Thyroglobulin type-I-like domains in invariant chain fusion proteins mediate resistance to cathepsin L digestion.

The MHCII associated invariant chain isoform Ii41 shows homology to a repeat in thyroglobulin (TgR). We show that the Ii31 isoform, which lacks the TgR-like domain, is sensitive to cathepsin L treatment whereas Ii41 displays substantial resistance. The TgR-like sequence of Ii41 was exchanged for thyroglobulin type-IA and -IB repeats, that contain six or four cysteine residues. Resistance to cathepsin L digestion was maintained upon substitution of the Ii41 TgR for homologous sequences from TgR type-IA. Mutation of a conserved cysteine in the TgR domain of an Ii fusion protein strongly reduced resistance to cathepsin L digestion.

Sodium dodecyl sulfate-resistant HLA-DR "superdimer" bands are in some cases class II heterodimers bound to antibody.

The detection of dimers of dimers in MHC class II crystals has excited speculation about their possible functions in T cell Ag recognition. Biochemical evidence for the existence of DR superdimers falls short of proof and is controversial. To monitor B lymphoma cells for high m.w. complexes of HLA-DR molecules, membrane preparations and cell lysates were screened by one- and two-dimensional Western blotting. Under these conditions, in which DRalpha beta heterodimers were readily detected, no DR complexes with an (alpha beta)2-chain composition could be identified. Two mAbs (L243 and D1-12) immunoprecipitated high m.w. DR complexes suspected to be superdimers. However, biochemical analysis revealed that, rather than superdimers, these were SDS-stable complexes of DR in combination with the Abs. Thus, previous observations of HLA-DR superdimer bands may also reflect complexes of DR molecules with bound Ab.

The invariant chain derived fragment CLIP is an efficient in vitro inhibitor of peptide binding to MHC class II molecules.

The invariant chain derived peptide CLIP inhibits association of peptides to the class II peptide binding site. Two DR3 specific peptides, the microbacterial heat shock protein 65 derived peptide hsp3-13 and the naturally occurring invariant chain derived peptide Ii131-149 were employed to study binding inhibition by CLIP (Ii82-102) in a series of combinations. Incubation of detergent solubilized DR polypeptides from Ii-free cells with 500 microM of synthetic CLIP almost completely prevents binding of 50 microM subsequently added DR3-specific peptides. When CLIP and the peptides were added simultaneously to DR3 molecules, binding of hsp3-13 was abolished, whereas binding of Ii131-149 was only partially blocked. This indicates apparent affinity differences of the peptides. The addition of CLIP to preformed DR-peptide complexes substantially reduced binding of hsp3-13,while there was little effect on the DR associated Ii131-149. The profound inhibitory ability of CLIP, which in vivo would diminish binding of antigenic peptides, suggests an intracellular mechanism that abrogates the persistence of the CLIP-DR complex. The HLA-DM molecules have been suggested as candidates for this function. The strong in vitro binding of the naturally occurring peptide Ii131-149 to DR3 may suggest that only limited amounts of this peptide are available in vivo for competition of exogenous peptide binding to class II molecules.

Acquisition of peptides by MHC class II polypeptides in the absence of the invariant chain.

Association of invariant chain with class II molecules has been suggested to inhibit binding of peptides that are available while the class II complex is present in the endoplasmic reticulum (ER) and subsequently transported to endosomes. We tested HLA-DR-transfected rat2 fibroblast cells lacking expression of invariant chain for their ability to form SDS stable class II dimers indicative of peptide binding in the class II cavity. No SDS-resistant class II dimers originating from short pulse-labeled immunoprecipitates can be identified. Prolonged ER retention of DR polypeptides by Brefeldin A treatment does not induce any stable class II dimers. In pulse-chase experiments, heat labile class II dimers are readily detectable after a 60-min chase, increasing in amounts by 4 h of chase. In vitro incubation of rat2DR cell lysate with DR3-binding peptides converts pulse-labeled class II molecules to SDS-resistant dimers. This indicates the ability of ER-resident DR dimers to bind peptides. Inhibition studies were conducted to define the intracellular site where stable class II complexes are formed in rat2DR cells. The lysosomotropic reagent chloroquine abrogates the appearance of SDS-resistant DR complexes in invariant chain-free rat2DR cells, which is consistent with the impact of chloroquine on peptide loading in other APCs. Leupeptin treatment strongly reduces the amount of heat-labile class II molecules but does not impair peptide loading when DR3-specific peptides were added to viable cells or to cell lysates. This result suggests that leupeptin inhibits intracellular degradation of polypeptides and thereby depletes the endocytic pathway of peptides available for class II binding.