Cell-free and cellular synthesis of chromogranin A and B of bovine adrenal medulla

doi:10.1016/0306-4522(85)90323-9

Neuroscience

Volume 14, Issue 2, February 1985, Pages 735-746

https://doi.org/10.1016/0306-4522(85)90323-9 Get rights and content

Abstract

We have studied the cell-free and cellular synthesis of chromogranins A and B, two immunologically distinct protein families of adrenal chromaffin granules. Two cell-free systems (wheat germ and reticulocyte lysate) were used for translating messenger RNA isolated from bovine adrenal medulla. Two primary translation products could be immunoprecipitated in case of chromogranin A. In the presence of microsomes the two chromogranin A precursors (pre-chromogranins A) were converted into a single protein, apparently by the removal of different signal peptides. For chromogranin B only one precursor (pre-chromogranin B) was translated. In isolated chromaffin cells only one chromogranin A protein was synthesized which corresponded to the processed cell-free translation product. During prolonged incubation this protein became slightly larger and more acidic, probably due to glycosylation in the Golgi region. Chromogranin B is post-translationally converted to a significantly more acidic protein.

It is concluded that proteolytic breakdown of newly synthesized chromogranin A and B in chromaffin granules is a slow process comparable to that of the enkephalin precursors. It is not yet known what function these chromogranins have and whether breakdown to smaller subunits is necessary for any function to evolve.

