Conformational variants of class II MHC/peptide complexes induced by N- and C-terminal extensions of minimal peptide epitopes

Abstract

Class II MHC molecules are known to exist in conformational variants. "Floppy" and "compact" forms of murine MHC molecules, for example, are discriminated by their migration behavior on SDS/PAGE and represent empty and ligand-loaded forms. Here we show that formation of distinctly faster-migrating ligand complexes (F-forms) rather than the normal compact (C-) forms of HLA-DR1 or -DR4 results from extensions of minimal peptide epitopes (such as HA306-318 or IC106-120) by approximately 10 amino acids at either the N or the C terminus. Two similar but distinct F-forms (FI and FII) were detected, depending on the site of the extension. Both F-forms were characterized by increased surface hydrophobicity and reduced SDS-stability. Native gel separations and size exclusion chromatography indicated that the F-forms had increased hydrodynamic radii compared with the C-form and an apparent size similar to that of empty MHC molecules. The regions on the ligand overhangs responsible for the effect began at a distance of approximately 5 amino acids on either side of the epitopes, comprised 4-8 amino acids (i.e., a total overhang of 9-14), and did not have a particular sequence preference. The possible functional significance of these forms is discussed.

Figure 1

Apparent molecular weight (MW_app) of class II MHC/ligand complexes loaded with monomeric or dimeric ligand constructs. (A) Soluble HLA-DR1 or -DR4 molecules loaded with HA306-318 peptide (P lanes) or dimeric ligand constructs consisting of two HA306-318 epitope units linked by a tetraethylene glycol spacer (D lanes) were separated by SDS/PAGE. The MW_app indicated in the figure refers to bands representing distinct MHC/ligand complexes. Gels were visualized by silver staining.

Figure 2

Induction of F_I- and F_II-complexes by amino acid overhangs of the ligand. Soluble HLA-DR1 molecules were loaded with ligand constructs containing the epitope HA306-318. The structure of the ligands is depicted schematically on the upper right panel. The box represents the HA306-318 binding epitope, and the line represents a tetraethylene glycol unit used to connect the epitopes. The cross in the N_YD- and the C_YD-ligand marks a substitution of the main anchor residue Y₃₀₈ with D in one of the two HA306-318 epitopes, which abrogates binding to the HLA-DR1 molecule. N₁₀-N₁ and C₁₀-C₁ represent dimeric ligand constructs with truncations in the N- or C-terminal epitope. (A) Formation of F_I- and F_II-ligand complexes with teg-linked peptide constructs with defined N- or C-terminal polypeptide extensions. HLA-DR1/ligand complexes, loaded with monomeric (lanes P, M, and M′) and dimeric HA306-318 ligand constructs (lanes D, N_YD, and C_YD), were separated by SDS/PAGE. The position of bands representing the compact C-form (associated with the monomeric ligands) as well as of the two distinct F_I- and F_II-forms (associated with the dimeric ligands) are indicated. (B) To estimate distance and size of the polypeptide region responsible for the formation of the F_I- and F_II-complexes, HLA-DR1 molecules were loaded with truncated forms of the dimeric HA306-318 construct. The series of N-terminal truncations (Left) consists of HA306-318 peptides linked by a teg spacer with the extension VKQNTLKLAT (lane N₁₀), LKLAT (lane N₅), and T (lane N₁). In addition, a ligand also was used in which the LKLAT overhang was connected by two instead of one teg-units (lane N₅′, shown on the right side of the gel). For the series of C-terminal truncations (Right), overhangs were used with the sequence GPKYVKQNTL (lane C₁₀), GPKYV (lanes C₅ and C₅′), and G (lane C₁). On both gels, empty HLA-DR1 molecules (−) and ligand complexes preformed with the HA306-318 peptide (P lanes), the monomer (M lanes), and the dimer (D lanes) are shown as a reference. (C) Formation of F-complexes with naturally extended polypeptide ligands. HLA-DR1 molecules were loaded with peptides extended by 20 amino acids of the natural sequence of the hemagglutinin protein on the N-terminal side (HA286-318; lane P_N) and on the C-terminal side (HA306-338; lane P_C) of the HA binding epitope. SDS/PAGE separation is shown in comparison to the HA306-318-loaded complex (lane P) and the complex loaded with the dimer (lane D). (D) Formation of an F_II-complex by a ligand with randomized overhang. HLA-DR1 molecules were loaded with an HA306-318 epitope, which (through a teg-spacer on the C-terminal side) was connected to a 15-aa-long peptide with random sequence (HA-X₁₅). The overhang was produced by a sequential synthesis using an amino acid pool (G, A, P, V, I, L, M, Y, F, S, T, N, Q, D, E, K, R, H). The HLA-DR1/HA-X₁₅ ligand complex (lane X₁₅) is shown in comparison to the complex loaded with the HA306-318-peptide (lane P), the dimer (lane D), and the HA306-338 peptide (lane P_C).

