Interaction of calcium with porcine adrenal chromogranin A (secretory protein-I) and chromogranin B (secretogranin I)

Secretory granules of endocrine cells contain one or more of the acidic secretory proteins chromogranin A (secretory protein-I), chromogranin B (secretogranin I), and secretogranin II (chromogranin C). It has been proposed that these proteins play a role in the packaging of secretory products. In the present study, lysates of purified porcine adrenal chromaffin granules containing chromogranins A and B and a putative chromogranin B fragment bound calcium and formed aggregates in the presence of 10–20 mM calcium at pH 5–6 and at 100 mM or less KCl, NaCl, or norepinephrine. The precipitates contained virtually all of the chromogranin B and the chromogranin B fragment and about one-third of the chromogranin A. The aggregates did not form or were dissociated at the pH and salt concentration of the extracellular fluid. Calcium precipitated purified chromogranin A and chromogranin B from pure solution to the same extent as from the granule lysates. Parathormone, added to the lysates, was incorporated in the precipitates, whereas the acidic secretory protein ovalbumin and norepinephrine were not. These findings suggest that secretory protein-I and secretogranin can exist in situ as aggregates that may include selected secretory products.

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An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

The Journal of Cell Biology ◽

10.1083/jcb.109.1.17 ◽

1989 ◽

Vol 109 (1) ◽

pp. 17-34 ◽

Cited By ~ 73

Author(s):

P Rosa ◽

U Weiss ◽

R Pepperkok ◽

W Ansorge ◽

C Niehrs ◽

...

Keyword(s):

Secretory Pathway ◽

Secretory Granules ◽

Intracellular Localization ◽

Secretory Protein ◽

Secretory Proteins ◽

Chromogranin B ◽

Double Labeling ◽

Protein Protein Interaction ◽

And Function ◽

Secretogranin I

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

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In vitro aggregation of the regulated secretory protein chromogranin A

Biochemical Journal ◽

10.1042/bj20021195 ◽

2002 ◽

Vol 368 (2) ◽

pp. 605-610 ◽

Cited By ~ 15

Author(s):

Renu K. JAIN ◽

Wen Tzu CHANG ◽

Chitta GEETHA ◽

Paul B.M. JOYCE ◽

Sven-Ulrik GORR

Keyword(s):

Chromogranin A ◽

Hydrophobic Interactions ◽

Divalent Cations ◽

Secretory Granules ◽

Nucleation Site ◽

Secretory Protein ◽

Secretory Proteins ◽

Triton X ◽

Induced Aggregation

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.

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Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice

The EMBO Journal ◽

10.1093/emboj/17.12.3277 ◽

1998 ◽

Vol 17 (12) ◽

pp. 3277-3289 ◽

Cited By ~ 19

Author(s):

S. Natori

Keyword(s):

Transgenic Mice ◽

Secretory Granules ◽

Secretory Protein ◽

Chromogranin B ◽

Secretogranin I

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Sorting and storage during secretory granule biogenesis: looking backward and looking forward

Biochemical Journal ◽

10.1042/bj3320593 ◽

1998 ◽

Vol 332 (3) ◽

pp. 593-610 ◽

Cited By ~ 363

Author(s):

Peter ARVAN ◽

David CASTLE

Keyword(s):

Secretory Pathway ◽

Molecular Mechanisms ◽

Secretory Granules ◽

Secretory Proteins ◽

Membrane Composition ◽

Granule Formation ◽

Granule Membrane ◽

Regulated Secretory Pathway ◽

Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

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Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

10.21203/rs.3.rs-90987/v1 ◽

2020 ◽

Author(s):

Jae Myoung Suh ◽

Kwang-eun Kim ◽

Isaac Park ◽

Jeesoo Kim ◽

Myeong-Gyun Kang ◽

...

Keyword(s):

Blood Plasma ◽

Mouse Liver ◽

Secretory Pathway ◽

Secretory Protein ◽

Protein Labeling ◽

Secretory Proteins ◽

Tissue Specific ◽

Dynamic Tracking

Abstract Here we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which labels secretory pathway proteins as they transit through the ER-lumen to enable dynamic tracking of tissue-specific secreted proteomes in vivo. We expressed iSLET in the mouse liver and demonstrated efficient in situ labeling of the liver-specific secreted proteome which could be tracked and identified within circulating blood plasma. iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

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Targeted Ablation of the Chromogranin A (Chga) Gene: Normal Neuroendocrine Dense-Core Secretory Granules and Increased Expression of Other Granins

Molecular Endocrinology ◽

10.1210/me.2005-0398 ◽

2006 ◽

Vol 20 (8) ◽

pp. 1935-1947 ◽

Cited By ~ 57

Author(s):

Geoffrey N. Hendy ◽

Tong Li ◽

Martine Girard ◽

Richard C. Feldstein ◽

Shree Mulay ◽

...

Keyword(s):

Secretory Granule ◽

Chromogranin A ◽

Chromaffin Cells ◽

Secretory Granules ◽

Hormone Secretion ◽

Proteolytic Processing ◽

Dense Core ◽

Secretory Process ◽

Chromogranin B ◽

Developmental Abnormalities

Abstract Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

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Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

The Journal of Cell Biology ◽

10.1083/jcb.140.6.1331 ◽

1998 ◽

Vol 140 (6) ◽

pp. 1331-1346 ◽

Cited By ~ 75

Author(s):

Andreas Krömer ◽

Michael M. Glombik ◽

Wieland B. Huttner ◽

Hans-Hermann Gerdes

Keyword(s):

Deletion Mutant ◽

Secretory Granules ◽

Full Length ◽

Structural Motif ◽

Secretory Proteins ◽

Sequence Motifs ◽

Chromogranin B ◽

Recombinant Vaccinia ◽

Vaccinia Viruses

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.

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Expression of Regulated Secretory Proteins Is Sufficient to Generate Granule-like Structures in Constitutively Secreting Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m310613200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 20242-20249 ◽

Cited By ~ 83

Author(s):

Nicole Beuret ◽

Hansruedi Stettler ◽

Anja Renold ◽

Jonas Rutishauser ◽

Martin Spiess

Keyword(s):

Protease Inhibitor ◽

Secretory Granules ◽

Secretory Proteins ◽

Ionophore A23187 ◽

Cell Periphery ◽

Chromogranin B ◽

Granule Formation ◽

Trans Golgi Network ◽

Secretogranin Ii ◽

Golgi Network

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein α1-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30–70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas α1-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.

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