Molecular characterization of two paralog genes encoding Gla-rich protein (Grp) in zebrafish By C. Fazenda 1,2 , I. A. L. Silva 1,2 , M. L. Cancela 1,3 and N. Conceic¸a˜o 1 1 Centre of Marine Science (CCMAR) University of Algarve, Faro, Portugal; 2 PhD Program in Biomedical Sciences, University of Algarve, Faro, Portugal; 3 Dept of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal Summary Gla-rich protein (GRP) is a novel vitamin K-dependent protein with the highest Gla content of any protein known to date that has been identified in all taxonomic groups of vertebrates (named GRP1) with a paralog in bony fish (named GRP2). In sturgeon, as well as during mouse development, cartilaginous tissues or their precursors are primary sites of GRP expression. In this article, we identify two grp isoforms, grp1 and grp2, encoded by two distinct genes localized in zebrafish chromo- somes 25 and 4, respectively. These two genes span, respectively, 6 kb and 9 kb of genomic DNA and are both composed of five exons. Within the coding regions, the overall amino acids identity is 48.6 and 42.0%, respectively, for Grp1 and Grp2 compared to the human GRP. We also have identified the presence of splice variants already previously described in mouse, and corresponding expression levels were determined during embryonic stages and in different adult tissues. The levels of grp expression appear to be inversely correlated, with grp1 being expressed first and remaining high during early develop- ment while expression of grp2 appears later and increases in late larval and juvenile stages, having greater prevalence in adult tissues. We conclude that in zebrafish, grp paralogs exhibited distinct patterns of expression suggesting different regulatory pathways for each gene. Introduction Gla-rich protein (GRP) is a novel vitamin K dependent (VKD) protein that contains 16 ?-carboxyglutamic acid (Gla) residues thus carrying the highest Gla percentage of any known VKD protein. It was recently identified in the calcified cartilage of Adriatic sturgeon (Viegas et al., 2008) but found to be present in other tissues. Vertebrate GRP genes are organized into 5 coding exons that code for a pre-propeptide of ~135 amino acids and were identified in most classes of vertebrates (revised in Cancela et al., 2012). The primary structure of GRP is highly conserved among vertebrates. Once the transmembrane signal peptide is removed, the remaining proGRP is cleaved by a furin-like protease (at RXXR site) into a propeptide (38-39 amino acids) and a mature peptide (67-74 amino acids). Although the molecular function for GRP is still under investigation, it was suggested to exert a role as modulator of calcium availability due to its high content in Gla residues known to bind calcium ions (revised in Cancela et al., 2012). Although no information is yet available about factors controlling GRP gene expression or its possible relevance for human diseases, GRP protein has been associated to sites of ectopic calcification (Viegas et al., 2009). Zebrafish is now an accepted model to study skeletal development and mechanisms of calcification. Being a vertebrate, it can be used to screen for mutants with abnormal tissue calcification including skeletal phenotypes, and for functional analysis of wild type and mutant skeletal proteins. Because development of zebrafish skeleton occurs by mechanisms similar to humans, it was successfully used for identification of gene mutations respon- sible for human pathologies such as osteogenesis imperfecta (Fisher et al., 2003) or craniofacial syndromes (Nissen et al., 2006), thus contributing to understand the molecular basis of human diseases affecting normal patterns of tissue calcifica- tion. In addition, zebrafish has many advantages over mam- malian models: easy to manipulate genetically, high resolution in vivo analysis, transparency of embryos and larvae, and increasing number of available molecular tools and techniques including (i) sequencing and annotation of its genome, (ii) availability of genetic mutants and transgenics (http://zfin.org/ and http://zebrafish.org/zirc/home/guide.php), and (iii) strate- gies for embryo manipulations allowing overexpres- sion ⁄ knock-down of selected genes (Morcos, 2001). In the present study we have used zebrafish to analyse the fine structure of the two grp paralog genes, grp1 and grp2, since zebrafish, as all other bony fish analysed, has two distinct isoforms, encoded by two different genes (Viegas et al., 2008). We have also compared the protein structures encoded by both zebrafish and human genes. The data obtained provides additional information towards unveiling the function of these genes. Materials and methods RNA preparation Total RNA was extracted from different embryo, larval and juvenile stages, ranging from 1-cell to 60 days post-fertilization (dpf) and from adult tissues (including bony, cartilaginous, and major soft tissues) as described by Chomczynski and Sacchi (1987). RNA integrity was checked by agarose-form- aldehyde gel electrophoresis, and concentration was deter- mined by spectrophotometry at 260 nm (NanoDrop 1000; Thermo scientific). Molecular cloning of grp cDNAs Complete coding region of cDNAs for zebrafish grp1 and grp2 (Accession Nos. JQ003911 and JQ003912, respectively) were amplified by RT-PCR from total RNA extracted from adult fishes by standard conditions using specific forward primers, grp1_f1 and grp2_f1 and specific reverse primers, grp1_r1 and J. Appl. Ichthyol. 28 (2012), 377–381 Ó 2012 Blackwell Verlag, Berlin ISSN 0175–8659 Received: November 03, 2011 Accepted: February 22, 2012 doi: 10.1111/j.1439-0426.2012.02004.x U.S. Copyright Clearance Centre Code Statement: 0175–8659/2012/2803–0377$15.00/0 Applied Ichthyology Journal of