Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan

The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein, much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000-100,000; pI 4.3-5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.     

Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence

A method is presented for the purification of human chromogranin from adrenal glands obtained at autopsy. The procedure involved homogenization of whole glands in aqueous buffer, salt precipitation, affinity chromatography using a highly specific monoclonal antibody (LK2H10) and reverse-phase high-pressure liquid chromatography. Chromogranin purified from autopsy adrenal glands revealed a high degree of polypeptide heterogeneity when analyzed by silver-stained SDS polyacrylamide gels. Greater than 90% of the protein was represented by a cluster of polypeptides with an Mr = 70 000 (i.e. chromogranin A), while the remaining protein was highly disperse in molecular weight. That these various polypeptides were in fact chromogranin was shown by Western blotting using monoclonal antibody LK2H10. About 6 nmol of chromogranin were obtained from 97 g of starting adrenals which was estimated to be a 25% yield and a 250-fold enrichment from adrenal homogenates. Critical to achieving reasonable yields of this protein was the need for particular low pH buffers for resuspension of chromogranin after solvent removal steps. Chromogranin purified from human adrenal glands was similar in amino acid composition, and identical in the N-terminal amino acid sequence (24 residues) to bovine chromogranin A. A secondary sequence representing 25% of the total protein and missing the first three residues of the N-terminus suggested the possibility of N-terminal processing of chromogranin in situ. The conservation of the N-terminal amino acid sequence of human and bovine chromogranin contrasts with the strong sequence variability predicted by antisera cross-reactivity and suggests that the N-terminus of chromogranin may be critical for its biological activity.     

Structural characterization of the proteins encoded by adenovirus early region 2A

Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.     

The isolation and characterization of the methyl acceptor protein from adrenal chromaffin granules

A methyl acceptor protein (MAP), which serves as a substrate for adrenal medullary protein carboxymethylase (PCM, E.C. 2.1.1.24), has been isolated from a hypotonic lysate of adrenal chromaffin granules. The isolated MAP was shown to be distinct from the adrenal chromaffin granule protein, dopamine β-hydroxylase (DBH). The properties of MAP, including its amino acid composition, were comparable to those reported for chromogranin A, a major acidic protein found in adrenal chromaffin granules.     

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion.     

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.     

Chromogranin, an integral membrane protein

Chromogranin is the major soluble protein of the adrenal medulla chromaffin granule and is secreted upon nervous stimulation. Using antisera to pure chromogranin in immunoblotting procedures, we show that chromogranin is the major integral membrane protein as well. Extraction of chromaffin granule membranes with low salt, high salt, chelating agents, or calcium-containing solutions does not remove the chromogranin from the membranes. The membrane form of chromogranin can be purified on a C-18 semi-preparative column using high pressure liquid chromatography. Amino-terminal sequence data indicate that the membrane and soluble forms of chromogranin are identical or very similar.     

Peptide Mapping Studies of the Chromogranins and of Two Chromaffin Granule Proteoglycans

Abstract: The chromogranins, a family of related acidic glycoproteins, and two chondroitin sulfate/dermatan sulfate proteoglycans were isolated from the soluble contents of bovine adrenal chromaffin granules by chromatography on DEAE-cellulose. These chromaffin granule matrix glycoconjugates were treated with trypsin, and the resulting peptides were fractionated by HPLC. The two proteoglycans, which differ in their concentration of glycosaminogly-cans and glycoprotein oligosaccharides, yielded almost identical peptide patterns and would both appear to have the same protein moiety. The peptide profile of the proteoglycans differs, however, from that of the chromogranins, which they closely resemble in terms of amino acid composition. The various chromogranin fractions obtained by gel filtration were also found to have significant differences in the chromatographic patterns of their tryptic peptides.     

Different processing of chromogranin B into GAWK-immunoreactive fragments in the bovine adrenal medulla and pituitary gland

Chromogranin B 420-493 (GAWK)-like immunoreactivity (chromogranin B (420-493)-LI) was determined by radioimmunoassay using two different rabbit antisera, one raised against chromogranin B (420-436) (GAWK 1-17) (Ab420-436) and the other against chromogranin B 439-457 (GAWK 20-38) (Ab439-457), in bovine and human tissues. Chromogranin B (420-493)-LI was present in the bovine adrenal medulla chromaffin granules as well as in the anterior pituitary gland and was released from the cultured bovine chromaffin cells by stimulation with high K+ or nicotine. Chromogranin B (420-493)-LI present in the bovine tissues was detected using Ab420-436 but was not detected using Ab439-457. In the human tissues, chromogranin B (420-493)-LI was detected using Ab420-436 as well as Ab439-457. This suggests that the amino acid sequence of this region (chromogranin B 439-457) is different between human and bovine. On the gel permeation chromatography, chromogranin B (420-493)-LI was eluted at the void volume in the bovine adrenal medulla and at an apparent molecular weight of 4000 in the anterior pituitary gland. On the reverse-phase high-performance liquid chromatography, multiple peaks of chromogranin B (420-493)-LI was detected in the bovine adrenal medulla while one component of chromogranin B (420-493)-LI was found in the anterior pituitary gland. These results suggest that chromogranin B is processed into small fragments of chromogranin B (420-493)-LIs and that this processing is tissue-specific.     

beta-Granins: 21 kDa co-secreted peptides of the insulin granule closely related to adrenal medullary chromogranin A

Three closely related forms of a 21 kDa protein which is co-secreted with insulin have been purified and analysed. These differed in behaviour on ion-exchange chromatography but were indistinguishable by their susceptibility to staphylococcal V8 proteinase digestion, amino acid composition or N-terminal amino acid sequence. Their amino acid composition and N-terminal sequences were remarkably similar to adrenal medullary chromogranin A, a much larger protein (72 kDa). Antibodies to chromogranin A also reacted strongly with the 21 kDa protein in isolated insulin granules. It is concluded that the 21 kDa proteins either represent a repeated domain within the chromogranin molecule or a closely related gene product. The name beta-granin is proposed for these proteins.     

