Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein.
Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62) is a major intestinal microvillar membrane glycoprotein that digests lactose, the main carbohydrate of milk. To investigate structure/function relationships of LPH and to assess the impact of intracellular processing on the function of LPH and on its transport to the cell surface, we have expressed a full-length cDNA encoding LPH in mammalian COS-1 cells. Analysis of the expressed protein by immunoprecipitation with monoclonal anti-LPH antibodies and treatments with endo-beta-N-acetylglucosaminidase H and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides with apparent molecular masses of 215 and 230 kDa, representing the mannose-rich (pro-LPHh) and complex (pro-LPHc) glycosylated forms of the precursor. By contrast to pro-LPH in human enterocytes, the expressed pro-LPH in COS-1 cells does not undergo intracellular proteolytic cleavage to generate a form similar to the mature enzyme of the brush-border membrane. Intracellular cleavage, however, is not essential for the molecule to acquire its enzymatic activity since pro-LPH in COS-1 cells is enzymatically as active as LPH isolated from intestinal brush-border membranes. Indirect immunofluorescent staining of transfected cells demonstrated that pro-LPH is expressed at the cell surface. This was further corroborated by the sensitivity of the complex glycosylated form (pro-LPHc) to trypsin in the medium. Our results provide the first conclusive evidence that pro-LPH is an enzymatically active molecule and that the intracellular proteolysis of pro-LPH is not essential for the generation of transport-competent forms of LPH.
Naim
H Y
HY
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9050.
Lacey
S W
SW
Sambrook
J F
JF
Gething
M J
MJ
D001483:Base Sequence; D001692:Biological Transport; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases; D006801:Humans; D007150:Immunohistochemistry; D007422:Intestines / Q000201:enzymology*; D043322:Lactase; D010694:Lactase-Phlorizin Hydrolase / Q000235:genetics* / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014357:Trypsin; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D011498:Protein Precursors; D004247:DNA; D006596:Hexosaminidases; D043322:Lactase; D001616:beta-Galactosidase; D010694:Lactase-Phlorizin Hydrolase; D014357:Trypsin