Structural hierarchy of regulatory elements in the folding and transport of an intestinal multidomain protein

J Biol Chem. 2010 Feb 5;285(6):4143-52. doi: 10.1074/jbc.M109.060780. Epub 2009 Dec 2.

Abstract

Human intestinal lactase-phlorizin hydrolase, LPH, encompasses four homologous domains, which presumably have evolved from two subsequent duplications of one ancestral gene. The profragment, LPHalpha, comprises homologous domains I and II and functions as an intramolecular chaperone in the context of the brush-border LPHbeta region of LPH. Here, we analyze the inter-relationship between homologous domains III and IV of LPHbeta and their implication in the overall structure, function, and trafficking of LPH. In silico analyses revealed potential domain boundaries for these domains as a basis for loop-out mutagenesis and construction of deletion or individual domain forms of LPH. Removal of domain IV, which contains lactase, results in a diminished phlorizin hydrolase activity, lack of dimerization in the endoplasmic reticulum (ER), but accelerated transport kinetics from the ER to the Golgi apparatus. By contrast, deletion of domain III, which harbors phlorizin hydrolase, generates a malfolded protein that is blocked in the ER. Interestingly, homologous domain III is transport-competent per se and sorted to the apical membrane in polarized Madin-Darby canine kidney cells. Nevertheless, it neither dimerizes nor acquires complete phlorizin hydrolase activity. Our data present a hierarchical model of LPH in which the homologous domain III constitutes (i) a fully autonomous core domain within LPH and (ii) another intramolecular chaperone besides the profragment LPHalpha. Nevertheless, the regulation of the trafficking kinetics and activity of domain III and entire LPH including elevation of the enzymatic activities require the correct dimerization of LPH in the ER, an event that is accomplished by the non-autonomous domain IV.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Catalytic Domain / genetics
  • Cell Line
  • Chlorocebus aethiops
  • Electrophoresis, Polyacrylamide Gel
  • Endoplasmic Reticulum / enzymology
  • Golgi Apparatus / enzymology
  • Humans
  • Immunoprecipitation
  • Intestinal Mucosa / enzymology*
  • Lactase-Phlorizin Hydrolase / chemistry*
  • Lactase-Phlorizin Hydrolase / genetics
  • Lactase-Phlorizin Hydrolase / metabolism*
  • Lactose / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Models, Molecular
  • Mutation
  • Phlorhizin / metabolism
  • Protein Folding*
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Protein Transport
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Transfection

Substances

  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Phlorhizin
  • Lactase-Phlorizin Hydrolase
  • Lactose