Human lactase-phlorizin hydrolase: evidence of dimerization in the endoplasmic reticulum.
Human lactase-phlorizin hydrolase [EC 3.2.1.23-3.2.1.62] is a disaccharidase located in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a precursor protein in the endoplasmic reticulum and in addition to being glycosylated is subsequently proteolytically processed to the mature microvillus membrane-bound form after passing the trans-Golgi compartment. We studied the oligomerization of human lactase-phlorizin hydrolase in transfected polarized Madin Darby canine kidney cells using metabolic labeling and sucrose-density centrifugation analysis. We detected high mannose dimers of the lactase-phlorizin hydrolase precursor molecule after metabolic labeling with [35S]methionine at 37 and 15 degrees C. In addition, both complex-glycosylated lactase-phlorizin hydrolase precursor molecule and the mature microvillus membrane-bound enzyme showed this oligomeric structure. Chemical crosslinking resulted in the detection of covalently crosslinked lactase-phlorizin hydrolase dimers after sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results provide evidence that oligomerization of lactase-phlorizin hydrolase is an early event and begins in the endoplasmic reticulum.
D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D010694:Lactase-Phlorizin Hydrolase / Q000737:chemistry*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry; D009924:Organ Culture Techniques; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D010694:Lactase-Phlorizin Hydrolase