Molecular cloning and characterization of a porcine UL16 binding protein (ULBP)-like cDNA

Abstract

UL16 binding proteins (ULBPs) are ligands for the NK cell activating receptor NKG2D. A cDNA encoding a porcine ULBP-like protein (PULBP) was cloned and the predicted amino acid sequence exhibited 35-52% identity to human ULBPs. Southern blot analysis suggested that there is only one ULBP-like gene in the pig genome. Transcripts of PULBP and another potential NKG2D ligand, MIC2, were detected by RT-PCR in a wide range of tissues. Recombinant PULBP-Fc and human ULBP2-Fc fusion proteins were made and used to examine PULBP binding to porcine PBMCs and a human NK cell line (NKL cells). PULBP-Fc bound to a subpopulation of porcine PBMCs but not NKL cells. Conversely, human ULBP2-Fc did not bind to porcine PBMCs but did stably interact with NKL cells.

Porcine UL16-Binding Protein 1 Expressed on the Surface of Endothelial Cells Triggers Human NK Cytotoxicity through NKG2D

“…Using an algorithm to score potential ligands (29), pULBP1 was predicted to bind human NKG2D using both the crystal structure of human NKG2D/human ULBP3 and human NKG2D/mouse Rae-1␤ interactions as template, whereas pMIC2 was predicted to bind NKG2D only using the structure of human NKG2D/human ULBP3 as template (data not shown). Intriguingly, a previous study showed no binding of pULBP1-Fc to the human NK cell line NKL by flow cytometry, whereas binding to porcine PBMC was revealed (33). It was concluded then that pULBP1 does not interact with human NKG2D.…”

“…Because both pULBP1 and pMIC2 transcripts were detected in a pEC (33), they represent potential ligands for human NKG2D. Therefore, the aim of the present study was to test whether pULBP1 and pMIC2 can act as functional ligands for human NKG2D, resulting in xenogeneic human anti-pig endothelial cell NK cytotoxicity.…”

“…Southern blot analysis suggested that only one pULBP exists in the pig genome, which is in sharp contrast to the much higher number of ULBP genes, at least six, that were described in humans. However, considering hybridization conditions in these studies (33), the possibility that other more distantly related porcine ULBP-like genes exist cannot be fully excluded.…”

“…Similarly to the human MIC genes, a consensus heat shock element, but no iron response element, was found upstream of the porcine exon one. Only recently, porcine ULBP1 (pULBP1) was cloned and characterized as a homologue of human ULBP (33). Phylogenetic analyses place pULBP1 evolutionarily close to the bovine ULBP-like genes MHCLA1 and MHCLA2.…”

Human NK Cytotoxicity against Porcine Cells Is Triggered by NKp44 and NKG2D

“…In contrast, it is conceivable that NCR and NKG2D as well as their ligands have not been subjected to the pressure that caused the evolution of MHC genes and their receptors. The porcine NCR and NKG2D ligands are unknown; however, comparison of porcine and human genomic sequences suggests the presence of one ULBP gene and one MIC-A/MIC-B-like gene (MIC2) in the pig genome (40,41). NKG2D ligands are generally poorly expressed by normal cells but are up-regulated in transformed, infected, and stressed cells in both mice and humans (25).…”