title: Impaired trafficking and subcellular localization of a mutant lactase associated with congenital lactase deficiency.
abstract: OBJECTIVE
  Congenital lactase deficiency (CLD) is a cause of disaccharide intolerance and malabsorption characterized by watery diarrhea in infants fed breast milk or lactose-containing formulas. The molecular basis of CLD is unknown. Mutations in the coding region of the brush border enzyme lactase phlorizin hydrolase (LPH) were found to cause CLD in a study of 19 Finnish families. We analyzed the effects of one of these mutations, G1363S, on LPH folding, trafficking, and function.
  
  
  METHODS
  We introduced a mutation into the LPH complementary DNA that resulted in the amino acid substitution G1363S. The mutant gene was transiently expressed in COS-1 cells, and the effects were assessed at the protein, structural, and subcellular levels.
  
  
  RESULTS
  The mutant protein LPH-G1363S was misfolded and could not exit the endoplasmic reticulum. Interestingly, the mutation creates an additional N-glycosylation site that is characteristic of a temperature-sensitive protein. The intracellular transport and enzymatic activity, but not correct folding, of LPH-G1363S were partially restored by expression at 20 degrees C. However, a form of LPH that contains the mutations G1363S and N1361A, which eliminates the N-glycosylation site, did not restore the features of wild-type LPH. Thus, the additional glycosyl group is not required for the LPH-G1363S defects.
  
  
  CONCLUSIONS
  This is the first characterization, at the molecular and subcellular levels, of a mutant form of LPH that is involved in the pathogenesis of CLD. Mutant LPH accumulates predominantly in the endoplasmic reticulum but can partially mature at a permissive temperature; these features are unique for a protein involved in a carbohydrate malabsorption defect implicating LPH.
authors.affiliation: Department of Physiological Chemistry, University of Veterinary Medicine, Hannover, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Behrendt
authors.forename: Markus
authors.initials: M
authors.lastname: Keiser
authors.forename: Melanie
authors.initials: M
authors.lastname: Hoch
authors.forename: Hassan Y
authors.initials: HY
authors.lastname: Naim
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism; D043322:Lactase / Q000172:deficiency* / Q000235:genetics; D010694:Lactase-Phlorizin Hydrolase / Q000235:genetics / Q000378:metabolism*; D007787:Lactose Intolerance / Q000235:genetics*; D008286:Malabsorption Syndromes / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D021381:Protein Transport / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D051381:Rats; D012680:Sensitivity and Specificity; D046509:Tissue Culture Techniques
chemical_list: D050506:Mutant Chimeric Proteins; D012333:RNA, Messenger; D043322:Lactase; D010694:Lactase-Phlorizin Hydrolase