Immunoelectron microscopic localization of dopamine beta-hydroxylase and chromogranin A in adrenomedullary chromaffin cells

Dopamine beta-hydroxylase (DBH) was purified from bovine adrenal medullae. Rabbit IgG raised against DBH inhibited its activity by 80%. In an immunoblot analysis, the IgG specifically recognized two subunits of DBH the 72 and 75 KD components. Chromogranin A (CGA) also was purified from bovine adrenal medullae, and rabbit IgG against CGA recognized this chromogranin A in the immunoblot analysis. The intracellular distribution of DBH and CGA in bovine chromaffin cells was determined quantitatively by immunoelectron microscopy using post-embedding protein A-gold technique. DBH and CGA were localized exclusively on chromaffin granules. The binding of gold particles to these granules was saturable. The maximum number of gold particles bound to the granules roughly corresponded to the number of DBH or CGA molecules in the granules estimated biochemically. DBH was observed evenly in the periphery and in the dense matrix of the chromaffin granules.     

Neurons and neuroendocrine cells contain chromogranin: detection of the molecule in normal bovine tissues by immunochemical and immunohistochemical methods

Gel-eluted bovine chromogranin (CG), the 75,000 dalton acidic protein abundantly present in adrenal chromaffin granules, was used as immunogen to prepare anti-CG serum. The specificity of the antiserum was demonstrated in immunoblots of electrophoresed bovine CG and in immunohistochemical studies of bovine adrenal medulla. In the immunoblots, the predominant immunoreactive band had a molecular weight of 75,000 daltons. Bands with a higher or lower molecular weight were also immunoreactive and may represent CG precursors or breakdown products. In the adrenal gland, only adrenal chromaffin cells contained CG immunoreactivity. Immunoblots and immunohistochemistry were also used to characterize the distribution of CG in bovine tissues. CG was expressed by cells of the diffuse neuroendocrine system (DNS) including: adrenal chromaffin cells, enterochromaffin cells, pancreatic islet cells, cells of the adenohypophysis, thyroid C cells, parathyroid cells, and submandibular gland. CG was also seen in four locations not previously recognized to express this antigen: thymic epithelial cells, neurons, the inner segment of rods and cones, and the submandibular gland. We demonstrate a wider distribution of CG than previously recognized and that the molecule detected in tissue by immunohistochemistry is indeed CG. We conclude that CG is expressed by neurons, cells of the DNS, and by a few other cells that may or may not be related to the DNS. The antiserum described here should prove valuable in developing an understanding of the function(s) of CG.     

Proteolytic processing of chromogranin A in cultured chromaffin cells

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.     

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.     

Co-localization of chromogranin A and B,secretogranin II and neuropeptide Y in chromaffin granules of rat adrenal medulla studied by electron microscopic immunocytochemistry

Summary The co-localization of various antigens in rat chromaffin granules was investigated by the immunogold staining procedure. In ultrathin serial sections staining of chromaffin granules was obtained with antisera against chromogranin A, chromogranin B, secretogranin II and neuropeptide Y. These results indicated that these antigens are costored within chromaffin granules. To further corroborate this point a double immunogold staining procedure was used. This method unequivocally established that chromogranin A, chromogranin B, secretogranin II and neuropeptide Y are co-localized in the same chromaffin granules. These results are relevant for studies demonstrating changes in the level of these peptides in adrenal medulla. The co-localization makes it likely that such changes lead to a different relative composition of the secretory quanta of chromaffin granules.     

Analysis of the carbon-13 and proton NMR spectra of bovine chromaffin granules

Natural abundance carbon-13 and proton NMR spectra of bovine chromaffin granules have been obtained and analyzed using computer simulation techniques. High resolution spectra show the presence of a fluid aqueous phase containing epinephrine, ATP and a random coil protein. The protein spectrum contains unusually intense resonances due to glutamic acid and proline and has been simulated satisfactorily using the known amino acid composition of chromogranin A.The lipid phase of chromaffin granules gives rise to intense, but very broad, resonances in the carbon-13 spectrum. Protons in the liquid phase are also observable as a very rapid component of the proton-free induction decay ($\text{T}{}_2\text{? 15 μ}\text{s}$).Linewidths of the carbon-13 spectra have been used to set upper limits on rotational correlation times and on the motional anisotropy in the aqueous phase. These limits show that the aqueous phase is a simple solution (not a gel) that is isotropic over regions much larger than solute dimensions. No gel transition is observed between ?3 and 25°C. The carbon-13 spectra are definitely inconsistent with a lipoprotein matrix model and chromaffin granules previously proposed by Helle and Serck-Hanssen ((1975) Mol. Cell. Biochem. 6, 127–146). Relative carbon-13 intensities of ATP and epinephrine are not consistent with the known 1:4 mol ration of these components. This fact suggests that epinephrine and ATP are not directly complexed in intact chromaffin granules.     

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.