Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes.
Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance energy transfer (FCET) showed that these class I MHC molecules self-associate in liposome membranes, forming small aggregates even at low surface concentrations. The lateral diffusion coefficient (Dlat) of Fl-HLA-A2 decreases with increasing surface protein concentration over a range of lipid:protein molar ratios (L/P) between 8000:1 and 2000:1. The reduction in Dlat of HLA molecules in DMPC liposomes is found to be sensitive to time and temperature. The rotational correlation time for Er-HLA-A2 in DMPC liposomes at 30 degrees C is 87 +/- 0.8 microseconds, at least 10 times larger than that expected for an HLA monomer. There is also significant quenching of donor (Fl-HLA) fluorescence at 37 degrees C in the presence of acceptor-labeled (sulforhodamine-labeled HLA) protein indicating proximity between HLA molecules even at L/P = 4000:1. FPR and FCET measurements with another membrane glycoprotein, glycophorin, give no evidence for its self-association. HLA aggregation measured by FPR, FCET, and TPA was blocked by beta 2-microglobulin, b2m, added to the liposomes. The aggregation of HLA-A2 molecules is not an artifact of their reconstitution into liposomes. HLA aggregates, defined by FCET, were readily detected on the surface of human lymphoblastoid (JY) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Chakrabarti
A
A
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Matko
J
J
Rahman
N A
NA
Barisas
B G
BG
Edidin
M
M
D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004058:Diffusion; D004923:Erythrosine / Q000737:chemistry; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D008081:Liposomes / Q000276:immunology*; D011510:Proteolipids
D015789:HLA-A2 Antigen; D007140:Immunoglobulin Fab Fragments; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D004923:Erythrosine