Analysis of Xenopus laevis RECK and Its Relationship to Other Vertebrate RECK Sequences

Abstract

Aims: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored protein that regulates cell migration by both inhibiting matrix metalloproteinases and modulating cellular pathways. Our study is the first to clone and examine the expression pattern of reck during early Xenopus laevis development. Study Design: Expression and sequence analysis. Place and Duration of Study: Department of Biology, University of Western Ontario, 2 years. Methodology: Xenopus laevis RECK cDNA was sequenced and used to make probes to analyze RECK temporal and spatial expression using PCR and in situ hybridization respectively. The X. laevis sequence was also compared to a breadth of vertebrate and a few invertebrate RECK amino acid sequences to ascertain conserved regions and domains that may be present in all vertebrate and other animal species. Results: Semi-quantitative PCR, whole mount in situ hybridization, and immunohistochemistry were used to examine the expression pattern of reck during early X. laevis development. reck expression is low during gastrulation but increases during neurulation and throughout organogenesis. RECK is also present in neural and vascular structures of the X. laevis embryo, consistent with its role in other vertebrates. The predicted full-length X. laevis RECK amino acid sequence was found to be highly conserved with other RECK proteins. All RECK proteins are found to have 3 inhibitory Kazal motifs, where the first is the most conserved in all animals. Furthermore, all RECK proteins contain 6 conserved cysteines in each of 5 knot motifs, with the 3rdknot being the most highly conserved. Knot domains were more conserved amongst birds, than amongst herptiles and mammalians. Conclusion: The conservation of expression and sequence suggest that RECK has a very conserved function in vertebrates and possibly all animals.