| Zebrafish (Danio rerio) is a small tropical fish that has been an important model organism for developmental biology and human diseases because of many inherent advantages: small size, high fecundity, early transparent embryos, in vitro development and physiological similarity to mammals and so on.Selectins are one of the family of cell surface molecules. Selectins includes P-,l- and E-selectin. P-selectin expressed on activated platelets and endothelial cells, L-selectin expressed on leukocytes and E-selectin expressed on activated endothelial cells. They play important roles in inflammation, thrombosis, tumor metastasis and so on. This researches on selectins will contribute to this study of the pathogenesis, diagnosing and treating of these diseases. This researches on mammalian selectins are intensive and comprehensive, and this researches on lower vertebrate selectins are absent.Here we firstly cloned the full-length cDNA of P-selectin and PSGL-1 from zebrafish, and analyzed their evolution and spatiotemporal expression patterns. And, we preliminary explored the biological function of zebrafish P-selectin. Here we firstly cloned the L-selectin partial cDNA from zebrafish including the complete open reading frame and forecast the E-selectin coding region from zebrafish, preliminary studied the expression pattern of L-selectin, and analyzed the evolution of L- and E- selectins. On the basis of this, we predicted the promoter regions of zebrafish P-selectin and PSGL-1, and analyzed the regulatory elements and potential transcription factor binding sites in these promoter regions by bioinformatics.The full-length cDNA sequence of zebrafish P-selectin is 2,800 bp and contains a 111-bp 5'-UTR, a 2,607-bp coding sequence and an 82-bp 3'-UTR. The zebrafish P-selectin gene contains 16 exons and 15 introns and is located on chromosome 20. Zebrafish P-selectin cDNA encodes a putative 868-amino acid protein with a theoretical molecular weight of 122.36 kDa and isoelectric point of 6.27. The putative protein has a signal peptide sequence of 25-amino acids and is rich in cysteine residues (n=69, 8% of total amino acids), which is similar to known P-selectin proteins. The amino acid sequence of zebrafish P-selectin shows 37% to 39% identity with that of known P-selectins. Smart analysis shows that the putative protein shares 5 distinct conserved structural domains involved in P-selectin function with known P-selectin proteins: an N-terminal lectin domain, an epidermal growth factor-like domain, a complement regulatory protein repeat domain, a transmembrane domain and a short cytoplasmic tail.Phylogenetic analysis shows zebrafish and mammalian P-selectins form distinct clades. This indicates a relatively distant evolutionary relationship between mammals and zebrafish,which is consistent with the different evolutionary ranks in biological classification.To clarify the role of zebrafish P-selectin, we analyzed its spatial and temporal expression patterns during early development using real-time quantitative PCR and whole-mount in situ hybridization. The results of real-time quantitative PCR showed that P-selectin was expressed at all developmental stages from 0.2 to 96 hpf, the expression increased gradually from the 1-cell stage to 72 hpf and then decreased at 96 hpf, and the expression level was highest at 72 hpf and the expression level at 72 hpf was significantly different from that at other developmental stages. The results of whole-mount in situ hybridization also showed that the expression existed at 4 hpf, the expression was evident in the anterior of the embryo at 90% epiboly and ubiquitous at 12 hpf, the expression was obvious in the head region and the dorsal aorta and axial veins in the trunk region at 18 hpf, and the expression was strong in the head region and circulatory system at 24-72 hpf. The results of real-time quantitative PCR and whole-mount in situ hybridization indicates that zebrafish P-selectin is expressed maternally, plays important roles in early development, and may be involved in many aspects of early embryonic development, especially vascular development.To investigate the biological function of zebrafish P-selectin, we analyzed its induced expression by ADP using real-time quantitative PCR. After zebrafish at 84 hpf was treated with 100μM ADP, P-selectin expression significantly upregulated within 30 min after induction as compared with controls. The expression was significantly upregulated at 2 min after ADP induction, reached the highest level at 8 min, and decreased at 15 and 30 min. This indicates that zebrafish P-selectin may involves in thrombosis.This study indicates that the