Enhanced binding of peptide antigen to purified class II major histocompatibility glycoproteins at acidic pH.

Abstract

SllmmsryHelper T lymphocytes recognize peptide antigens stably associated with class II major histocompatibility complex (MHC) glycoproteins on the surface of antigen-presenting cells and serve to regulate a wide variety of immune responses. A previous study from our laboratory had demonstrated that the functional association of various peptide antigens with the antigen-presenting cell membrane was increased at pH 5 as compared to pH 7, consistent with the potential role of acidic endosomal compartments in antigen processing. The mechanism for this effect was not determined. In the present study, assays using purified class II glycoprotein were used to further define this mechanism. The potential requirement for pH-dependent interactions involving non-MHC membrane components was excluded in functional assays with purified class II reconstituted in artificial membranes containing only neutral phospholipids and cholesterol. The association of HEL(104-120) with I-E a, and OVA(323-339) with I-A a, was increased at pH 5, as measured by activation of specific T cell hybridomas. An enzyme immunoassay was developed to measure the binding of biotin-labeled peptides to purified class II in detergent micelles. The pH dependence of binding paralleled our previous functional results. Optimum binding of biotin-HEL(104-120) to I-E d was observed at pH "~4.5, whereas maximum binding of biotin-Myo(106-118) to I-A d occurred at pH ~5.5. The latter peptide also bound weakly to I-E d, but with a pH dependence similar to that observed using HEL(104-120). Further experiments with biotin-HEL(104-120)/I-E d indicated that both the apparent af~nity and the apparent concentration of peptide-binding sites are increased as hydrogen ion concentration is increased from pH 7 to pH 5. The effect of pH in this range was largely reversible and was not associated with a change in peptide dissociation that could be measured with our assay system. Binding was not inhibited in the presence of 1.5 M NaC1, suggesting that electrostatic interactions between HEL(104-120) and I-E d are not essential for binding. It is proposed that protonation of a critical group(s) in the class II molecule regulates its capacity to form stable complexes with peptide. However, this effect alone does not fully account for the rapid kinetics ofpeptide binding observed in experiments with intact antigen-presenting cells.

Specific and general HLA-DR binding motifs: comparison of algorithms

“…It has been suggested that the absence of "negative" amino acids may be more important than the presence of amino acids giving a "positive" interaction with MHC [58]. Also, the reported enhanced binding of peptide HEL(104 -120) at acidic pH [59], where the net charge is less negative, is in agreement with the disrupting effect on binding of Asp and Glu at several positions of the peptide at higher pH [60] where the net charge is more negative.…”

Transmembrane Domain-Mediated Colocalization of HLA-DM and HLA-DR Is Required for Optimal HLA-DM Catalytic Activity

“…Unilamellar liposomes containing purified DR1 and/or DM were generated as described in Materials and Methods. Samples containing identical quantities of DM and DR1 distributed together or separately into liposomes were incubated for 3 h at pH 5 with 1 M biotin-MAT (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) peptide. After chilling on ice, liposomes were solubilized in 1% NP40 and biotin-MAT-DR1 complexes were quantified by europium fluorescence immunoassay (f).…”

“…DR1-biotin CLIP complexes were formed by incubation of 500 nM DR1 with 5 M biotin-CLIP for 18 h at 37°C. For dissociation, preformed complexes were diluted 10-fold and incubated in 20-l volumes with 100 M unlabeled MAT (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). After neutralization, the complexes were captured on L243-coated microtiter wells at 4°C and biotin complexes were measured with streptavidin-europium as previously described (5).…”

“…CLIP peptide was biotinylated as previously described (25). All binding and dissociation reactions were done in binding buffer (0.1 M citrate-phosphate buffer at pH 5.0, 0.2% NP40, containing protease inhibitors) (5).…”

Processing and presentation of tetanus toxin by antigen-presenting cells from patients with chronic granulomatous disease (CGD) to human specific T cell clones are not impaired

“…Antigen processing by APC can be inhibited with reagents including chloroquine, monensine and NH4CI that elevate intracellular pH [8]. These acidic conditions are required for efficient antigen presentation, partly to provide the optimum conditions for acidic enzymes which process both invariant chain (Ii) [9,10] and antigen [7,10], and partly to facilitate peptides binding to MHC class II [11 ]. Early studies employing mutant hamster ovary cells suggested that acidification of early endosomes was an important event in antigen processing [12].…”