Human lactase-phlorizin hydrolase expressed in COS-1 cells is proteolytically processed by the lysosomal pathway.
Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62), a major glycoprotein of the microvillus membrane of human small intestinal epithelial cells (enterocytes), is vital for the digestion of lactose during early infancy. The enzyme is synthesized in enterocytes as a single-chain precursor and subsequently proteolytically processed to the mature microvillus membrane-bound form. Because it has been reported that COS-1 cells were not able to proteolytically process LPH to the mature protein, these cells have been used as a model system to study potential roles of different proteases. COS-1 cells transfected with a full-length cDNA for human LPH synthesized enzymatically active enzyme. Immunoprecipitation of the expressed glycoproteins and their subsequent analysis by SDS-PAGE showed synthesis of two polypeptide species having apparent molecular masses of 210 and 220 kDa, respectively, corresponding to the high-mannose (pro-LPHh) form and the complex glycosylated (pro-LPHc) form of the LPH precursor. Surprisingly, an additional polypeptide species corresponding in size to the mature LPH found in human intestinal cells was also detected after longer chase periods. The source of this species was clearly pro-LPH, as its formation was inhibited by Brefeldin A. The cleaved form of LPH was not found on the cell surface; furthermore, its formation was prevented by an inhibitor of lysosomal function. We conclude from these data that in transfected COS-1 cells pro-LPH is transported to the cell surface, from which it is internalised and enters the lysosomal pathway, where proteolytic cleavage leads to a molecule not unlike mature LPH.
Wüthrich
M
M
Department of Paediatrics, University of Berne, Switzerland.
Sterchi
E E
EE
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D020126:Brefeldin A; D019556:COS Cells; D003517:Cyclopentanes / Q000494:pharmacology; D006801:Humans; D010694:Lactase-Phlorizin Hydrolase / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects
D003517:Cyclopentanes; D000643:Ammonium Chloride; D020126:Brefeldin A; D010694:Lactase-Phlorizin Hydrolase