Proteolytic processing of human lactase-phlorizin hydrolase is a two-step event: identification of the cleavage sites

Archives of Biochemistry and Biophysics
M WüthrichE E Sterchi

Abstract

Human lactase-phlorizin hydrolase (EC 3.2.1.23/62) is a major disaccharidase in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a single-chain precursor protein and undergoes proteolytic processing during maturation. We studied proteolytic processing of human lactase-phlorizin hydrolase in transfected COS-1, Caco-2, and MDCK cells using metabolic labeling, surface immunoprecipitation, protease sensitivity assays, and microsequencing. Furthermore, we generated mutated forms of the enzyme to alter potential proteolytic cleavage sites and expressed these in Caco-2 and COS-1 cells. Since the N-terminal amino acid of microvillus lactase-phlorizin hydrolase corresponds to Ala869 in the precursor protein, it has been speculated that processing occurs at position Arg868-Ala869. Substitution of Arg868 with isoleucine, lysine, or glutamic acid had no effect on the proteolytic processing of pro-LPH in Caco-2 cells. As in wild-type enzyme a processed 160-kDa form was generated. These data are not consistent with a primary proteolytic processing at position Arg868-Ala869. Using amino-terminal amino acid sequencing of this processed form isolated from stable transfected MDCK cells we identified the...Continue Reading

Citations

Dec 4, 2009·The Journal of Biological Chemistry·Marc BehrendtHassan Y Naim
Mar 8, 2005·Postgraduate Medical Journal·S B MatthewsA K Campbell
Mar 22, 2005·Biochimica Et Biophysica Acta·Jesper T Troelsen
Jan 10, 2006·American Journal of Human Genetics·Mikko KuokkanenIrma Jarvela
Dec 26, 2001·The Journal of Biological Chemistry·Ralf JacobHassan Y Naim
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Apr 18, 1998·The Journal of Biological Chemistry·J OuwendijkJ A Fransen
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