Figure 3

Formation of HLA-DR1/ligand complexes with ligand constructs containing the IC106-120 epitope. (A) Comparison of HLA-DR1/ligand complexes formed with the HA306-318 and IC106-120 ligand constructs. The MHC molecules were loaded with the peptides HA306-318 and IC106-120 (P lanes) or with dimeric teg-linked tandem constructs HA-HA or IC-IC (D lanes). The position of bands representing the C-form or the F_I- or F_II-form is indicated. (B) SDS/PAGE separation of ligand complexes loaded with IC106-120 binding epitopes with defined N- and C-terminal overhangs. For the left gel, IC ligands were used, which, through the teg-spacer, were extended by the nonbinding Y308D-substituted HA306-318 epitope [N-terminal, YD-IC (lane N_YD); C-terminal, IC-YD (lane C_YD)]. The right gel shows complexes formed with natural polypeptide ligands extended by the amino acid sequence of the invariant chain protein [N-terminal, IC97-120 (lane P_N); C-terminal, IC106-135 (lane P_C)]. (C) Comparison of the migration of extended IC106-120 ligands in the absence of SDS. Gel separations are shown that were carried out in the presence (Upper) or absence (Lower) of SDS.

Figure 4

Characterization of the F- and C-forms of HLA-DR1/ligand complexes. (A) Surface hydrophobicity. The migration behavior of HLA-DR1/ligand complexes was compared under native conditions and in the presence of SDS. Soluble HLA-DR1 molecules were loaded with the HA306-318 or the IC106-120 peptide (P lanes) or with dimeric constructs HA-HA or IC-IC (D lanes). Gels of the MHC/ligand complexes are shown that were separated in the presence (Upper) or in the absence (Lower) of SDS. (B) pH stability. HLA-DR1/ligand complexes were loaded with HA306-318 peptide (P lanes) or the dimer (D lanes) at a pH ranging from 5.0–7.0 (indicated above the lanes) and were analyzed by SDS/PAGE. (C) SDS stability. HLA-DR1 molecules, preloaded with the biotinylated ligands HA306-318, HA-HA, or IC97-120, were incubated for 3 h at 37°C in the absence (open bars) or presence (hatched bars) of 0.1% SDS. The amount of stable MHC/ligand complex was determined after the incubation by ELISA.

Figure 5

Determination of the apparent size of F- and C-forms of HLA-DR1/ligand complexes by size exclusion chromatography. The elution profiles of HLA-DR1/ligand complexes formed with ligands containing the HA306-318 (Upper) and IC106-120 epitope (Lower) are shown. In the left panels, the elution of peptide-loaded C-forms is compared with the elution of the F-forms generated with the teg-linked YD hybrid dimers (indicated in the figure). As a reference, elution volumes of marker proteins and their molecular weights are indicated. The right panels show a comparison of the elution of the ligand-loaded forms (solid lines) to the elution of empty HLA-DR1 preparations (dashed lines).

Figure 6

Effect of cathepsin S and HLA-DM on C- and on F-forms of HLA-DR1/ligand complexes. HLA-DR1/ligand complexes were incubated with cathepsin S or soluble HLA-DM. (A) Conversion of an F-complex into the C-form by cathepsin S digest. F_II-complexes, preformed by loading the HA-X₁₅ ligand onto the HLA-DR1 molecules, were subjected to cathepsin S digest to remove the random peptide overhang. The gel shows the SDS/PAGE analysis of HLA-DR1/HA-X15 ligand complexes (lanes X₁₅) before (−) and after (+) the treatment with cathepsin S. Also shown are the complexes formed with the HA306-318 peptide (lane P) and the HA306-338 peptide (P_C). (B) HLA-DM mediated release of ligands from C- or F-ligand complexes. F- (closed square) or C-HLA-DR1/ligand complexes (closed circle) were formed with biotinylated ligands containing the HA306-318 (Left) or the IC106-120 binding epitope (Right). HA306-318 and IC106-120 were used for the formation of C-complexes, and the F complexes were generated with the HA teg-dimer or the IC97-120 peptide. The ligand complexes were incubated with the indicated amounts of soluble HLA-DM, and the release of ligands was measured by ELISA by determining the amount of stable MHC ligand complexes left after the incubation.