Abstract
The folding of human intestinal prolactase-phlorizin hydrolase (pro-LPH) has been analyzed in a cell-free transcription/translation system. In the presence of the thiol oxidant GSSG, disulfide bond formation in pro-LPH can be promoted concomitant with the binding of the molecule to a conformation-specific monoclonal anti-LPH antibody. Under these conditions, pro-LPH does not bind to the molecular chaperone BiP. In the absence of GSSG, on the other hand, pro-LPH does not bind to the monoclonal anti-LPH antibody, but can be immunoprecipitated with a polyclonal antibody that is directed against a denatured form of the enzyme. In this case, interaction of pro-LPH with immunoglobulin heavy chain binding protein can be discerned. The results demonstrate the existence of intramolecular disulfide bonds that are essential for the promotion of pro-LPH to a native conformation. Furthermore, BiP is involved in the folding events of pro-LPH.
Highlights
The pathways leading to the promotion of a protein to its final native configuration are still not completely defined
We investigated the folding of pro-Lactase-phlorizin hydrolase (LPH) with particular emphasis on disulfide bond formation
S DS,PAGE-Samples were prepared for e lec t ro ph ores is by mix in g wit h 5 volumes of S DS -I'AGE s a m ple huffer in th e pres -nce of 1i0 ms: D'PT for reducing conditions und in the presence of 100 m~1 iodonce tami de for nonreducing conditions lind boil ed for Ii m in
Summary
Vol 270, No 31, Issue of August 4, pp. 18678-18684, 1995 Printed in U.S.A. From the tF'rotein Secretion Group, Institute of Microbiology, Heinrich Heine University, Universitiitsstrasse 1, D-40225 Dusseldorf, Federal Republic of Germany and the §Division of Biological Sciences, Department of Biochemistry and Molecular Biology, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. In the presence of the thiol oxidant GSSG, disulfide bond formation in proLPH can be promoted concomitant with the binding of the molecule to a conformation-specific monoclonal anti-Lj'H antibody Under these conditions, pro-LPH does not bind to the molecular chaperone RiP. Several modification reactions are initiated during entry of the extending polypeptide chain into the ER lumen and may facilitate the folding of the protein into native conformation These include signal sequence cleavage, attachment of mannose-rich carbohydrate chains, and disulfide bond formation. Of note is the presence of a large profragment (LPHa; 868 amino acids) at the N-terminal end that precedes brush border LPHm. Recent identification of LPHO' in intestinal biopsy samples (Nairn et aZ., 1994) and analysis of its possible function have led to the hypothesis that LPHO' may playa crucial role in the folding of pro-LPH, perhaps as an intramolecular chaperone (Oberholzer et al, 1993; Nairn et aZ., 1994).
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