Involvement of the carboxyl-terminal propeptide of beta-glucuronidase in its compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach.
The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.
Roswell Park Memorial Institute, Department of Molecular and Cellular Biology, Buffalo, New York 14263.
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Medda
R M
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Chemelli
J L
JL
Martin
L R
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Pohl
R T
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Swank
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002265:Carboxylic Ester Hydrolases; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004792:Enzyme Precursors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D004792:Enzyme Precursors; D008562:Membrane Glycoproteins; D010455:Peptides; D002265:Carboxylic Ester Hydrolases; C009751:egasyn; D005966:Glucuronidase