A Complex Dance: The Importance of Glycosaminoglycans and Zinc in the Aggregation of Human Prolactin.
The zinc binding hormone pituitary human prolactin (hPRL) is stored in secretory granules of specialized cells in an aggregated form. Glycosaminoglycans (GAGs) are anionic polysaccharides commonly associated with secretory granules, indicating their involvement in granule formation. Here we, for the first time, study the impact of GAGs in combination with Zn(2+) on the reversible hPRL aggregation across the pH range of 7.4-5.5. Zn(2+) alone causes hPRL aggregation at pH 7.4, while aggregation between pH 7.4 and 5.5 requires both Zn(2+) and GAGs. GAGs alone cause hPRL aggregation below pH 5.5. Comprehensive thermal stability investigations show that hPRL is particularly destabilized toward thermal denaturation at pH 5.5 and that GAGs increasingly destabilize hPRL at decreasing pH values. We propose that Zn(2+) causes hPRL aggregation through low-affinity Zn(2+) binding sites on hPRL with GAGs facilitating Zn(2+) binding by neutralizing repulsive positive charges of hPRL in the acidic environments of the TGN and mature secretory granules. In a manner independent of the aggregation-causing agent(s), the different hPRL aggregates show very similar secondary structure and amorphous morphology. We speculate that this may be a recognizable sorting signal in the formation of hPRL granular vesicles.
Center for Insoluble Protein Structures, Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, Aarhus University , 8000 Aarhus C, Denmark.
Line Friis Bakmann
LF
Christensen
Center for Insoluble Protein Structures, Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, Aarhus University , 8000 Aarhus C, Denmark.
Kirsten Gade
KG
Malmos
Department of Biomedicine-Medical Microbiology and Immunology, Aarhus University , 8000 Aarhus C, Denmark.
Gunna
G
Christiansen
Center for Insoluble Protein Structures, Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, Aarhus University , 8000 Aarhus C, Denmark.
Daniel Erik
DE
Otzen
D001665:Binding Sites; D002942:Circular Dichroism; D003594:Cytoplasmic Granules / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D011388:Prolactin / Q000737:chemistry* / Q000378:metabolism; D066329:Protein Aggregates; D011485:Protein Binding; D022142:Secretory Vesicles / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D006025:Glycosaminoglycans; D066329:Protein Aggregates; D011388:Prolactin; D015032:Zinc