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Identification of protease-sensitive sites in Human Endothelial-Monocyte Activating Polypeptide II protein
1Department of Surgery, Division of Surgical Sciences, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA.
Abstract:
The cleaved approximately 22-kDa form of Endothelial-Monocyte Activating Polypeptide [mature (m)EMAP II] functions as a potent inhibitor of tumor growth. Although the anti-tumor effect of mEMAP II has been described, little is known regarding the cleavage of mEMAP II from its precursor form (pEMAP II). We determined that pEMAP II is expressed at the cell membrane surface and proteinases MMP-9, elastase, and cathepsin L release protein fragments consistent with mEMAP II molecular mass. MMP-9 and elastase generate a approximately 25-26 kDa spanning fragments, while cathepsin L generates a approximately 22 kDa fragment. Although several fragments are processed from pEMAP II within a 44 AA residue stretch, cathepsin L cleaves pEMAP II within 4 amino acids of the determined N-terminal sequence, suggesting that this region is sensitive to proteinases.
Insights
Mature Endothelial-Monocyte Activating Polypeptide (mEMAP II) inhibits tumor growth. Proteinases MMP-9, elastase, and cathepsin L cleave the precursor form (pEMAP II) at the cell membrane, releasing active mEMAP II.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Mature Endothelial-Monocyte Activating Polypeptide (mEMAP II) is a known tumor growth inhibitor.
- The mechanism of mEMAP II release from its precursor (pEMAP II) is not well understood.
Purpose of the Study:
- To investigate the cleavage process of pEMAP II and identify the proteinases involved.
- To characterize the fragments generated during pEMAP II processing.
Main Methods:
- Cell surface expression analysis of pEMAP II.
- Enzymatic cleavage assays using purified proteinases (MMP-9, elastase, cathepsin L).
- Analysis of protein fragment molecular mass.
Main Results:
- pEMAP II is expressed at the cell membrane.
- MMP-9 and elastase generate ~25-26 kDa fragments from pEMAP II.
- Cathepsin L cleaves pEMAP II to produce a ~22 kDa fragment, consistent with mEMAP II.
- Cathepsin L cleavage occurs within 4 amino acids of the N-terminus, indicating a sensitive region.
Conclusions:
- Specific proteinases, including MMP-9, elastase, and cathepsin L, are responsible for processing pEMAP II.
- Cathepsin L efficiently generates the mature, biologically active mEMAP II form.
- The N-terminal region of pEMAP II is a key site for proteinase activity.
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