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In vitro aggregation of the regulated secretory protein chromogranin A
- Publication Year :
- 2002
-
Abstract
- Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation.
- Subjects :
- Octoxynol
chemistry.chemical_element
Protein aggregation
Calcium
Biochemistry
Divalent
Hydrophobic effect
Epitopes
Cations
Chromogranins
Animals
Histidine
Magnesium
Molecular Biology
Edetic Acid
chemistry.chemical_classification
biology
Chromogranin A
Cell Biology
Alkaline Phosphatase
In vitro
Recombinant Proteins
Zinc
Secretory protein
Spectrometry, Fluorescence
chemistry
Chaperone (protein)
biology.protein
Cattle
Research Article
Molecular Chaperones
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....fef09f7b257035f48a5e16789e722c22