Elsevier

Gene

Volume 297, Issues 1–2, 4 September 2002, Pages 151-158
Gene

Identification of a novel calreticulin isoform (Crt2) in human and mouse

https://doi.org/10.1016/S0378-1119(02)00880-6Get rights and content

Abstract

Calreticulin is a Ca2+-binding chaperone localized mainly in the endoplasmic/sarcoplasmic reticulum in all higher organisms. To date, only one calreticulin isoform has been identified in human and mouse. Here we report a novel calreticulin isoform (Crt2) in human and mouse, with 53 (human) and 49% (mouse) identity to the previously identified calreticulin in respective species. The gene encoding the novel human calreticulin isoform spans 17 kb of genomic DNA and is expressed in testis, showing a similar expression as the chaperone calmegin. Phylogenetic analysis shows that two or more calreticulin (crt) genes are present both in plants and in mammals. The duplication of the crt gene in human and mouse suggests functional diversity, and variations in expression patterns among calreticulins. Two novel calreticulin (Crt2) isoforms, with high homology to the human and mouse calreticulin isoform (Crt2), were also identified in pig and rat via expressed sequence tags.

Introduction

Calreticulin (Crt) is a multi-functional protein localized mainly in the endoplasmic/sarcoplasmic reticulum (ER/SR) in most cells of all higher organisms (for review see; Michalak et al., 1992, Krause and Michalak, 1997, Michalak et al., 1999, Corbett and Michalak, 2000, Johnson et al., 2001). Although Crt has been suggested to be involved in over 40 intra- and extracellular processes, the research on Crt has mainly focused on its role in protein folding (Denecke et al., 1995, Hebert et al., 1996, Saito et al., 1999, Nakamura et al., 2001), and Ca2+-signaling (Camacho and Lechleiter, 1995, Mery et al., 1996, John et al., 1998, Nakamura et al., 2001, Persson et al., 2001).

Crt is an evolutionary highly conserved protein typically containing an HDEL/KDEL C-terminal ER-retention signal, and three major internal domains (Michalak et al., 1992, Michalak et al., 1999). These consist of a globular N-domain and two Ca2+-binding domains; a high-affinity but low-capacity P-domain, and a low-affinity but high-capacity C-domain. The P-domain has furthermore been shown to contain two triplets of conserved amino acid sequences necessary for the lectin-like chaperone function and potentially involved in the high-affinity Ca2+-binding properties of the protein (Baksh and Michalak, 1991). In addition, Crt typically contains an N-terminal signal sequence for ER-targeting, and three conserved cysteine residues involved in forming disulfide bridges to maintain proper folding of the protein (Matsuoka et al., 1994, Højrup et al., 2001).

Two or more genes encoding Crt isoforms have been found in Arabidopsis thaliana, Zea mays, Hordeum vulgare, Xenopus laevis and Bos taurus, but only one gene encoding Crt has previously been identified in human and mouse (McCauliffe et al., 1990, McCauliffe et al., 1992, Rooke et al., 1997). Furthermore, only one messenger RNA (mRNA) product had been identified in human and mouse, suggesting that there is no alternative splicing. The previously identified crt gene in human is located on chromosome 19 (19p13.2) (McCauliffe et al., 1990), and in mouse on chromosome 8 (Rooke et al., 1997). These two crt genes consist of nine exons with similar intron-exon organizations, and span a relatively short amount of genomic DNA (4.2 and 4.8 kb for human and mouse, respectively) (McCauliffe et al., 1992, Waser et al., 1997).

Here we report a novel crt isoform (crt2) expressed in testis in human, with an orthologue in mouse. The genomic structure, and chromosomal localization of the human crt2 is discussed, and the phylogenetic relationship of Crt proteins is described. Two additional crt2 isoforms, based on expressed sequence tag (EST) analysis, were discovered in pig and rat.

Section snippets

Sequence comparison of Crt proteins

Protein sequences corresponding to Homo sapiens Crt (denoted Crt1), Mus musculus Crt (denoted Crt1), B. taurus Crt1, Drosophila melanogaster Crt, A. thaliana Crt3, H. sapiens Crt2, and M. musculus Crt2 (GenBank Accession numbers AAA51916, CAA33053, AAB30209, CAA45791, AAC49697, XP_058951, and BAB24660, respectively), obtained from the GenBank database via NCBI (National Center for Biotechnology Information) (www.ncbi.nlm.nih.gov), were used for sequence analysis. ClustalW multiple alignments of

Identification of novel Crt isoforms

Performing standard BLASTP analysis at NCBI against Crt protein sequences from various species, we identified two novel Crt isoforms in human and mouse (GenBank Accession numbers: XP_058951 and BAB24660, respectively). The novel isoforms were for human 384 amino acids, and for mouse 380 amino acids in length. The sequences showed 53 and 49% identity, and 69 and 66% similarity to the previously identified Crt's in human and mouse, respectively [GenBank Accession numbers: AAA51916 (human) and

Acknowledgements

We thank Dr Urban Johanson and Magnus Alsterfjord for valuable discussions and advice regarding various analyzes. We also thank The Swedish Research Council for financial support.

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