The pro region of human intestinal lactase-phlorizin hydrolase.
Human small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a single-chain polypeptide precursor, prepro-LPH, that undergoes two sequential cleavage steps: the first in the endoplasmic reticulum to pro-LPH (215-kDa) and the second, following terminal glycosylation in the Golgi apparatus, to mature 160-kDa LPH (denoted LPH beta). The LPH beta molecule is subsequently targetted to the brush-border membrane. Characterization of the N-terminal profragment (denoted LPH alpha) of pro-LPH using an epitope-specific, anti-peptide polyclonal antibody reveals that LPH alpha (i) has an apparent molecular weight of approximately 100,000, (ii) is not associated with LPH beta after cleavage of pro-LPH has occurred, and (iii) is not transported to the cell surface or secreted into the extracellular medium. In biosynthetic labeling experiments, a clear precursor/product relationship could be demonstrated between pro-LPH and the LPH alpha and LPH beta polypeptides. Further, LPH alpha has a significantly shorter half-life than LPH beta. LPH alpha is neither N- nor O-glycosylated, despite the presence of 5 potential N-glycosylation sites. LPH alpha, which is rich in cysteine and hydrophobic amino acid residues, may fold rapidly into a tight and rigid globular domain in which carbohydrate attachment sites are no longer accessible to glycosyltransferases. When expressed independently in COS-1 cells, the LPH beta polypeptide forms a misfolded, transport-incompetent molecule. We propose a role for the LPH alpha domain within the pro-LPH molecule as an intramolecular chaperone during folding in the ER.
Naim
H Y
HY
Institute of Microbiology, Heinrich Heine University of Düsseldorf, Germany.
Jacob
R
R
Naim
H
H
Sambrook
J F
JF
Gething
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D001706:Biopsy; D002451:Cell Compartmentation; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology*; D010694:Lactase-Phlorizin Hydrolase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D004792:Enzyme Precursors; D000939:Epitopes; D018832:Molecular Chaperones; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010694:Lactase-Phlorizin Hydrolase