Pera EM et al. (2005), Exploration of the extracellular space by a lar...

XB-ART-1346

Int J Dev Biol 2005 Jan 01;497:781-96. doi: 10.1387/ijdb.052003ep.

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Exploration of the extracellular space by a large-scale secretion screen in the early Xenopus embryo.

Pera EM , Hou S , Strate I , Wessely O , De Robertis EM .

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Secreted proteins play a crucial role in intercellular communication during embryogenesis and in the adult. We recently described a novel method, designated as secretion cloning, that allows identifying extracellular proteins exclusively based on their ability to be secreted by transfected cells. In this paper, we present the results of a large-scale screening of more than 90,000 clones from three cDNA expression libraries constructed from early Xenopus embryos. Of 170 sequenced clones, 65 appeared to encode secreted proteins; 26 clones (40%) were identical to previously known Xenopus genes, 25 clones (38%) were homologous to other genes identified in various organisms and 14 clones (22%) were novel. Apart from these bona fide secreted proteins, we also isolated lysosomal or other secretory pathway proteins and some cytoplasmic proteins commonly found in body fluids. Among the novel secreted proteins were two putative growth factors of the Granulin family, termed xGra1 and xGra2; they are structurally similar to EGF and TGFalpha and show a spotted expression pattern in the epidermis. Another secreted protein, designated xSOUL, belongs to the family of heme-binding proteins and exhibits distinct expression in the early brain. A third protein, termed Xystatin, is related to cysteine proteinase inhibitors. Our results indicate that secretion cloning is an effective and generally useful tool for the unbiased isolation of secreted proteins.

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Species referenced: Xenopus laevis
Genes referenced: acp5 actb actg1 admp adsl akr1b1 aldh1a2 aldoc cer1 chmp1b chmp2a cndp2 cst3 ctsb ctsd ctsl ctsv darmin dctpp1 dnaja2 dnajb11 egf enpp2 fbp1 frzb frzb2 fth1 gamt gdi2 glul gpc4 grn has1 hebp2 hexb hnrnpa1 hsbp1 htra1 igfbp5 ism1 kdelr2 ldhb lefty1 lfng lman1 lman2 man2b1 manf mdh1 nbl1 nodal3 nog nucb1 pabpc1 pdia3 pepd pgam1 pkm plbd2 plg ppib ppp4c psap rdh10 rhoa rpe serpine2 serpinh1 sfrp2 shmt1 smarcc1 spint2 stmn1 szl taldo1 tinf2 tmem170a tor1a tpm3 tpt1 ubqln4 urod vcp ykt6 ywhab ywhae ywhaq ywhaz zp4

