Processing of human pro-lactase-phlorizin hydrolase at reduced temperatures: cleavage is preceded by complex glycosylation.
Intracellular processing of human intestinal lactase-phlorizin hydrolase (LPH) includes an essential proteolytic cleavage step that generates the mature brush border enzyme from the single-chain polypeptide precursor (pro-LPH). Previous work in organ culture of small intestinal biopsy samples [Naim, Sterchi & Lentze (1987) Biochem. J. 241, 427-434] has demonstrated that this cleavage occurs intracellularly. Since no intermediate forms of pro-LPH (trimmed or complex glycosylated) could be discerned in pulse-chase analyses it was suggested that the cleavage process occurs at a fast rate. To identify intermediate forms of pro-LPH prior to cleavage, I studied the biosynthesis of LPH by employing a pulse-chase protocol in mucosa explants (or biopsies) at reduced temperatures (22 degrees C). Here, I could identify by immunoprecipitation with monoclonal anti-LPH antibodies four LPH forms that exhibited a product-precursor relationship:mannose-rich precursor (pro-LPHh), trimmed pro-LPH (LPHt), complex glycosylated pro-LPH (pro-LPHc) and cleaved, mature LPH (LPHm). The results clearly indicate that the generation of mature LPH is preceded by complex glycosylation of the precursor form. The fact that this was not previously observed in the same experimental system under normal biosynthetic labelling conditions (37 degrees C) demonstrates that the cleavage process of pro-LPH occurs at a fast rate in the human small intestine.
Naim
H Y
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Institute of Microbiology, Heinrich-Heine-University of Düsseldorf, Germany.
D016521:Electrophoresis, Gel, Pulsed-Field; D004792:Enzyme Precursors / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D007583:Jejunum / Q000201:enzymology; D043322:Lactase; D010694:Lactase-Phlorizin Hydrolase / Q000096:biosynthesis / Q000378:metabolism*; D009924:Organ Culture Techniques; D011499:Protein Processing, Post-Translational; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism*
D004792:Enzyme Precursors; D043322:Lactase; D001616:beta-Galactosidase; D010694:Lactase-Phlorizin Hydrolase