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 thetrans-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°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.