CHEMICAL DENATURATION AND MODIFICATION OF OVALBUMIN ALTERS ITS DEPENDENCE ON UBIQUITIN CONJUGATION FOR CLASS-I ANTIGEN PRESENTATION

Class I presentation of microinjected native OVA by a temperature-sens itive ubiquitin conjugation mutant, ts85, but not wild-type murine cel ls, was markedly inhibited following incubation at a nonpermissive tem perature. In contrast, the nonpermissive temperature did not affect cl ass I presentation of a minimal OVA peptide expressed in the cytosol. Therefore, these results provide a second example in which a temperatu re sensitive mutation in the ubiquitin conjugation pathway inhibits MH C class I presentation of native OVA. Surprisingly, incubation at the nonpermissive temperature did not inhibit class I presentation of chem ically denatured and alkylated OVA microinjected into the cytosol of m utant cells. Similarly, the presentation of endogenously synthesized O VA (which is expressed from a recombinant vaccinia virus and, presumab ly, is misfolded in the cytosol) was also not inhibited in both mutant cell lines. Methylation of the lysine groups in denatured OVA, which blocks ubiquitin conjugation, reduced but did not eliminate the presen tation of denatured OVA, providing evidence for both ubiquitin-depende nt and ubiquitin-independent pathways for class I presentation. In con trast, a proteasome inhibitor blocked class I presentation of all form s of OVA, while a control peptide aldehyde was not inhibitory. These r esults indicate that modification of the structure of a protein can in fluence its requirements for ubiquitin conjugation for efficient class I presentation, with the key alteration possibly being the loss of pr oper conformation. However, regardless of the form of the Ag, the prot easome appears to be required for generating peptides from both endoge nously synthesized and microinjected OVA for class I presentation.