Evidence that HLA-G is the functional homolog of mouse Qa-2, the Ped gene product

Abstract

Qa-2, a murine class Ib major histocompatibility complex (MHC) molecule, is a possible functional homolog of human leukocyte antigen G (HLA-G). Both molecules have been implicated in immunoregulation and embryonic development and both occur in membrane-bound and soluble isoforms that arise by alternative splicing. Soluble splice variants have been implicated in the reproductive functions of HLA-G. While soluble variants of Qa-2 have been previously detected in T lymphocytes, we now demonstrate the presence of mRNA for one of the two known soluble forms of Qa-2 in eight-cell embryos and in blastocysts. Qa-2 is glycosylphosphatidylinositol (GPI) linked in the outer leaflet of the cell membrane and is found in lipid raft microdomains where other raft-associated proteins transduce signals into the cell. In contrast, HLA-G has a truncated six amino acid cytoplasmic tail. By fluorescence co-localization in JEG-3 cells, using fluorescent cholera toxin beta subunit (a lipid raft marker) and anti-HLA-G antibody, we have demonstrated that membrane-bound HLA-G also localizes to lipid rafts, consistent with functional homology between the two molecules. Finally, our experiments in which we have purified Qa-2 and transferred it via a process known as protein painting to Qa-2 negative cells represent a model for potential therapy involving HLA-G.

FIGURE 1

(A) Membrane bound and soluble isoforms of human leukocyte antigen G (HLA-G) and Qa-2. The membrane bound isoforms of HLA-G and Qa-2 have a 6-amino acid cytosolic tail and a glycosylphosphatidylinositol (GPI) tail in the outer leaflet of the membrane, respectively. The figure is derived from mRNA and protein data [4, 16]. The S1 Qa-2 isoform is structurally similar to the soluble HLA-G5 isoform, while S2 Qa-2 would correspond with a soluble HLA-G4 isoform. (B) Expression of Qa-2 mRNA in preimplantation embryos (M = 100 bp ladder; + = C57BL/ 6J; − = CBA/CaJ; 8C = 8-cell embryos, B = blastocysts). The positive and negative controls are from activated T cells, prepared as described in the text. GPI-linked Qa-2 appears at 292 bp while S1 Qa-2 appears at 175 bp.

FIGURE 2

Qa-2 and HLA-G co-localize in lipid rafts. EL-4 cells (top row a–e) and JEG-3 cells (bottom row f–j). Differential interference contrast (a and f), Hoechst 33342 nuclear staining (b and g), anti-Qa-2 (c) and anti-HLA-G (h) labeled with Alexa Fluor488 goat anti-mouse IgG, GM1 ganglioside raft marker labeled with cholera toxin subunit β conjugated with Alexa Fluor594 (d) and (i). Overlay images of anti Qa-2 and CTβ (e) and HLA-G and CTβ (j). Yellow color is indicative of colocalization of Qa-2 or HLA-G and lipid rafts. HLA-G expression in JEG-3 cells was heterogeneous, consistent with previous reports [17].

FIGURE 3

Protein painting of Qa-2-negative (CBA/CaJ) splenic mononuclear cells with immunoaffinity-purified Qa-2. Cells were incubated in either medium (pale histogram) or medium containing Qa-2 (dark histogram), washed, and stained with anti-Qa-2-biotin and streptavidin-TriColor.