functional structure domains of P-selectin protein are highly conserved among species, zebrafish P-selectin plays important roles during early development and has potential roles in thrombosis. This data first indicates that P-selectin plays important roles during early development. So, this study provides a novel insight into the understanding of P-selectin structure and function.The partial cDNA sequence of zebrafish L-selectin is 1,005 bp, including the complete open reading frame. zebrafish L-selectin gene is located on chromosome 20. Zebrafish L-selectin partial cDNA encodes a putative 324-amino acid protein with a theoretical molecular weight of 35.93 kDa and isoelectric point of 6.21. The amino acid sequence of zebrafish L-selectin shows 28 to 32% identity with that of known L-selectins. Smart analysis shows that the putative protein shares 5 distinct conserved structural domains involved in L-selectin function with known L-selectin proteins: an N-terminal lectin domain, an epidermal growth factor-like domain, a complement regulatory protein repeat domain, a transmembrane domain and a short cytoplasmic tail.Phylogenetic analysis shows zebrafish and mammalian L-selectin proteins form distinct clades. This indicates a relatively distant evolutionary relationship between mammals and zebrafish,which is consistent with the different evolutionary ranks in biological classification.To clarify the role of zebrafish L-selectin, we analyzed its expression during embryonic development using RT-PCR. The results of RT-PCR showed L-selectin was expressed at all developmental stages from 0.2 to 72 hpf. This indicates that zebrafish L-selectin is expressed maternally and plays important roles in embryonic development.This study indicates that the functional structure domains of L-selectin protein are highly conserved and plays important roles during embryonic development. This data first indicates that L-selectin plays important roles during embryonic development. So, this study provides a novel insight into the understanding of L-selectin structure and function.The coding region sequence of predicted E-selectin from zebrafish is 2,214 bp, encoding a putative 746-amino acid protein with a theoretical molecular weight of 82.47 kDa and isoelectric point of 4.60. The predicted E-selectin gene from zebrafish is located on chromosome 20. The amino acid sequence of predicted zebrafish E-selectin shows 39% to 41% identity with that of known E-selectins. Smart analysis shows that the putative protein shares 5 distinct conserved structural domains involved in E-selectin function with known E-selectin proteins: an N-terminal lectin domain, an epidermal growth factor-like domain, a complement regulatory protein repeat domain, a transmembrane domain and a short cytoplasmic tail.Phylogenetic analysis shows zebrafish and mammalian E-selectin proteins form distinct clades. This indicates a relatively distant evolutionary relationship between mammals and zebrafish,which is consistent with the different evolutionary ranks in biological classification.This study indicates that the functional structure domains of E-selectin protein are highly conserved. This data contributes to the further study on the structure and function of zebrafish E-selectin.The full-length cDNA sequence of zebrafish PSGL-1 is 1,594 bp and contains a 182-bp 5'-UTR, an 855-bp coding sequence and a 557-bp 3'-UTR. The zebrafish PSGL-1 gene contains 3 exons and 2 introns and is located on chromosome 5. Zebrafish PSGL-1 cDNA encodes a putative 284 amino acid protein with a theoretical molecular weight of 30.33 kDa and isoelectric point of 7.96. The putative protein has a signal peptide sequence of 27amino acids and is a secretory protein.In this putative P-selectin binding region of zebrafish PSGL-1 (residues 29–93), there is 1 tyrosine (Tyr86) for potential sulfation site, 12 threonines for potential O-glycosylation sites and 5 consensus sequences (NXS/T) for potential N-glycosylation sites. The whole extracellular domain of zebrafish PSGL-1 has 31 threonine and 22 Serine residues for potential O-glycosylation sites and 8 consensus sequences (NXS/T) for potential N-glycosylation sites. The 160-residue extracellular domain is rich in serine, threonine and proline (22Sers/31Thrs/13Pros), characteristic of mucin-like domains, but the striking repeat pattern is not observed in the whole extracellular domain. Zebrafish PSGL-1 has 5 cysteine residues, and they are located in 11, 174, 188, 208 and 232. Among them, one (Cys11) is in the putative signal peptide region, two (Cys174 and Cys188) is in the extracellular region, one (Cys208) is in the transmembrane region, and