Maturation of human intestinal lactase-phlorizin hydrolase: generation of the brush border form of the enzyme involves at least two proteolytic cleavage steps.
Human lactase-phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine, is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH (LPHbeta) by a trypsin-like protease (Naim et al., 1987, 1991). Arg868-Ala869 has been previously proposed to be the putative cleavage site for this processing step. Site-directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHbeta species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N-terminal end of brush border LPH at Lys851-Arg852 and Arg830-Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868-Ala869 represents the final cleavage site that generates LPHbeta. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868-Ala869, to generate LPHbeta initial; LPHbeta is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868-Ala869 to mature brush border LPH (LPHbeta initial).
Jacob
R
R
Protein Secretion Group, Institute of Microbiology, Heinrich Heine University of Düsseldorf, Germany.
Radebach
I
I
Wüthrich
M
M
Grünberg
J
J
Sterchi
E E
EE
Naim
H Y
HY
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D003062:Codon; D006224:Cricetinae; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D007421:Intestine, Small / Q000201:enzymology*; D007668:Kidney; D010694:Lactase-Phlorizin Hydrolase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D003062:Codon; D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001120:Arginine; D010694:Lactase-Phlorizin Hydrolase; D010450:Endopeptidases