A panel of 11 region-specific radioimmunoassays for measurements of human chromogranin A

INTRODUCTION: The primary structure of human chromogranin A (CgA) not only contains 10 pairs of basic amino acids, which are potential cleavage sites for specific endogenous proteases, but also other sites in the molecule can be subjected to cleavage. Several CgA-related peptides have been identified in tissue, and many of the biological effects attributed to CgA seem to be mediated by these peptides. MATERIALS AND METHODS: Peptides homologous to defined parts of the human CgA molecule were selected and synthesised. Antibodies were raised, and 11 specific radioimmunoassays were developed. Plasma samples from 20 patients with neuroendocrine tumours were collected and measured in all assays. RESULTS: All assays measured circulating levels of CgA-derived peptides. Only four of the assays measured concentrations that correlated with that of total CgA. However, concentrations of the individual CgA-related peptides were generally lower than the concentration of total CgA. Different neuroendocrine tumours seem to process CgA differently. The ratio between a given region-specific assay and total CgA is inversely correlated to tumour activity. CONCLUSION: The assays presented allow measurements of defined regions of CgA and will thus become important tools for further studies of processing of CgA.     

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.     

Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours

INTRODUCTION: Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. MATERIALS AND METHODS: Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. RESULTS: Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154-165 (N-terminal secretoneurin), the SgII 172-186 (C-terminal Secretoneurin) and the SgII 225-242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. CONCLUSION: Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.     

Proteolytic processing of chromogranin A by the prohormone convertase PC2

The neuroendocrine secretory protein chromogranin A (CgA) is a precursor for various biologically active peptides. Several single and paired basic residues are present within its primary amino acid sequence comprising cleavage sites for prohormone convertases. In this study, SH-SY5Y human neuroblastoma cells were stably transfected with the prohormone convertase PC2 to analyse the proteolytic processing of endogenous chromogranin A and, in particular, the formation of the chromogranin-A-derived peptide GE-25. Our analyses revealed a significant change in the pattern of proteolytic conversion of chromogranin A in cells expressing PC2. Mock-transfected control cells contained mainly the intact chromogranin A molecule and hardly any shorter products were found. On the other hand, PC2-transfected cells showed extensive processing of chromogranin A, resulting in significantly lower amounts of the intact precursor and especially high levels of the free peptide GE-25.     

Region-specific antibodies to chromogranin B display various immunostaining patterns in human endocrine pancreas.

Chromogranin (Cg) B is an acidic glycoprotein present in neuroendocrine tissue. The sequence shows several dibasic amino acid positions susceptible to proteolytic cleavage. The purpose of this study was to elucidate the expression of CgB epitopes in the human endocrine pancreas. Tissue sections of six human pancreata were immunostained with 16 different region-specific antibodies to the CgB molecule, using double immunofluorescence techniques. The CgB epitope pattern varied in the four major islet cell types. B (insulin)-cells expressed immunoreactivity to all region-specific antibodies. The antibodies to the N-terminal and mid-portions of CgB showed moderate immunoreactivity, the C-terminal antibodies weak. A (glucagon)-cells were reactive only to the N-terminal and mid-portion antibodies but, after microwave pretreatment, to all antibodies, whereas D (somatostatin)-cells expressed only the sequence CgB 244-255 and a subpopulation CgB 580-595. PP (pancreatic polypeptide) cells were immunostained with antibodies between CgB 1-417 and a few with CgB 580-593. The fragment CgB 244-255 was expressed in all four cell types. The cause of these differences may be cell-specific cleavage or masking of the molecule, but varying translation of CgB mRNA is also possible. The extent to which these epitopes reflect fragments having biological functions remains to be evaluated.     

Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells.

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.     

Measurements of chromogranin B can serve as a complement to chromogranin A

OBJECTIVE: CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. METHODS: Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours (N=86), patients with decreased renal function (N=35) and patients on treatment with proton pump inhibitors (N=29). RESULTS: The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. CONCLUSIONS: We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.     

