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Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein
M McGrogan1, C Simonsen, R Scott
1Invitron Corporation, Redwood City, California 94063.
Abstract:
A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2-terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2-terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic.
Insights
Human neutrophils contain a 14-kD protein identical to eosinophil major basic protein (MBP). This study characterizes its cDNA, revealing a 23-kD precursor (pro-MBP) that yields the mature, bipolar MBP.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Human neutrophils (PMNs) possess a 14-kD protein.
- This protein's N-terminal sequence is identical to eosinophil major basic protein (MBP).
Purpose of the Study:
- To characterize the human neutrophil 14-kD protein, identified as MBP.
- To elucidate the structural and functional properties of MBP.
Main Methods:
- Purification of the 14-kD protein from human PMNs.
- N-terminal sequencing of the purified protein.
- Isolation of cDNA clones from an HL-60 cell library.
- Northern blot analysis of HL-60 cells.
- Sequence analysis of full-length cDNA clones.
Main Results:
- The 14-kD neutrophil protein is identical to eosinophil MBP.
- MBP is constitutively expressed and highly transcribed in HL-60 cells from a single-copy gene.
- Full-length cDNA predicts a 23-kD precursor (pro-MBP) cleaved to mature 14-kD MBP.
- Pro-MBP is a bipolar molecule with distinct hydrophilic (N-terminal) and hydrophobic (C-terminal) halves.
Conclusions:
- Human neutrophils express MBP, previously thought to be specific to eosinophils.
- The precursor form, pro-MBP, exhibits a unique bipolar structure.
- Understanding MBP's structure provides insights into its function in inflammatory responses.