Biosynthesis of protease nexin-I

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Protease nexin-I (PN-I) is a secreted serine protease inhibitor from human fibroblasts, forming complexes with proteases and binding to cell receptors.
• PN-I targets proteases like thrombin, urokinase, trypsin, and plasmin; heparin accelerates its binding to thrombin.
• A previously developed polyclonal antibody against PN-I can block PN-I-protease complex binding and immunoprecipitate PN-I.

Purpose of the Study:

• To investigate the biosynthesis and regulation of Protease nexin-I (PN-I) in human fibroblasts using a specific anti-PN-I antibody.
• To characterize the molecular properties and processing of PN-I during its secretion.

Main Methods:

• Utilized a polyclonal antibody against PN-I for immunoprecipitation and competition assays.
• Employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to analyze PN-I and its precursor.
• Performed amino-terminal sequencing to confirm protein identity and endoglycosidase H treatment to assess glycosylation.

Main Results:

• Demonstrated that unlabeled PN-I competes with metabolically labeled PN-I for binding to the anti-PN-I antibody, confirming antibody specificity.
• Identified a lower molecular weight intracellular precursor of PN-I, sensitive to endoglycosidase H, indicating high mannose N-linked carbohydrates.
• Mature PN-I, also appearing as a doublet, is resistant to endoglycosidase H but contains 3 kDa of N-linked carbohydrate; it is constitutively secreted and regulated by cellular uptake, reaching a steady state in conditioned media within 48 hours.

Conclusions:

• Protease nexin-I (PN-I) undergoes post-translational modification, including N-linked glycosylation, with distinct intracellular precursor and mature forms.
• PN-I is constitutively secreted by fibroblasts and its levels in the medium are regulated by a receptor-mediated uptake mechanism.
• The findings provide insights into the biosynthesis, processing, and regulation of PN-I, a key serine protease inhibitor.