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Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells
J Ouwendijk1, W J Peters, R A van de Vorstenbosch
1Department of Cell Biology and Histology, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands.
The Journal of Biological Chemistry
|April 18, 1998
Summary
Human lactase-phlorizin hydrolase (LPH) undergoes cleavage, yielding LPHbeta and LPHalpha. This study suggests LPHalpha is rapidly degraded after its release, impacting enzyme function.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Human lactase-phlorizin hydrolase (LPH) is a key digestive enzyme in the small intestine.
- Pro-LPH (230 kDa) is processed into LPHbeta (160 kDa) and LPHalpha (100 kDa) at the Golgi apparatus.
- LPHalpha's role after cleavage was previously unknown, though it was hypothesized to act as an intramolecular chaperone.
Purpose of the Study:
- To investigate the fate and localization of the LPHalpha profragment in polarized epithelial cells.
- To determine if LPHalpha has a role after its cleavage from LPHbeta.
- To analyze the degradation pathway of LPHalpha.
Main Methods:
- Stable expression of wild-type and epitope-tagged LPH in Caco-2 cells.
- Utilized antibodies against LPH, a profragment epitope, and a VSV tag for detection.
- Analyzed protein processing rates and localization of cleaved forms.
Main Results:
- Both wild-type and tagged LPH proteins were processed and cleaved similarly.
- The LPHbeta form was successfully localized to the apical cell surface.
- LPHalpha could not be recovered using specific antibodies, indicating it was not stable after cleavage.
Conclusions:
- The LPHalpha profragment is likely degraded immediately after its cleavage from LPHbeta.
- This rapid degradation suggests LPHalpha does not have a sustained role after its chaperone function.
- Understanding LPHalpha's fate is crucial for comprehending LPH maturation and function.