Subunit interactions of class I histocompatibility antigens

Abstract

The kinetics of dissociation of iodinated beta 2-microglobulin (beta 2m) from the papain-solubilized class I histocompatibility antigen HLA-B7 have been investigated. In the presence of unlabeled beta 2m, most of the HLA dissociates according to a single rate constant, whereas in the absence of unlabeled beta 2m, the system approaches an equilibrium dependent upon the initial HLA concentration. When iodinated beta 2m is incubated with unlabeled HLA-B7, the rate of incorporation of beta 2m into the complex is much less dependent on the concentration than is expected for a simple association/dissociation system; instead, the system behaves as if the "activity" (in a thermodynamic sense) of the HLA heavy-chain intermediate cannot surpass a critical concentration. The dissociation rate for each class I specificity is a function of temperature, ionic strength, pH, and the status of the heavy chain (papain solubilized vs. detergent solubilized). High temperature, high ionic strength, and extremes of pH promote dissociation. The intact molecule dissociates about 10 times more slowly than the papain-solubilized molecule. In contrast, the rate of dissociation of all papain-solubilized class I antigens tested falls within the range of about a factor of 2. The presence of the carbohydrate has no effect on the rate of dissociation. The possibility that HLA class I antigen dissociation may occur in vivo within acidic internal vesicles is discussed.

β2-Microglobulin as a negative regulator of the immune system: high concentrations of the protein inhibit in vitro generation of functional dendritic cells

“…Tuberculin purified protein derivative (PPD) was purchased from Statens Serum Institut (Copenhagen, Denmark). 3 [H]-Thymidine and Ficoll-Hypaque were from Amersham Pharmacia Biotech (Piscataway, NJ).…”

“…MoDCs at various numbers were added and cultured at 37°C in 5% CO 2 for 6 days. Sixteen hours before harvest, 1 Ci (0.037 MBq) 3 [H]-thymidine was added to each well. Cells were harvested, and radioactivity was measured in a ␤-liquid scintillation analyzer (Packard, Meriden, CT).…”

“…MoDCs were pulsed with 2.5 g/mL PPD for 2 hours, washed 3 times with PBS, and cultured with purified autologous T cells for 6 days. T-cell proliferative response was measured by using overnight incubation with 3 [H]-thymidine (1.0 Ci [0.037 MBq]/well), as described in MLR assay.…”

“…1 Because it is noncovalently associated with the ␣-chain and has no direct attachment to the cell membrane, ␤ 2 M on the cell surface can exchange with free ␤ 2 M present in serum-containing medium. 2,3 Free ␤ 2 M is found in body fluids under physiologic conditions as a result of shedding from cell surfaces or intracellular release. ␤ 2 M is almost exclusively catabolized within the kidney; 95% to 100% of circulating ␤ 2 M is eliminated through glomerular filtration.…”

Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression.

“…Although the biosynthesis of major histocompatibility complex molecules has been studied in great detail over the past few years, the exact events and the requirements for the association of both subunits (40) in the surface expression and stability of the complex are still of interest, since very little is known concerning the molecular mechanisms of their intracellular transport (26). The importance of the unique asparagine-N-linked glycosylation site in HLA molecules has been previously investigated by an enzymatic approach with glycosidases or by treatment in vivo with tunicamycin (an inhibitor of N-linked glycosylation).…”