Inhibition of peptide binding to DR molecules by a leupeptin-induced invariant chain fragment.
Loading of peptides onto DR molecules was studied by characterizing precursors of the mature peptide-DR complexes expressed at the surface of B cells. Since invariant chain (Ii) prevents binding of peptides by DR molecules, it was speculated that analysis of complexes between DR heterodimers and proteolytic fragments of Ii offers the possibility to examine how DR molecules and peptides assemble. Using a procedure combining a two-step affinity chromatography and gel filtration, we isolated from leupeptin-treated B cells complexes between DR molecules and N-terminal Ii fragments previously called "leupeptin-induced polypeptides" (LIP; Blum and Cresswell, 1988, Proc. natn. Acad. Sci. U.S.A. 85, 3975-3979). It was observed that the most prominent LIP fragment has a relative molecular mass (M(r)) of 16 kDa. In addition, we show that this polypeptide species does not bear N-linked glycans, indicating that this fragment does not extend beyond residue 129 of Ii. Similarly to DR alpha beta heterodimers associated with the full length 33 and 35 kDa Ii forms, DR alpha beta heterodimers associated with LIP fragments are unstable in sodium dodecyl sulfate (SDS) at ambient temperature, whereas mature DR alpha beta heterodimers are resistant to dissociation with SDS. These results are indirect evidence that LIP-DR complexes are devoid of bound peptides. This possibility was supported by showing that LIP-DR complexes fail to bind a radioiodinated tetanus toxin peptide (125I-p2), while DR molecules, which are spontaneously released from complexes with LIP fragments, bind the labeled peptide. These results demonstrate that association with LIP fragments is sufficient to prevent binding of peptides by DR molecules. This notion was further documented by showing that binding of 125I-p2 on DR heterodimers is inhibited by preparations of LIP fragment. By contrast, a soluble recombinant fragment corresponding to the extracytoplasmic region of Ii did not block 125I-p2 binding. The results presented in this study indicate that the cytoplasmic and/or transmembrane region of Ii is required to prevent peptide binding by DR molecules, while the extracytoplasmic portion of Ii, though capable of associating with DR molecules, lacks the capacity to block peptide binding.
Demotz
S
S
Basel Institute for Immunology, Switzerland.
Danieli
C
C
Wallny
H J
HJ
Majdic
O
O
D000595:Amino Acid Sequence; D000944:Antigens, Differentiation, B-Lymphocyte; D001402:B-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D006684:HLA-DR Antigens / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010316:Particle Size; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects
D000944:Antigens, Differentiation, B-Lymphocyte; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D007976:Leupeptins; D010446:Peptide Fragments; C045084:invariant chain; C032854:leupeptin