title: Dimerization of lactase-phlorizin hydrolase occurs in the endoplasmic reticulum, involves the putative membrane spanning domain and is required for an efficient transport of the enzyme to the cell surface.
abstract: Analysis of the quaternary structure of human intestinal lactase-phlorizin hydrolase (LPH) by chemical cross-linking and sucrose-gradient centrifugation reveals that the brush border form of LPH (LPH beta; 160-kDa) is a homodimeric molecule. Dimerization ensures in the ER when LPH is still exclusively found as an uncleaved mannoserich precursor (pro-LPHb; 215-kDa). This is supported by the following observations. (i) Biosynthetically labeled intestinal biopsy specimens as well as transfected COS-1 cells expressing pro-LPH contain monomeric and dimeric forms of pro-LPHb; the complex glycosylated pro-LPH (pro-LPHc; 230-kDa) as well as the cleaved mature LPH beta species in intestinal biopsy samples are discerned exclusively as dimers. (ii) Dimeric forms of pro-LPHh could be also detected when cells were biosynthetically labeled at 15 degrees C, at which temperature the egress of pro-LPH from the ER is blocked. Dimerization is essential for the transport competence of pro-LPH and is strongly associated with the presence of an intact transmembrane domain. Mutant pro-LPH-mact lacking the complete transmembrane domain persists as a monomeric, mannose-rich and transport-incompetent molecule that is not secreted into the exterior milieu, accumulates most likely in the ER and is ultimately degraded. Further, deletion of the cytoplasmic tail in the pro-LPH-ct mutant leads to marked reduction in the proportion of dimeric as well as complex glycosylated pro-LPH-ct. Finally, dimerization is linked to the acquisition of LPH to its biological function, since only dimers of wild type pro-LPH or pro-LPH-ct are enzymatically active, while their monomeric counterparts as well as pro-LPH-mact are not.
authors.affiliation: Institute of Microbiology, Heinrich Heine University of Düsseldorf, Germany.
authors.forename: H Y
authors.initials: HY
authors.lastname: Naim
authors.forename: H
authors.initials: H
authors.lastname: Naim
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001692:Biological Transport / Q000502:physiology; D001706:Biopsy; D019556:COS Cells / Q000201:enzymology / Q000648:ultrastructure; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018604:Epitope Mapping; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D007422:Intestines / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D010694:Lactase-Phlorizin Hydrolase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008358:Mannose / Q000032:analysis; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology; D016296:Mutagenesis / Q000502:physiology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D007527:Isoenzymes; D008565:Membrane Proteins; C096385:pro-lactase-phlorizin hydrolase; D010694:Lactase-Phlorizin Hydrolase; D010450:Endopeptidases; D008358:Mannose