Biosynthesis of N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase: disulfide-linked dimers are formed at the site of synthesis in the rough endoplasmic reticulum.
N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH) is a metalloendopeptidase found in mucosal epithelial cells of the human small intestine. The purification and characterization of this enzyme was described in the preceding paper (E. E. Sterchi et al. (1988) Arch. Biochem. Biophys. 265, 105-118). In this paper, we report on the biosynthesis and posttranslational processing of PPH in organ cultures of human small intestinal mucosa. Continuous labeling for 6 h with L-[35S]methionine, immunoprecipitation with monoclonal antibody 3/716/36, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a polypeptide with Mr 100,000. This molecule was highly glycosylated as treatment with endo-beta-N-acetylglucosaminidase F resulted in a reduction to Mr 70,000. This was also the size of the species isolated after culture in the presence of tunicamycin, an inhibitor of N-linked glycosylation. Pulse-chase labeling showed that the first detectable form of PPH had a Mr 90,000 which corresponded to the high-mannose precursor as assessed by its sensitivity to endo-beta-N-acetylglucosaminidase H. Within 15 min of chase and prior to complex glycosylation, dimerization due to the formation of interchain disulfide bonds occurred (Mr 180,000). Dimerization thus took place within the rough endoplasmic reticulum and might play an important role in the transport through to the cell surface. After 2 h of chase, PPH started to appear in the culture medium, indicating that the enzyme was secreted from the cells, a finding not observed with other microvillus membrane hydrolases.
Sterchi
E E
EE
Department of Gastroenterology, University Children's Hospital, Berne, Switzerland.
Naim
H Y
HY
Lentze
M J
MJ
D010129:4-Aminobenzoic Acid / Q000378:metabolism*; D062365:Aminobenzoates / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002648:Child; D003470:Culture Media / Q000032:analysis; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D062366:para-Aminobenzoates
D062365:Aminobenzoates; D003470:Culture Media; D004220:Disulfides; D062366:para-Aminobenzoates; C006717:bentiromide; D008985:Monensin; D008666:Metalloendopeptidases; C053833:meprin A; D010129:4-Aminobenzoic Acid