Related Experiment Videos
Biochemical and functional characterization of aminopeptidase N expressed by human melanoma cells
A Menrad1, D Speicher, J Wacker
1Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Abstract:
A cell surface protein expressed on melanoma cells, but not on normal melanocytes, was biochemically and functionally characterized. Microsequencing of the M(r) 143,000 affinity-purified protein revealed amino acid sequence identity to aminopeptidase N (EC 3.4.11.2). In situ expression, indirect immunofluorescence, and Western blotting demonstrated that aminopeptidase N is tightly associated with extracellular matrix components. A specific polyclonal antiserum and the competitive inhibitors of aminopeptidase N, bestatin and amastatin, inhibited invasion of an aminopeptidase N-expressing metastatic melanoma cell line through the reconstituted basement membrane Matrigel in a dose-dependent manner. In vitro digestion of Matrigel with affinity-purified aminopeptidase N revealed an enzyme-sensitive M(r) 160,000 protein. These experiments suggest a role for aminopeptidase N in melanoma invasion of basement membranes.
Insights
Aminopeptidase N, a protein found on melanoma cells, aids in cancer invasion. Inhibiting this enzyme blocks melanoma cells from penetrating basement membranes, suggesting it is a potential therapeutic target.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Melanoma cells express unique cell surface proteins not found on normal melanocytes.
- Understanding these proteins is crucial for identifying melanoma-specific targets.
- Aminopeptidase N (APN) is a cell surface enzyme implicated in various cellular processes.
Purpose of the Study:
- To biochemically and functionally characterize a cell surface protein on melanoma cells.
- To investigate the role of aminopeptidase N (APN) in melanoma cell invasion.
- To determine if APN is associated with extracellular matrix components and contributes to basement membrane degradation.
Main Methods:
- Affinity purification and microsequencing to identify the cell surface protein.
- In situ expression, indirect immunofluorescence, and Western blotting to assess APN localization.
- In vitro invasion assays using a reconstituted basement membrane (Matrigel).
- Enzyme activity assays to assess APN's ability to degrade extracellular matrix components.
Main Results:
- The identified protein was confirmed to be aminopeptidase N (APN).
- APN was found to be tightly associated with extracellular matrix components.
- Inhibition of APN activity with specific antiserum or competitive inhibitors significantly reduced melanoma cell invasion through Matrigel.
- Purified APN degraded a M(r) 160,000 protein within Matrigel.
Conclusions:
- Aminopeptidase N (APN) is expressed on melanoma cells and plays a significant role in their invasive capabilities.
- APN's association with the extracellular matrix and its enzymatic activity facilitate melanoma cell invasion of basement membranes.
- Targeting APN may represent a novel therapeutic strategy for inhibiting melanoma metastasis.