Fragmentation of bovine chromogranin A by plasma kallikrein

Chromogranin A has been reported to be processed in vivo by an as yet undefined proteinase(s) suggesting that it is a precursor of biologically active peptides such as pancreastatin. In this study, plasma kallikrein was used as a model proteinase to identify the cleavage sites exposed in bovine parathyroid chromogranin A. Purified bovine parathyroid chromogranin A was digested with human plasma kallikrein. The proteolytic fragments produced were isolated by HPLC and chemically characterized by amino acid composition and sequence analysis. The combined results indicate that the enzyme has preference for specific single Arg residues, cutting C-terminal to this amino acid, although certain pairs of basic sites were also cleaved. The characterized fragments were released in a selective manner from the whole molecule with rapid production of the fragments covering positions 1-247 and 352-358.     

The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects

The chromogranins A (CgA) and B (CgB) and secretogranin II (SgII) constitute the main members of a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These genetically distinct proteins, CgA, CgB, SgII and the less well known secretogranins III-VII are collectively referred to as 'granins' and characterised by numerous pairs of basic amino acids as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC 1/3 and PC2. This review is directed towards comparative and functional aspects of the granins with emphasis on their phylogenetically conserved sequences. Recent developments provide ample evidence of widely different effects and targets for the intact granins and their derived peptides, intracellularly in the directed trafficking of storage components during granule maturation and extracellularly in autocrine, paracrine and endocrine interactions. Most of the effects assigned to the granin derived peptides fit into patterns of direct or indirect inhibitory modulations of major functions. So far, peptides derived from CgA (vasostatins, chromacin, pancreastatin, WE-14, catestatin and parastatin), CgB (secretolytin) and SgII (secretoneurin) are the most likely candidates for granin-derived regulatory peptides, of postulated relevance not only for homeostatic processes, but also for tissue assembly and repair, inflammatory responses and the first line of defence against invading microorganisms.     

The primary structure of human chromogranin A and pancreastatin

A full-length clone encoding human chromogranin A has been isolated from a lambda gt10 cDNA library of a human pheochromocytoma. The nucleotide sequence reveals that human chromogranin A is a 439-residue protein preceded by an 18-residue signal peptide. Comparison of the protein sequence of human chromogranin A with that of bovine chromogranin A shows high conservation of the NH2-terminal and COOH-terminal domains as well as the potential dibasic cleavage sites, whereas the middle portion shows remarkable sequence variation (36%). This part of human chromogranin A contains a sequence homologous to porcine pancreastatin at residues 250-301. The sequence variation in this part of human chromogranin A compared to porcine pancreastatin is 32% and thus of the same magnitude as that between human and bovine chromogranin A. Therefore, the difference between porcine pancreastatin and the corresponding portions of bovine or human chromogranin A can be explained by species variation, suggesting that pancreastatin is derived from chromogranin A itself rather than a protein that is only similar to chromogranin A. Moreover, the pancreastatin sequence contained in human chromogranin A is flanked by sites for proteolytic processing. Together, these observations suggest that human chromogranin A may be the precursor for a human pancreastatin molecule and possibly for other, as yet unidentified, biologically active peptides.     

Peptide microarrays for the characterization of antigenic regions of human chromogranin A

Microarraying peptides is a powerful proteomics technique for studying molecular recognition events. Since peptides have small molecular mass, they are not easily accessible when adsorbed onto solid supports. Moreover, peptides can lack a well-defined three-dimensional structure, and therefore a correct orientation is essential to promote the interaction with their target. In this work, we investigated the suitability as a peptide array substrate of a glass slide coated with a copolymer of N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and [3-(methacryloyl-oxy)propyl]trimethoxysilyl. This polymeric surface was used as substrate for peptides in the characterization of linear antigenic sites of human chromogranin A, a useful tissue and serum marker for neuroendocrine tumors and a precursor of many biologically active peptides. The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which linear epitopes are freely exposed despite peptide random orientation. The results reported in this article are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.     

Defective Secretion of Islet Hormones in Chromogranin-B Deficient Mice

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to β-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.     

Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues

During the course of reversed-phase high-pressure liquid chromatography (RP-HPLC) purification of the 7B2 peptide originally isolated in our laboratory from human pituitary gland extracts, two novel peptides were identified and purified to homogeneity. The complete amino acid sequence of the first one was established in 1985 and recently found to be entirely homologous to positions 420-493 of the just published chromogranin B sequence. This peptide, denoted GAWK, could originate from chromogranin B following specific cleavage at the basic amino acids flanking both termini of GAWK. Moreover, another peptide isolated in our laboratory from the same source and denoted CCB has been discovered and its sequence is also part of the same chromogranin B molecule. Here again, this peptide, occupying positions 597-653 and located at the COOH-terminal region of chromogranin B, could derive from specific processing at basic amino acids, Arg-Lys-Lys, present at positions 594-596. In a manner reminiscent of the relationship between pancreastatin and chromogranin A, it is proposed that both GAWK and CCB are produced from chromogranin B after specific processing at basic amino acids. These data are thus in favor of a putative role of chromogranins as precursors to potentially bioactive peptides.     

Immunohistochemical and Biochemical Studies with Region-Specific Antibodies to Chromogranins A and B and Secretogranins II and III in Neuroendocrine Tumors

This short review deals with our investigations in neuroendocrine tumors (NETs) with antibodies against defined epitopes of chromogranins (Cgs) A and B and secretogranins (Sgs) II and III. The immunohistochemical expression of different epitopes of the granin family of proteins varies in NE cells in normal human endocrine and non-endocrine organs and in NETs, suggesting post-translational processing. In most NETs one or more epitopes of the granins were lacking, but variations in the expression pattern occurred both in benign and malignant NETs. A few epitopes displayed patterns that may be valuable in differentiating between benign and malignant NET types, e.g., well-differentiated NET types expressed more CgA epitopes than the poorly differentiated ones and C-terminal secretoneurin visualized a cell type related to malignancy in pheochromocytomas. Plasma concentrations of different epitopes of CgA and CgB varied. In patients suffering from carcinoid tumors or endocrine pancreatic tumors the highest concentrations were found with epitopes from the mid-portion of CgA. For CgB the highest plasma concentrations were recorded for the epitope 439–451. Measurements of SgII showed that patients with endocrine pancreatic tumors had higher concentrations than patients with carcinoid tumors or pheochromocytomas. SgIII was not detectable in patients with NETs.     

2',3'-Dialdehydo-UDP-N-acetylglucosamine inhibits UDP-N-acetylglucosamine 2-epimerase,the key enzyme of sialic acid biosynthesis

Chromogranin B (CgB) is a member of the granin family of neuroendocrine secretory proteins, which has been proposed to play a role in secretory granule biogenesis and as a precursor to bioactive peptides. The cloning of CgB in a phylogenetically distant vertebrate, the frog Rana ridibunda, reveals a modest overall homology (35-40%) with mammalian CgB. However, the sequences of the N- and C-terminal regions are more highly conserved (57-65% amino acid identity) and may give rise to novel regulatory peptides. In frog, intense expression of CgB mRNA was observed in particular structures of the brain and in the distal lobe of the pituitary.     

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

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.     

The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules

The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.     

The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera

Summary The distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELATE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.     

Do neuroendocrine cells in human prostate cancer express androgen receptor?

The presence of androgen receptors (AR) in neuroendocrine cells was investigated in benign tissue of 10 prostatectomy specimens, in 12 prostatic adenocarcinomas with focal neuroendocrine differentiation and in 1 case of a pure neuroendocrine small cell carcinoma of the prostate. Neuroendocrine cells were defined by their reactivity with an antibody to chromogranin A. Monoclonal antibody F39.4 directed against the amino-terminal domain of the AR molecule was used to detect AR. AR and chromogranin A were simultaneously visualized with a double immunofluorescence technique. The results indicate that chromogranin positive cells in both benign and malignant prostatic tissue lack detectable expression of AR. No effect of endocrine therapy was noted. These results are in agreement with the hypothesis that prostatic neuroendocrine tumour cells represent an androgen insensitive cell population, which incidentally may expand to replace the androgen-sensitive tumour cell population during androgen ablation therapy.     

The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera.

The distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELTAE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.