to controls (1, 2). Quantification of 3 different experiments is reported in (3). Hours post-fertilization are indicated.
Figure 6. Temporal and spatial expression profile of SpH protease gene during sea urchin embryo development.Results of whole-mount in situ hybridization with anti-sense (columns A, C) and sense (columns B, D) probes are displayed for each developmental stage listed at the lower right corner of each panel.
Figure 7. Inhibition of SpHp activity during gastrulation.Sea urchin zygotes were treated after hatching with E64d (10–50 µM) and embryonic development was recorded till 72 h p.f.
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