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Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase
Abstract:
The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H as judged by its increased electrophoretic mobility (Mr 210 000 after treatment). The labelling of this form decreased during a chase of 120 min and instead two polypeptides of Mr 245 000 and 160 000 occurred, which both barely had their electrophoretic mobility changed by treatment with endo H. The Mr 160 000 polypeptide is of the same size as the mature lactase-phlorizin hydrolase and was the only form expressed in the microvillar membrane. Together, these data are indicative of an intracellular proteolytic cleavage during transport. The presence of leupeptin during labelling prevented the appearance of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence of leupeptin.
Insights
Pig small intestinal lactase-phlorizin hydrolase undergoes intracellular processing. Proteolytic cleavage is not essential for transport to the microvillar membrane.
Area of Science:
- Biochemistry
- Molecular Biology
- Gastroenterology
Background:
- Lactase-phlorizin hydrolase (LPH) is a key enzyme in small intestinal digestion.
- Understanding LPH biosynthesis is crucial for comprehending nutrient absorption and related disorders.
Purpose of the Study:
- To investigate the biosynthesis and processing of pig small intestinal lactase-phlorizin hydrolase.
- To elucidate the intracellular transport and maturation pathway of LPH.
Main Methods:
- Organ culture of mucosal explants from pig small intestine.
- Metabolic labeling with [35S]methionine.
- Endo H sensitivity assays to assess glycosylation.
- Analysis of polypeptide molecular weights (Mr) by electrophoresis.
- Inhibition of proteolytic cleavage using leupeptin.
Main Results:
- An intracellular, membrane-bound precursor polypeptide (Mr 225,000) sensitive to endo H was identified.
- During a 120-minute chase, this precursor was processed into Mr 245,000 and Mr 160,000 polypeptides, resistant to endo H.
- The Mr 160,000 polypeptide corresponds to mature LPH and is localized in the microvillar membrane.
- Leupeptin treatment blocked the formation of the Mr 160,000 form but not the Mr 245,000 form, indicating cleavage occurs post-glycosylation.
- Proteolytic cleavage was not required for microvillar membrane transport.
Conclusions:
- Pig LPH biosynthesis involves intracellular proteolytic cleavage during its transport.
- This cleavage occurs after trimming and complex glycosylation.
- The proteolytic step is not essential for the enzyme's delivery to the microvillar membrane.