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Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein
Krzysztof B Wicher1, Erik Fries
1Department of Medical Biochemistry and Microbiology, Uppsala University, P.O. Box 582, S-751 23 Uppsala, Sweden. krzysztof.wicher@imbim.uu.se
Summary
Complement C1r-like protein (C1r-LP) cleaves haptoglobin (Hp) proform in the endoplasmic reticulum. This newly identified proteolytic activity of C1r-LP is crucial for Hp maturation before entering the Golgi apparatus.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Secretory proteins often require proteolytic cleavage for activation.
- Haptoglobin (Hp) undergoes cleavage in the endoplasmic reticulum (ER), an unusual step in the secretory pathway.
Purpose of the Study:
- To identify the protease responsible for cleaving haptoglobin (Hp) proform in the ER.
- To characterize the proteolytic activity of complement C1r-like protein (C1r-LP).
Main Methods:
- Coexpression of pro-haptoglobin (proHp) and C1r-LP in COS-1 cells.
- Site-directed mutagenesis of the putative active site of C1r-LP.
- In vitro cleavage assays using purified proteins.
- RNA interference to suppress C1r-LP expression in HepG2 cells.
Main Results:
- C1r-LP coexpression with proHp resulted in proHp cleavage within the ER.
- Cleavage was dependent on C1r-LP's proteolytic activity, as shown by a catalytically inactive mutant.
- Purified C1r-LP cleaved proHp in vitro at the expected site.
- C1r-LP demonstrated specificity, not cleaving the proform of complement C1s.
- Reduced C1r-LP expression decreased proHp cleavage in HepG2 cells.
Conclusions:
- Complement C1r-like protein (C1r-LP) is a novel ER-resident protease that cleaves haptoglobin (Hp) proform.
- This finding reveals a new function for C1r-LP and clarifies an unusual step in Hp processing.
- C1r-LP accounts for a significant portion of endogenous proHp cleavage activity.
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