The invariant chain forms complexes with class II major histocompatibility complex molecules and antigenic peptides "in vivo".
The binding of a chicken ovalbumin peptide (residues 323-339), Ova-(323-339), to I-Ad molecules was investigated in vitro and in vivo. By using antigenic peptides labeled either with a hapten or with fluorescein, complexes formed in vitro between I-Ad and antigenic peptides were detected by Western blot analysis with an antibody recognizing the hapten 7-nitrobenzo-2-oxa-1,3-diazole and by scanning gels for fluorescence emitted by fluoresceinated peptide. Both techniques reveal that Ova-(323-339) binds not only to I-Ad alpha/beta heterodimers and separated alpha and beta chains but also to complexes of higher molecular mass. Additional analysis shows that one of these additional complexes contains I-Ad heterodimers, antigenic peptides, and also invariant chain. To explore the physiological role of these complexes, cells were incubated with haptenated peptide and the I-Ad-peptide complexes formed in vivo were purified by affinity chromatography using hapten-specific antibody. The complexes formed migrate with a significantly higher apparent molecular mass than the alpha/beta heterodimers. A band at 180 kDa contained the alpha/beta heterodimer, the antigenic peptide, and the invariant chain. These results show that in vivo high molecular mass complexes formed by the I-Ad heterodimer and the invariant chain bind antigenic peptides.
Viguier
M
M
Stauffer Laboratory of Physical Chemistry, Stanford University, CA 94305.
Dornmair
K
K
Clark
B R
BR
McConnell
H M
HM
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D000949:Histocompatibility Antigens Class II / Q000276:immunology* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008970:Molecular Weight; D010047:Ovalbumin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D010047:Ovalbumin