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	Fig. 1. Secretion Cloning: a novel approach to isolate secreted proteins. (A) Overview of the method. Bacterial colonies from expression cDNA libraries are picked from agar plates and individually grown in 96 well blocks. Culture media from 16 clones are pooled and plasmid DNA prepared by miniprep. Human embryonic kidney 293T cells are co-transfected with the cDNA pools and pAdvantage (pAdv) DNA. After 36 hours, the transfected cells are labeled with 35S-Methionine/35S-Cysteine and incubated for another 36 hours. Proteins in the cell supernatant are separated by SDS-PAGE and detected by autoradiography. Once a candidate pool is identified, the procedure is repeated using subpools to individualize the positive cDNA clone. (B) Method of sib-selection. Left panel, bacteria from selected pools were regrown in a 96 well block and the 16 clones of each pool arrayed in 4x4 wells. The molecular weight of the candidate protein in each pool is indicated on the side. Note that pools grouped next to each other should differ in their molecular weight. Right panel, autoradiogram of protein gels. Each lane is loaded with the supernatant of cells transfected with a subpool of eight cDNA clones, prepared from one column (letter) or one row (number) of the 96 well block. Note that a positive clone is identified, when the bands in two lanes have the same molecular weight as the original pool. (C) Example of an SDS-PAGE of radioactive supernatants of 293T cells transfected with pools of 16 cDNA clones from a Xenopus gastrula library. Note in lanes 12 and 14 bands of 34 and 45 kD, respectively, which led to the initial identification of secreted Frizzled-related Protein-2 (sFRP2) and Protease inhibitor Nexin-1 (PN-1) in Xenopus. (D) Optimization of transfection conditions; cDNA clones encoding Protein Phosphatase X (PPX) or Xenopus Cystatin (xCys) were transfected alone (lanes 1-3) or with 10% pAdvantage DNA (pAdv, lanes 4-6). The time between transfection and the addition of 35S-methionine/35S-cysteine is indicated in hours. Note that the protein yield is highest in the presence of pAdvantage and when metabolic labeling was started 36 hours after transfection. (E) Detection of Sizzled, PPX and xCys protein in the medium of transfected cells after varying lengths of radioactive labeling. Note that conditioning for 36 hours yields strong signals of the desired proteins, while longer incubation increases non-specific background.
	Fig. 2. Overview of the proteins isolated by secretion cloning. (A) Positive clones identified in three expression cDNA libraries from early Xenopus embryos. The number on top of each bar indicates the number of proteins isolated as bands by SDS-PAGE in the supernatant of transfected cells. The black portion of the bars indicates unique sequences. (B) Number of known proteins that have functionally been characterized in Xenopus, related proteins characterized in other vertebrates but not in Xenopus and novel proteins not characterized yet. (C) Unique sequences isolated in the individual expression cDNA libraries. (D) Predicted subcellular localization of cDNAs related to those of other species or to known Xenopus cDNAs. Proteins are subdivided into secreted, lysosomal/ endoplasmatic reticulum (ER)/Golgi apparatus and cytoplasmatic/nuclear; numbers of cDNAs identified are indicated. (E) Subcellular localization of known and related sequences in each cDNA library. M, maternal library (32- cell stage); vG, ventral gastrula stage library, dG, dorsal gastrula stage library.
	Fig. 3. xGra1 and xGra2 belong to the Granulin family of secreted growth factors. (A) Supernatant culture medium of human embryonic kidney (293T) cells transfected with cDNA encoding nonsecreted green fluorescent protein as control (GFP), Xenopus Granulin-1 (xGra1) or Xenopus Granulin-2 (xGra2). Cells were labeled with 35S-methionine and - cysteine and their supernatants were analyzed by SDS-PAGE and autoradiography. Note that xGra1 is secreted as a 105 kD protein and xGra2 as an 82 kD protein. (B) Diagrams of xGra1 and xGra2. The signal peptide cleavage sites are indicated by triangles. The black boxes indicate conserved granulin repeats (Pfam accession number PF00396). The numbers and guidelines indicate repeats conserved in both proteins and are based on sequence similarities. Note that the granulin repeats 3, 8 and 9 of xGra1 are missing in xGra2. (C) Signature of the granulin repeat. Note the conserved spacing of four cysteine doublets flanked by two cysteine singletons on each side. (D) Sequence alignment of xGra1 and xGra2. xGra1 corresponds to the Granulin provisional protein sequence previously published for Xenopus laevis (GenBank accession number AAH48224) and xGra2 is novel (GenBank accession number DQ004683). Identical amino acid residues are shaded in black and similar or conserved residues in gray. Dots represent gaps introduced into the amino acid sequence in order to obtain optimal alignment. The signal peptide cleavage site as predicted by SignalP is indicated with an arrowhead. Black bars indicate the granulin repeats and the stars the conserved cysteine residues. The overall number of amino acids is indicated at the end of each sequence. (E-G) Expression of xGra2 analyzed by whole-mount in situ hybridization. (E) Four-cell stage embryo in animal view. Note the high level of maternal transcripts. (F) Embryo at late neurula stage in dorsal view showing spotted expression in the epidermis. (G) Early tail bud stage embryo in lateral view. Note expression in the pronephric glomus (gl).
