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Regulation and processing of a precursor form of eosinophil granule major basic protein (ProMBP) in differentiating
P Popken-Harris1, J Checkel, D Loegering
1Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA.
Abstract:
The cDNA for eosinophil granule major basic protein (MBP) encodes a prepromolecule with a total length of 222 amino acids (preproMBP). PreproMBP includes a secretory leader of 15 amino acids, an acidic propiece of 90 amino acids, and a basic MBP portion of 117 amino acids. The function of the propiece, which has a predicted pI of 3.9, is unknown, but it gives proMBP an overall acidic charge. Because proMBP is not found in mature eosinophils, we analyzed eosinophil differentiation in interleukin-5 (IL-5)-stimulated umbilical cord stem cells cultured for 24 days. By immunofluorescence, proMBP appeared by day 6 and peaked on day 18, whereas MBP was prominent at days 12 to 24. By day 6, Western blots detected heterogeneous glycosylated 33-kD proMBP; its peak expression occurred on day 12. Western blots showed sequential processing of 33-kD proMBP to an 18-kD intermediate form and finally to 14-kD MBP. By dual label immunoelectron microscopy, proMBP was localized primarily to large uncondensed eosinophil granules, whereas MBP was localized to granules containing a condensed central area. Thus, proMBP is likely expressed and processed as the granule condenses in a multistep process to 14-kD MBP in differentiating eosinophils.
Insights
Eosinophil differentiation involves the processing of eosinophil granule major basic protein (MBP) precursor (proMBP). ProMBP is expressed and processed through intermediate forms into mature MBP within developing eosinophils.
Area of Science:
- Cell Biology
- Immunology
- Protein Biochemistry
Background:
- Eosinophil granule major basic protein (MBP) is a key effector molecule.
- The precursor form, proMBP, and its processing are not fully understood.
- ProMBP's function and presence in mature eosinophils remain unclear.
Purpose of the Study:
- To investigate the expression and processing of eosinophil granule major basic protein (MBP) during eosinophil differentiation.
- To elucidate the role of the proMBP precursor in eosinophil granule formation.
Main Methods:
- Culture of human umbilical cord stem cells stimulated with interleukin-5 (IL-5).
- Immunofluorescence and Western blot analysis to detect proMBP and MBP.
- Dual-label immunoelectron microscopy to localize proteins within eosinophil granules.
Main Results:
- ProMBP expression begins by day 6 and peaks on day 18 in differentiating eosinophils.
- Sequential processing of 33-kD proMBP to intermediate forms and finally to 14-kD MBP was observed.
- ProMBP localizes to uncondensed granules, while mature MBP is found in condensed granule centers.
Conclusions:
- ProMBP is expressed and processed in a multistep manner during eosinophil differentiation.
- Granule condensation is associated with the processing of proMBP to mature MBP.
- This study clarifies the developmental pathway of eosinophil granule proteins.