HUMAN LACTASE-PHLORHIZIN 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 glycoprote in of the microvillus membrane of human small intestinal epithelial ce lls (enterocytes), is vital for the digestion of lactose during early infancy. The enzyme is synthesized in enterocytes as a single-chain pr ecursor and subsequently proteolytically processed to the mature micro villus membrane-bound form. Because it has been reported that COS-1 ce lls were not able to proteolytically process LPH to the mature protein , these cells have been used as a model system to study potential role s of different proteases. COS-1 cells transfected with a full-length c DNA for human LPH synthesized enzymatically active enzyme. Immunopreci pitation of the expressed glycoproteins and their subsequent analysis by SDS-PAGE showed synthesis of two polypeptide species having apparen t molecular masses of 210 and 220 kDa, respectively, corresponding to the high-mannose (pro-LPH(h)) form and the complex glycosylated (pro-L PH(c)) form of the LPH precursor. Surprisingly, an additional polypept ide species corresponding in size to the mature LPH found in human int estinal cells was also detected after longer chase periods. The source of this species was clearly pro-LPH, as its formation was inhibited b y Brefeldin A. The cleaved form of LPH was not found on the cell surfa ce; furthermore, its formation was prevented by an inhibitor of lysoso mal function. We conclude from these data that in transfected COS-1 ce lls pro-LPH is transported to the cell surface, from which it is inter nalised and enters the lysosomal pathway, where proteolytic cleavage l eads to a molecule not unlike mature LPH. (C) 1997 Federation of Europ ean Biochemical Societies.