	Fig. 4. xSOUL is a novel secreted protein of the SOUL/Heme-binding protein family. (A) Culture medium of 293T cells transfected with green fluorescent protein cDNA as control ( GFP) or Xenopus SOUL cDNA (xSOUL ). xSOUL is secreted as a 23 kD protein. (B) Evolutionary relationship of xSOUL and other members of the SOUL/heme binding protein (HBP) family. Proteins with the greatest sequence similarity cluster together and branch lengths are proportional to distance (TreeTop-Phylogenetic Tree Prediction; http://www.genebee.msu.su/services/ phtree_reduced.html). Only the mature proteins have been considered. Note that xSOUL is most closely related to a derived family member in the plant Arabidopsis thaliana (aSOUL). The listed proteins have the following GenBank accession numbers: xSOUL (GenBank accession number DQ004682); aSOUL (NM101570); human SOUL (AF117616); mouse SOUL (AF117614); zebrafish SOUL (BC045936); human HBP (AF117615); mouse HBP (AF117613); chick SOUL (AF117612). (C) Comparison of xSOUL with other SOUL/HBP sequences. Note that only xSOUL and aSOUL have cleavable signal peptides (triangle). The putative hemebinding region as reported by Zylka and Reppert (1999) is overlined. (D) Four-cell stage embryo in animal view showing maternal expression of xSOUL. (E) Anterior view of advanced neurula. Note weak expression in the anterior brain (arrowhead). (F) Tailbud stage embryo with distinct expression in the forebrain, lens, somites and gut.
	Fig. 5. Xystatin is a novel secreted cysteine proteinase inhibitor. (A) Supernatant of 293T cells non-transfected (control) or transfected with Xenopus Cystatin cDNA (xCys ). Note that xCys is secreted as an 18 kDa protein. (B) Phylogenetic tree indicating the relationship of xCys with other members of the cystatin superfamily. Only the mature proteins lacking the signal peptide have been aligned. GenBank accession numbers are as follows: Xystatin (DQ004681); salmon Cystatin (D86628); chick Cystatin (JO5077); murine Cystatin-C (NM009976); human Cystatin-C (BC013083). (C) Sequence alignment of Xcys and related members of the cystatin superfamily. Four conserved cysteine residues known to form two disulphide bonds are labeled with stars. The conserved active site sequence QxVxG and pro-trp (PW) sequences are underlined (Brown and Dziegielewska, 1997).
	Fig. 6. Whole-mount in situ hybridization of xPN-1, xNuc and xEDJ. Embryos are shown in animal (A), dorsal (B,D), lateral (C,H,I) or anterior view (E-G). (A-C) Xenopus Proteinase Nexin-1 (xPN-1). (A) Four-cell stage embryo showing high level of maternal transcripts. (B) Late neurula. Note expression in the notochord (n) and ear placode (e). (C) Early tail bud stage with additional expression domains in the lens (le) and ectoderm of the branchial arches (ba). (D-F) Xenopus Nucleobindin (xNuc). (D) Midgastrula showing expression in the notochord (n). (E) Early neurula with additional signal in the cement gland (cg). (F) Late neurula. Note strong expression in the cement gland and hatching gland (hg). (G-I) Xenopus ER-associated DNAJ chaperone (xEDJ). (G) Late neurula with expression in cement gland (cg) and hatching gland (hg). (H) Late neurula depicting faint signal on the ventral side (arrowhead). (I) Early tail bud stage. Note expression in the pronephros (pn) and in the hatching gland in the dorsal head.
	grn (granulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
	grn (granulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
	grn (granulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	hebp2 (heme binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
	hebp2 (heme binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
	hebp2 (heme binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
	dnajb11 (DnaJ heat shock protein family (Hsp40) member B11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	dnajb11 (DnaJ heat shock protein family (Hsp40) member B11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	serpine2 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
	serpine2 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior up.
	serpine2 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.