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Differential regulation of chromogranin A, chromogranin B and secretogranin II in rat brain by phencyclidine treatment
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Chromogranin A, chromogranin B and secretogranin II belong to the chromogranin family which consists of large protein molecules that are found in large dense core vesicles. Chromogranins are endoproteolytically processed to smaller peptides.
This study was designed to elucidate the regulation of chromgranin expression by acute and subchronic phencyclidine administration. The behavioral syndrome produced by phencyclidine represents a pharmacological model for some aspects of schizophrenia [Jentsch and Roth (1999) Neuropsychopharmacology 20, 201–225]. Tissue concentrations of chromogranins were measured with specific radioimmunoassays. Alterations in secretogranin II gene expression were investigated by in situ hybridization. A single dose of phencyclidine (10 mg/kg) led to a transient decrease in secretoneurin tissue levels in the prefrontal cortex after 4 h followed by an increase in secretoneurin tissue levels after 12 h. Repeated phencyclidine treatment (10 mg/kg/day) for five days resulted in elevated secretoneurin levels in cortical areas whereas chromogranin A and chromogranin B tissue levels were unchanged. After the same treatment, a significant increase in the number of secretoneurin containing neurons was found in cortical layers II–III, and V–VI as revealed by immunocytochemistry. The increases in secretoneurin levels were paralleled by an increased number of secretogranin II messenger RNA containing neurons as well as by an increased expression of secretogranin II by individual neurons.
The present study shows that secretoneurin II tissue concentration and secretogranin II messenger RNA expression is distinctly altered after acute and subchronic phencyclidine application. From these results we suggest that phencyclidine may induce synaptic alterations in specific brain areas and may contribute to a better understanding of synaptic dysfunction which may also occur in schizophrenia.
Co-distribution patterns of chromogranin B-like immunoreactivity with chromogranin A and secretoneurin within the human brainstem
2000, Brain Research
Citation Excerpt :
Hypothesized to function as a sorting and processing mechanism prior to exocytosis, these proteins are believed to act as peptide precursors, peptide hormones, and neurotransmitters following secretion [13]. Chromogranin A, chromogranin B, and secretogranin II, which are post-translationally glycosylated, phosphorylated, and sulfated [7], have been identified as suitable markers for normal and neoplastic endocrine cells [11] and neuronal cells. Several peptides derived from chromogranin B have been detected in various tissues [2, 5, 8].
As members of the chromogranin family, chromogranin A, chromogranin B, and secretogranin II are acidic proteins found in large, dense core vesicles. They are endoproteolytically processed to smaller peptides and released after neuronal stimulation. Using immunocytochemistry, this study closely examines chromogranin B-like immunoreactivity within the human brainstem and then takes a comparative view of co-distribution patterns by chromogranin B, chromogranin A, and secretogranin II. We used an antiserum raised against a synthetic peptide (PE-11) present in the chromogranin B molecule. Secretogranin II was localized with an antiserum against secretoneurin, a 33 amino acid peptide, found within the secretogranin II precursor. Like chromogranin A and secretoneurin, chromogranin B is expressed through all levels of the human brainstem. Chromogranin B was exclusively detected in neuronal structures. The medial part of the substantia nigra pars reticulata, the nucleus interpeduncularis, the area of the central gray, and the raphe complex displayed a high density of PE-11-like immunoreactivity. Furthermore, a prominent staining was found in the medial, dorsal and gelatinous subnuclei of the solitary tract and the dorsal motor nucleus of vagus. The substantia gelatinosa of the caudal trigeminal nucleus and spinal cord were also very strongly PE-11-immunopositive. In conclusion, chromogranin B and secretogranin II showed similar distributions while neuronal localization typically differed from chromogranin A aside from a few exceptions. These findings may provide a framework for future research in revealing a functional role of chromogranin peptides in the human brainstem.
Chromogranin A from bovine adrenal medulla: Molecular characterization of glycosylations, phosphorylations, and sequence heterogeneities by mass spectrometry
1999, Analytical Biochemistry
Chromogranin A (CGA) is a member of a family of acidic glycoproteins present in endocrine and neuroendocrine tissues. One of its suggested physiological roles is being a precursor molecule for several peptide hormons. Further interest in this protein has recently originated from its potential role in pathophysiological processes of Alzheimer's disease. The concentration of CGA in the brain has been used for diagnosis of this disease, and CGA as an insoluble deposit has been found in the extracellular β-amyloid plaques. By developing a new purification procedure we were able to isolate abundant CGA in high purity from bovine chromaffin cells. A MALDI-MS analysis of the intact protein revealed a heterogeneous molecular mass of ca. 50 kDa, indicating several structure modifications. By use of several subsequent proteolytic/chemical cleavage steps, HPLC isolation, a newly developed deglycosylation procedure, and several MS and MS-MS fragmentation approaches, the complete primary structure of CGA including four sequence heterogeneities, two O-glycosylations, five phosphorylations, and one disulfide bridge could be characterized. For both glycans six different forms could be identified. Ser167 was found to be mainly glycosylated by a trisaccharide, and Thr231 was found to be mainly glycosylated by a tetrasaccharide. Ser81, Ser124, and Ser297 residues were partially phosphorylated, whereas Ser372 and Ser377 were found completely phosphorylated. Sequence heterogeneities were identified in positions 293 (H/R), 301 (K/E), and 373 (Q/R) and at the partly missing C-terminal residue. Furthermore, a disulfide bridge between Cys17 and Cys38 was ascertained.
Bovine chromaffin cells: Studies on the biosynthesis of phospholipids in chromaffin granules
1994, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
We have studied the biosynthesis of chromaffin granules by labelling primary cultures of bovine chromaffin cells with either [³⁵S]methionine or various precursors for lipids. After labelling the cells were subjected to subcellular fractionation including density gradient centrifugation. After [³⁵S]methionine significant label (mainly represented by labelled chromogranin A) was found in the soluble proteins of chromaffin granules, whereas the membranes were relatively little labelled. However incorporation into membrane bound dopamine β-hydroxylase and cytochrome b-561 could be demonstrated. Neither of the used lipid precursors ([³H]glycerol, [³H]choline, [³H]palmitic acid or [³H]arachidonic acid) was incorporated to any significant extent into the soluble components of chromaffin granules. Thus there is no evidence that this secretory compartment contains any lipids or acylated proteins. Incorporation of lipid precursors into the membranes of chromaffin granules was apparently low. After short chases labelled lysolecithin was not present in these organelles. However with prolonged chase times labelled lysolecithin apparently appeared in chromaffin granules irrespective of whether the cells were stimulated or not. We can conclude that the reusable membranes of chromaffin granules have a very low lipid turnover. Lysolecithin is not transferred into these organelles during biosynthesis but is formed in them during their long life span. This formation of lysolecithin is independent of stimulation of these cells and therefore unlikely to be involved in exocytosis.
Peptides derived from the granins (chromogranins/secretogranins)
1994, Biochimie
Posttranslational processing of proenkephalins and chromogranins/secretogranins
1993, Neurochemistry International
Posttranslational processing of peptide-precursors is nowadays believed to play an important role in the functioning of neurons and endocrine cells. Both proenkephalins and chromogranins/secretogranins are considered as precursor molecules in these tissues, resulting in posttranslationally formed degradation products with potential biological activities.
Among the proteins and peptides of neuronal and endocrine secretory granules, the enkephalins and enkephalin-containing peptides have been most extensively studied. The characterization of the posttranslationally formed degradation products of the proenkephalins have enabled the understanding of their processing pathway.
Chromogranins/secretogranins represent a group of acidic glycoproteins, contained within hormone storage granules. The biochemistry, biogenesis and molecular properties of these proteins have already been studied for 25 years. The chromogranins/secretogranins have a widespread distribution throughout the neuroendocrine system, the adrenal medullary chromaffin granules being the major source of these storage components. Recent data provide evidence for a precursor role for all members of the chromogranins/secretogranins family although also several other functions have been proposed.
In this review, some of the methods applied to study proteolytic processing are described. In addition, the posttranslational processing of chromogranins/secretogranins and proenkephalins, especially the biochemical aspects, will be discussed and compared. Recent exciting developments on the generation and identification of potential physiologically active fragments will be covered.

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Cell-free and cellular synthesis of chromogranin A and B of bovine adrenal medulla

Abstract

Cell

J. biol. Chem.

Analyt. Biochem.

Archs Biochem. Biophys.

J. biol. Chem.

Biochim. biophys. Acta

J. biol. Chem.

J. biol. Chem.

Life Sci.

J. biol. Chem.

J. biol. Chem.

Archs Biochem. Biophys.

Neuroscience

Neuroscience

Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts

Eur. J. Biochem.

Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose

Secretion of a chromaffin granule protein, chromogranin from the adrenal gland after splanchnic stimulation

Nature, Lond.

Transfer of proteins across membranes. I. Presence of proteolytically processed and nonprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

J. Cell Biol.

Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterogenous components

J. Cell Biol.

A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels

Eur. J. Biochem.

Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease

Biochemistry

Similarity of secretory protein I from parathyroid gland to chromogranin A from adrenal medulla

Functional and morphological characterization of isolated bovine adrenal medullary cells

J. Cell Biol.