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The pro region of human intestinal lactase-phlorizin hydrolase
1Institute of Microbiology, Heinrich Heine University of Düsseldorf, Germany.
Insights
The N-terminal profragment (LPH alpha) of lactase-phlorizin hydrolase (LPH) acts as an intramolecular chaperone, aiding the proper folding of LPH beta in the endoplasmic reticulum for brush-border membrane targeting.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Human small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a precursor protein, prepro-LPH.
- This precursor undergoes sequential cleavage events to yield mature LPH beta and a profragment, LPH alpha.
Purpose of the Study:
- To characterize the N-terminal profragment (LPH alpha) of human lactase-phlorizin hydrolase (LPH).
- To elucidate the role of LPH alpha in the processing and folding of LPH beta.
Main Methods:
- Biosynthetic labeling experiments were employed to trace protein processing and half-lives.
- Characterization of LPH alpha involved molecular weight determination and assessment of its association with LPH beta.
- Functional analysis included expressing LPH beta independently in COS-1 cells.
Main Results:
- LPH alpha (approx. 100,000 Da) is distinct from LPH beta after pro-LPH cleavage and is not cell surface-targeted.
- A clear precursor-product relationship exists between pro-LPH, LPH alpha, and LPH beta.
- LPH alpha has a shorter half-life than LPH beta and is not glycosylated, suggesting rapid, rigid folding.
Conclusions:
- LPH alpha functions as an intramolecular chaperone, facilitating the correct folding of LPH beta in the endoplasmic reticulum.
- This chaperone activity is crucial for the proper transport and function of LPH beta at the brush-border membrane.
- Independent expression of LPH beta results in misfolding and transport incompetence, highlighting the essential role of LPH alpha.
Abstract:
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.