one (Cys232) is in the cytoplasmic tail region. Cys188 is at the proposed junction of the extracellular and transmembrane domains. The amino acid sequence of zebrafish PSGL-1 is 19-22% identical to that of mammalian PSGL-1. The sequence of the putative mature protein contains the following structural feature: a large extracellular domain of 160 residues, a transmembrane domain of 23 residues and a cytoplasmic tail of 73 residues by smart, as observed in mammals. The signal peptide, extracellular, transmembrane and cytoplasmic tail regions of zebrafish PSGL-1, which share 23-35%, 13-16%, 39-47% and 33-37% sequence homologies with that of human, mouse, horse, rat, bovine and pig .Phylogenetic analysis shows zebrafish and mammalian PSGL-1 proteins form distinct clades. This indicates a relatively distant evolutionary relationship between zebrafish and mammals,which is consistent with the different evolutionary ranks in biological classification.To clarify the role of zebrafish PSGL-1, we analyzed its temporal and spatial expression patterns during embryonic development using RT-PCR and whole-mount in situ hybridization. The results showed that zebrafish PSGL-1 was expressed in embryonic development and the expression increased gradually from 0.2 (1-cell stage) to 72 hpf. The weak expression was observed at 0.2 hpf (1-cell stage), then, the expression outstandingly increased from 12 to 26 hpf and 26 to 48 hpf and the expression was maintained at a relatively high level at 48 and 72 hpf. PSGL-1 was expressed in the embryo anterior at 90% epiboly and ubiquitously expressed at 12 hpf, the expression was strong in the head region, the whole myotome region of ventral trunk, intermediate cell mass region and the ventral vascular system at 26 hpf, the expression was in the whole ventral trunk, and still strong in the head region, the ventral vascular system and the intermediate cell mass region at 48 hpf, and the expression was confined to the cardiovascular system in the head region, the signal existed in the whole trunk, the strong signal was persisted in the intermediate cell mass region and the ventral vascular system, and there was no signal in the caudal region at 72 hpf. This indicates that PSGL-1 is expressed maternally, plays important roles in early embryonic development, and may be involved in many aspects of early embryonic development, especially muscl segment development.This study indicates that the functional structure domains of zebrafish PSGL-1 protein are highly conserved among species, despite a poor conservation of the extracellular domain, and the potential tyrosine sulfation and threonine O-glycosylation sites in the putative P-selectin binding region of zebrafish PSGL-1, and a single cysteine at the transmembrane and extracellular domain junction suggesting a disulfide-bonding pattern are evolutionary conserved between fish and mammals. This data first indicates that PSGL-1 plays important roles during embryonic development. So, this study provides a novel insight into the understanding of PSGL-1 structure and function.The 873-3000 bp of ATG upstream serves as the potential promoter region of P-selectin gene, and the 2012-3500 bp of ATG upstream serves as the potential promoter region of PSGL-1 gene. In the potential promoter region of zebrafish P-selectin gene, 7 TATA boxes are present, but CAATbox and GC box are absent; in the potential promoter region of zebrafish PSGL-1 gene, 1 TATA box and 1 CAAT box are present, but GC box is absent. CpG island is absent in the promoter regions of this two genes. The transcription factor binding sites in the potential promoter region of zebrafish P-selectin and PSGL-1 were analyzed by TFSEARCH on circumstances of scores of 90, the transcription factor binding sites (CdxA, SRY, Nkx-2, AML-1a, Sox-5, S8, Oct-1, HFH-2, HNF-3b,Pbx-1, HFH-1, STATx, Brn-2, HSF2, GATA-1, TATA, c-Myc/, C/EBP, c-Rel, XFD-1, AP-1 and YY ) are present in the potential promoter regions of zebrafish P-selectin gene, and the transcription factor binding sites (SRY, CdxA, Nkx-2, AML-1a, Sox-5, S8, Oct-1, HNF-3b, GATA-1, C/EBP, Evi-1, MZF1 and AP-1) are present in the potential promoter regions of zebrafish PSGL-1 gene.This study helps to this understanding on selectins and PSGL-1 evolutions, provides information for revealing zebrafish developmental mechanism, and further deepens this understanding on the molecular mechanism of zebrafish embryonic development, and This study may provide novel disease models for cardiovascular and cerebrovascular diseases and so on, and multi new dates for the diagnosis and treatment of these diseases.
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