Porcine Fc gammaRIII isoforms are generated by alternative splicing.

The Fc gammaRIII plays an essential role in antibody-mediating effector functions of immune cells. Here, we report that transcripts encoding porcine Fc gammaRIII isoforms are generated by alternative splicing. Fc gammaRIII a.1 is expressed on the cell surface while Fc gammaRIII a.2 is secreted from the transfected cells due to a partial deletion of the transmembrane domain. Interestingly, a putative soluble Fc gammaRIII (sCD16) is detected in circulating plasma. Both Fc gammaRIII a.2 and sCD16 exhibit a similar molecular mass (approximately 35 kDa) and contain the extracellular D2 domains that are structurally intact. Despite the D2 domain deletion, Fc gammaRIII a.3 associates with Fc gammaRIII a.1. Hence, these results suggest one possibility that three Fc gammaRIII isoforms differentially modulate Fc gammaRIII-mediated immune responses in the porcine system. Furthermore, we demonstrate that a cytolytic triggering G7 monoclonal antibody recognizes the D2 domain that is responsible for interacting with the immune complexes and subsequent activations of porcine innate immune cells. This result suggests that the D2 domain is a target region to develop therapeutic antibodies that regulate the FcR-mediated immune responses.

Identification of a novel Fc gamma RIIIa alpha-associated molecule that contains significant homology to porcine cathelin.

The following studies are the first to demonstrate the association of porcine FcgammaRIIIaalpha with a molecule that contains significant homology to the cathelin family of antimicrobial proteins. We performed immunoprecipitation of the porcine FcgammaRIIIaalpha multisubunit complex from Brij 96 lysates of polymorphonuclear leukocytes using the G7 mAb, which binds to FcgammaRIIIaalpha on the surface of porcine NK cells and phagocytes. Previous results indicate that the transmembrane alpha subunit of the FcgammaRIIIa complex is associated with the gamma subunit on the surface of porcine polymorphonuclear leukocytes and with several other unique proteins that surface iodinate and migrate at approximately 15, 20, and 25 kDa when analyzed by reducing SDS-PAGE. Through characterization of the porcine FcgammaRIIIa complex, we identified the 15-kDa molecule as a unique FcgammaR-associated protein that has not been described in other systems. We now report an association between FcgammaRIIIaalpha and a 15-kDa molecule that shares homology to cathelin, a protein of undetermined function initially identified in porcine leukocytes. A domain with a high degree of homology to cathelin is found in the proregions of a family of antibiotic proteins referred to as cathelicidins. The results of our studies indicate the presence of a novel FcgammaRIIIa complex in the porcine system, and may provide new insights into the function of this antimicrobial protein homologue in relation to the variety of responses mediated through FcgammaRs.

Molecular cloning, expression pattern and chromosomal mapping of pig CD69.

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.