Expression of CLEC4A in porcine tissues and leukocyte populations and characterization of mRNA splice variants

Abstract

CLECs are a group of molecules of the superfamily of C-type lectin domain containing receptors. Several receptors of this group have been described in humans and mice, as well as in other species. Many of them are expressed in immune cells, and have been shown to be involved in immune response modulation. Several molecules of this family have been proposed as targets for antigen delivery, which is what drew our interest in studying them in the pig. In this species only CLEC7A (also named Dectin-1) had been studied in some detail, and we have characterized two other members, CLEC12A and CLEC12B. Another molecule of interest was CLEC4A, for which we could not find any expression studies in pigs, although up to 5 different mRNA variants had been predicted by bioinformatics analysis of the genomic sequence in databases. Here we present an analysis of the expression of CLEC4A mRNA isoforms in tissues and blood cell subsets by PCR. Cloning and sequencing of cDNA of the different isoforms allowed us to identify all 5 predicted isoforms, in addition to characterizing 5 additional isoforms expressed in alveolar macrophages, of which the form coding for a canonical CLEC4A molecule was apparently the most abundant isoform. Analysis of the sequences of the 10 cDNA cloned allowed us to determine their exon usage and to identify their potential for protein coding. Only 8 of the characterized variants contain a recognizable coding sequence. The sequences of the coded proteins show both proteins with a putative transmembrane segment, and others without this feature, as well as proteins with the complete CLEC domain or with diverse truncated forms. We also prove that most of the cDNA can drive protein expression upon transfection in the CHO cell line. We discuss the relevance that the diversity of products of the porcine CLEC4A gene may have in the regulation of the expression and function of these molecules.