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Gene
2006 Feb 15;367:135-41. doi: 10.1016/j.gene.2005.10.014.
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Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development.
Devader C
,
Webb RJ
,
Thomas GM
,
Dale L
.
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We have characterized a cDNA encoding a Xenopus laevis apyrase (XAPY) that is expressed during embryogenesis. XAPY is highly homologous to two recently described mammalian apyrases, human SCAN-1 and rat Ca2+-NDPase, and to a lesser extent the salivary apyrase of the blood-feeding arthropod Cimex lectularis. RT-PCR analysis shows that Xapy is expressed at all the developmental stages tested, from oocytes through to tadpoles. Xapy transcripts are widely distributed in the embryo, but from late neurulae through to late tailbud stages they are highly enriched in the cement gland, an adhesive organ in the epidermis of the head. When expressed in HEK 293 cells, XAPY is largely retained in the endoplasmic reticulum, although some is also secreted. XAPY conditioned media hydrolyses UDP and UTP, confirming that it is a functional apyrase.
Fig. 3.
RT-PCR analysis of Xapy mRNA expression during Xenopus development. Polydenylated RNA was prepared from eggs (1), early gastrulae (10), late gastrulae (13), neurulae (17), late neurulae (20), tailbud (30), and tadpole (40). − RT is stage 20 poly (A)+ mRNA minus reverse transcriptase. Xapy mRNA is expressed at all the stages tested. Ubiquitously expressed odc mRNA was used as a control.
Fig. 4.
Whole mount in situ hybridisation analysis of Xenopus embryos. (A) Frontal view of stage 18 embryo showing that Xapy is enriched in the developing cement gland (cg), a patch of anteriorepidermis just ventral to the neural tube (nt). (B) Lateral view of stage 24 embryo showing that Xapy continues to be expressed the cement gland. (C–D) Lateral view of a stage 30 (C) and a stage 40 (D) embryo showing Xapy expression in the head (h) and notochord (n). Xapy continues to be expressed in the cement gland, but is restricted to the basal region. In (B–D), the head of the embryo is on the right. (E–F) Sections through the head of a stage 20 (E) and a stage 30 (F) embryo, showing that expression is highly enriched in the cement gland. Note that in (F) staining is localized to the basal region of the cement gland. p = pharynx, b = brain.
Fig. 5.
Western blot analysis of transfected HEK cells and culture media. Cell extracts and concentrated media from Xapy and Xapy-mt transfected HEK293 cells were separated by SDS-PAGE in both the absence (A) and presence (B) of reducing agent (β-mercaptoethanol). C = cell extracts, M = conditioned media. Myc-tagged XAPY was detected using the 9E10 monoclonal antibody. Multiple bands are seen in both media and cell extracts under non-reducing conditions, but only one band under reducing conditions. The result demonstrates that XAPY-MT can be secreted. However, since the media is 15× more concentrated that the cell extracts it is clear that most of the protein is detected in cell extracts.
Fig. 6.
Confocal images of XAPY-MT expressing HEK cells. (A) Brightfield and (B) fluorescent images. The Myc epitope was detected using the 9E10 monoclonal antibody and fluorescein conjugated second antibody. The fluorescent image shows strong perinuclear staining and uneven cytoplasmic staining of XAPY-MT, indicative of localization to the endoplasmic reticulum. The nuclei (n) of three XAPY-MT expressing cells are indicated.
Fig. 7.
Secreted XAPY is a functional Apyrase. Conditioned media from transfected, and mock transfected, HEK 293 cells was incubated with the indicated substrates and production of phosphate (PPi) measured. The basal level of phosphate production from mock transfected media was subtracted from that of XAPY conditioned media and the data normalized to UDP being 100%. The results show that UDP is the preferred substrate for XAPY.
cant1(calcium activated nucleotidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
cant1(calcium activated nucleotidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anteriorright, dorsal up.
cant1(calcium activated nucleotidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, laterial view, anteriorright.
cant1(calcium activated nucleotidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anteriorright.
cant1(calcium activated nucleotidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, transverse section.
cant1(calcium activated nucleotidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, transverse section.
Fig. 1.
Nucleotide and deduced amino acid sequence of Xenopus Apyrase (XAPY). Putative translational start sites (×3) are indicated (View the MathML source). The first initiation codon for translation of Xapy is preceded by an in frame stop codon (TAA). A single putative transmembrane domain is indicated (__), as are two potential N-linked glycosylation sites (– – – –). A putative ER retention domain is also indicated (----). This sequence appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ632336.
Fig. 2.
Comparison of amino acid sequences of Apyrases from Xenopus laevis (Xl), Xenopus tropicalis (Xt), human (Hs), rat (Rn), and Cimex lectularius (Cl). The proteins were aligned with the Clustal W software program in MacVector. Minor modifications were made by the authors. Conserved amino acids are indicated by shading. Amino acids that may be critical for binding substrates and for the Ca2+ dependency of the enzyme are indicated (*). The DDBJ/EMBL/GenBank accession numbers for these genes are AJ632336 (X. laevis), AAH61377 (X. tropicalis), AF328554 (human), AJ312207 (rat), AAD09177 (C. lectularius).
