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.

Introduction

The human UL16 binding proteins (ULBPs) function as activating ligands for the NK cell receptor NKG2D (Cosman et al., 2001, Sutherland et al., 2002). Upon infection with human cytomegalovirus, the viral protein UL16 binds some ULBPs (ULBP1 and ULBP2) thereby preventing their cell-surface expression as one mechanism for immune evasion (Rolle et al., 2003). In humans, four members of this MHC class I-extended gene family have been described (Cosman et al., 2001, Jan et al., 2003). Human ULBP1–3 are GPI-linked molecules while ULBP4 is a transmembrane protein. Members of the MHC class I-extended gene family, human ULBP1–4 contain α1 and α2 domains, but not α3 (and they do not bind β2-microglobulin). The expression of these molecules varies; ULBP4 has been reported to be expressed predominantly in the skin (Jan et al., 2003) while other ULBPs are expressed in a wide variety of tissues including lung, liver, heart and testis (Cosman et al., 2001). All four ULBPs are potent ligands for the activating NKG2D receptor found on NK cells, NKT cells, and on a subset of CD8+ T cells. Ligands for NKG2D, like the ULBPs, appear to have significant weight in the balance between activation and inhibition of NK cell activity (reviewed in Vivier et al., 2002). The interaction of ULBPs with NKG2D is capable of activating NK cells even in the presence of inhibitory signals (Cosman et al., 2001).

ULBP-like genes have also been identify in the mouse. In mouse, these are known as the Raet1 gene family (retinoic acid early transcripts 1) and include four highly related genes. Raet1 gene product, RAE-1, and H60, another mouse homologue of ULBP (Cerwenka et al., 2000, Cerwenka et al., 2002) are similar in structure as the ULBPs, containing extracellular domains with distant similarity to the α1 and α2 domains of the MHC class I molecules, but contain a low level of sequence conservation. Both of these proteins have been shown to interact with the mouse NKG2D receptor (Cerwenka et al., 2000, Diefenbach et al., 2001). More recently, two ULBP-like transcripts have been identified in cattle and they have been termed MHCLA1 and MHCLA2.

Human NKG2D also binds another class of MHC class I-extended gene family members, namely the MHC-encoded MICA and MICB proteins. These are also members of the MHC class I gene superfamily but unlike ULBPs, MICA/B contain α3 domains and are anchored to the membrane by transmembrane domains (Vivier et al., 2002). MICA and MICB are stress-inducible proteins whose expression is up-regulated in certain tumors and infected cells (Groh et al., 1996, Groh et al., 1999, Groh et al., 2001). Mice apparently lack orthologs of MICA and MICB but the presence of MICA- and MICB-like genes are clearly apparent in primates (Steinle et al., 1998, Seo et al., 2001) and cattle (based on est sequences in GenBank). In Sus scrofa (the pig), two MICA/B-like genes have been identified (dubbed MIC1 and MIC2) from genomic sequencing efforts (Chardon et al., 2001). At least in the pig H01 haplotype, MIC1 appears to be a pseudogene while MIC2 seems to possess features of a transcribed and functional MICA/B gene (Chardon et al., 2001).

The molecular cloning of a porcine ULBP-like cDNA and initial analysis of its functional properties are presented here. The evolution of ULBP genes and genes encoding other NKG2D ligands is an interesting aspect of the tailoring and tuning of immune responses during mammalian speciation. Characterization of genes encoding NKG2D ligands from swine may have clinical ramifications as well given the documented role of human NK cells in the rejection of porcine xenografts (Lin et al., 1997, Itescu et al., 1998). Ligands expressed by pig cells which are capable of interacting with human NKG2D potentially could be targets to minimize human NK cell-mediated killing of porcine xenografts.