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H H Gerdes

Publications and source records attributed to H H Gerdes.

30 records · Page 2Linked to original sources
PubMed1996

Identification of a functional cAMP response element in the secretogranin II gene.

Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis-acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to beta-globin as a reporter was constructed. Transfection analysis revealed that cAMP-induced transcription of the secretogranin II promoter/beta-globin gene in septal and insulinoma cells. DNA-protein binding assays showed that recombinant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4-CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as cAMP-stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB-2, a negative-acting CRE-binding protein. These results indicate that competitive effects are likely to occur between CRE-bound transcriptional activators and repressors. We conclude that cAMP-stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell-type-specific manner, and is likely to depend on the balance between positive and negative CRE-binding proteins in a particular cell type.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Visualization of protein transport along the secretory pathway using green fluorescent protein.

We have expressed green fluorescent protein (GFP) from A. victoria in the secretory pathway of HeLa cells by fusing it to the C-terminus of a secretory protein, chromogranin B. Under normal culture conditions at 37 degrees C maturation of GFP to the fluorescent form was not detectable. However, fluorescent GFP was observed when biosynthetic protein transport was arrested at the intermediate compartment or the trans-Golgi network by temperature blocks (15 degrees C and 20 degrees C, respectively). Reversal of the temperature blocks allowed the visualization of secretion of fluorescent GFP and offers the possibility to analyse transport in the secretory pathway in living cells.

Amino Acid Sequence↗
PubMed1994

Differential regulation of chromogranin B and synapsin I gene promoter activity by cAMP and cAMP-dependent protein kinase.

cAMP has neutrotrophic effects in the nervous system. We have investigated whether there is a correlation between cAMP-induced neurite outgrowth and induction of chromogranin B and synapsin I gene expression. These genes encode marker proteins of distinct populations of vesicles in neurons, neuroendocrine and endocrine cells, and in addition, they contain a cAMP response element (CRE) in their upstream regions, making it likely that cAMP-induced neuronal differentiation might be accompanied by increased transcription of these genes. We increased intracellular cAMP levels in neuronal and neuroendocrine cells and analyzed the levels of chromogranin B and synapsin I mRNA. Our data revealed that, while chromogranin B mRNA was in fact induced following cAMP stimulation, synapsin I mRNA was not affected. To analyze the cis-acting sequences, we constructed hybrid genes containing the upstream region of the mouse chromogranin B gene fused to a reporter gene. Similar plasmids containing the synapsin I or the glucagon promoter were constructed. Transfections of neuronal and endocrine cells, together with deletion mutagenesis, revealed that the CRE of the chromogranin B gene mediated the effect of cAMP upon transcription. This effect was mimicked by overexpression of the catalytic subunit of the cAMP-dependent protein kinase. In addition, overexpression of the negative-acting CRE-binding protein CREB-2 revealed that the chromogranin B CRE functions as a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Synapsin I gene expression, however, was not induced by either elevated intracellular cAMP concentration or by overexpression of protein kinase A, although a similar pattern of proteins, including CREB, bound to the synapsin I and chromogranin B CRE in vitro. Thus while the CRE element in the chromogranin B gene promoter is responsive to cAMP, the same element, when present in the synapsin I promoter, does not confer cAMP inducibility.

1-Methyl-3-isobutylxanthine↗
PubMed1992

The organisation of the mouse secretogranin II gene.

We have characterized the gene which encodes mouse secretogranin II (previously also referred to as chromogranin C), a tyrosine-sulfated secretory protein belonging to the granin (chromogranin/secretogranin) family which is found in secretory granules of most endocrine cells and neurons. The secretogranin II gene was found to contain 2 exons. In contrast to chromogranin A and chromogranin B, the two previously characterized granin genes, the entire secretogranin II protein is encoded by a single exon, exon 2, with exon 1 containing only a 5'-untranslated sequence. Consistent with previous data on the expression of secretogranin II, the putative promoter region was found to contain a cAMP-responsive element and a potential AP-1 binding site.

Amino Acid Sequence↗
PubMed1991

The granin (chromogranin/secretogranin) family.

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.

Amino Acid Sequence↗
PubMed1990

The organisation of the mouse chromogranin B (secretogranin I) gene.

A cosmid clone containing the gene for mouse chromogranin B (secretogranin I), a secretory protein found in secretory granules of most endocrine cells and neurons, was isolated and sequenced. The chromogranin B protein was found to be encoded by 5 exons which correspond to the cleaved signal peptide, the short N-terminal sequence preceding the disulfide-bonded loop structure, the disulfide-bonded loop structure itself, the large, variable region comprising approximately 90% of the protein, and the conserved C-terminal sequence. The promoter region of the chromogranin B gene is very GC-rich and contains a CATAA motif, a cAMP-responsive element and an Sp1 binding site.

Amino Acid Sequence↗
PubMed1989

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation.

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.

Amino Acid Sequence↗
PubMed1988

Gene response upon illumination in forming mRNA encoding peroxisomal glycollate oxidase.

Glycollate oxidase is a constituent of leaf peroxisomes. Its biosynthesis is, like the biosynthesis of many chloroplastic proteins, controlled by light, via phytochrome. The level of mRNA coding for glycollate oxidase was determined at different stages of greening of etiolated plant cells. The appearance of glycollate oxidase mRNA in the cytoplasm was measured by hybridization with cDNA containing part of the coding sequence for glycollate oxidase. cDNA was prepared from enriched mRNA, inserted into the Pst I site of pBR 322, and cloned in Escherichia coli DH-1. By differential colony hybridization and hybrid selection, a clone containing a 670 bp sequence complementary to mRNA encoding glycollate oxidase was selected and identified. Northern blot hybridization was used to investigate mRNA levels induced by light. It was found that continuous light affected the formation of glycollate oxidase mRNA. When a large population of microbodies was present in the cells being induced, the immediate mRNA increase was very pronounced, and was detectable as little as 20 min after the beginning of the light treatment. In contrast, a lag period in the mRNA increase was observed when the induction was performed with etiolated leaves which are characterized by the occurrence of a rather small population of microbodies. For comparison, we measured the time-course of formation of mRNA coding for a light-induced chloroplastic protein, i.e., a protein of the light-harvesting complex. The time-courses of levels of the two mRNAs indicate that the program of gene expression differs between the two particular proteins destined either for chloroplasts or for peroxisomes. The formation of glycollate oxidase mRNA could also be stimulated by a short pulse of light, a treatment of 15 s being a sufficient trigger.

Alcohol Oxidoreductases↗
PubMed1987

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.

Amino Acid Sequence↗