Sequencing of full-length canine platelet GPIbBETA and characterization of the promoter region

Sequencing of full-length canine platelet GPIbBETA and characterization of the promoter region Corresponding author: fabio.gentilini@unibo.it The platelet membrane glycoprotein complex GPIb/GPV/GPIX is composed of four membrane-spanning polypeptide subunits: GPIbα, GP Ibβ, GP IX and GP V in 2:2:2:1 stoichiometry. The complex plays a key role in the initial step of haemostatic process mediating adhesion of platelets to the exposed subendothelium by binding von Willebrand factor under the condition of high shear stress. All four GP subunits are encoded by different genes. The predicted canine GPIbβ locus is not annotated in the Ensembl database. Using 5’ RACE PCR and sequencing of the mRNA purified from canine platelets, the entire gene was identified on the chromosome 26 syntenic with the human chromosome 22. Homologies with the human ortholog are 87% and 84% at the nucleotide and aminoacid level, respectively. The gene codes for a 205 aminoacids protein with many leucine-rich repeat motifs and an overall GC content of 80%. The gene is composed by two exons: the leader exon includes 5’UTR not exceeding 40 nt with respect with the 600 nt of the human counterpart. Two different transcription start sites were found. The putative promoter region was predicted by Proscan software v1.7 in the 300 nt upstream of the 5’ cap site. The core promoter harbours different regulatory motifs: many SP1 and AP-2 but also T-Ag, GCF, CREEB, SIF and GATA. Notably, two GATA motifs are highly conserved when compared with humans. The cloning of the mRNA could eventually confirm the two different transcription start sites found with direct sequencing.