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

Abstract

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 TGFα 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.

The serpin PN1 is a feedback regulator of FGF signaling in germ layer and primary axis formation

“…In a screen for secreted proteins from gastrula stage Xenopus laevis embryos, we previously isolated five non-redundant full-length cDNA clones that encode the serpin family member PN1 (Pera et al, 2005). PN1 appeared as a 45 kDa protein in the supernatant of transfected and [ 35 S]methionine/cysteine-labeled HEK293T cells (Fig.…”

“…We compared the gene expression of PN1.b, the previously described PN1.a (Pera et al, 2005;Onuma et al, 2006) and the secreted serine protease HtrA1 (Hou et al, 2007). RT-PCR and whole-mount in situ hybridization analyses reveal maternal and zygotic transcription of PN1.a and PN1.b, whereas HtrA1 is robustly expressed only after the midblastula transition ( Fig.…”

“…In a direct screen for secreted proteins in early Xenopus embryos, we identified Protease nexin-1 (PN1) (Pera et al, 2005). PN1, which is also known as Glial-derived nexin or SerpinE2, is a serine protease inhibitor (serpin) that contains an exposed reactive center loop (RCL) that covalently binds to and blocks proteases such as thrombin, plasminogen activator, trypsin, urokinase and factor XIa (Baker et al, 1980;Stone et al, 1987;Knauer et al, 2000).…”

Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system

“…Full-length cDNA clones of XRDH10 and XRALDH2 in the expression vector pCS2 were obtained by secretion cloning (Pera et al, 2005). XRDH10 was fully sequenced (Gene Accession Number FJ213456).…”

“…In a screen for secreted proteins, we have recently identified the Xenopus homologue of RDH10 (Pera et al, 2005). XRDH10 expression partially overlaps with that of XRALDH2 in the early embryo and is subject to negative-feedback regulation by endogenous RA.…”