Peptide binding by protein disulfide isomerase, a resident protein of the endoplasmic reticulum lumen.
Previously we had demonstrated by photoaffinity labeling that a 57-kDa protein of the endoplasmic reticulum can bind and become covalently linked to glycosylatable photoreactive peptides containing the sequence-Asn-Xaa-Ser/Thr-. Subsequently, it was found that this protein, called glycosylation site-binding protein, was a multifunctional protein, i.e. it was identical to protein disulfide isomerase (PDI), the beta-subunit of prolyl hydroxylase and thyroid hormone-binding protein. In this study, the peptide specificity for binding to this 57-kDa protein, hereafter called PDI, has been investigated in more detail using photoaffinity probes. The results reveal that although N-glycosylation by oligosaccharyl transferase in the endoplasmic reticulum has an absolute requirement for an hydroxyamino acid in the third amino acid residue of the glycosylation site sequence, no such specificity is observed in the binding of such peptides to PDI. In addition to the lack of specificity for an hydroxyamino acid in the third residue position, no specificity was observed for the asparagine residue in the first position. Thus, binding is not restricted to peptides containing N-glycosylation sites. We have investigated the discrepancy between this apparent lack of sequence specificity and earlier results indicating that binding of peptides to PDI was specific for N-glycosylation site sequences. We now demonstrate that PDI in the lumen of microsomes is more efficiently labeled by peptides containing photoreactive-Asn-Xaa-Ser/Thr- sequences than by nonacceptor site sequences because the former become glycosylated. This increased labeling does not occur because the glycosylated form of the probes are preferentially recognized by PDI. Rather, it appears that increased polarity of the affinity probe after attachment of the oligosaccharide chain prevents its exit from the sealed microsomes, in effect concentrating it within the lumen of the microsome. These results, coupled with other studies on the multifunctional nature of PDI, suggest that the observed peptide binding may be a manifestation of the ability of PDI to recognize the backbone of polypeptides in the lumen of the endoplasmic reticulum.
Department of Biochemistry and Molecular Biology, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
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Lennarz
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006031:Glycosylation; D007535:Isomerases / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010777:Photochemistry; D019704:Protein Disulfide-Isomerases; D051381:Rats
D000345:Affinity Labels; D010455:Peptides; D007535:Isomerases; D019704:Protein Disulfide-Isomerases