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A Hille

Publications and source records attributed to A Hille.

At least 55 records · Page 3Linked to original sources
PubMed1990

Assembly of the ligand-binding conformation of Mr 46,000 mannose 6-phosphate-specific receptor takes place before reaching the Golgi complex.

The early steps in the biosynthesis of Mr 46,000 mannose 6-phosphate-specific receptor (MPR 46) have been studied by in vivo labeling of transfected BHK cells. The acquisition of phosphomannan-binding activity was compared with changes in protein structure and posttranslational modifications of MPR 46. Intramolecular disulfide bonds were formed before MPR 46 acquired a ligand-binding conformation. A conformational change that resulted in increased trypsin resistance, formation of highly immunogenic epitopes and assembly to noncovalently linked homodimers was observed almost simultaneously with the acquisition of ligand-binding activity. MPR 46 was shown to acquire ligand-binding activity before N-linked oligosaccharides were processed to complex-type forms. Maturation of the ligand-binding conformation was observed under conditions where transport to the Golgi was blocked by lowering the temperature to 16 degrees C, or by addition of brefeldin A or dinitrophenol to the medium at 37 degrees C. This suggests that receptor maturation and assembly take place before reaching the Golgi complex. The affinity towards phosphomannan-containing ligands was shown to be similar for the high-mannose and complex-glycosylated forms of MPR 46.

Animals↗
PubMed1990

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells.

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.

Animals↗
PubMed1989

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin).

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.

Amino Acid Sequence↗
PubMed1989

The ligand-binding conformation of Mr 46,000 mannose 6-phosphate-specific receptor. Acquisition of binding activity during in vitro synthesis.

Purified Mr 46,000 mannose 6-phosphate-specific receptor (MPR 46) lost its ligand-binding activity after reductive alkylation and after enzymatic deglycosylation. Deglycosylated MPR 46 did not assemble to homodimers. Therefore, we investigated the role of N-glycosylation, intrasubunit disulfide bonds, and subunit assembly for the acquisition of ligand-binding activity during in vitro synthesis of MPR 46. Up to 21% of MPR 46 synthesized in a reticulocyte lysate supplemented with dog pancreas microsomes acquired ligand-binding activity provided that 1-5 mM glutathione was present during translation and during a chase following translation. Acquisition of ligand-binding activity after cotranslational membrane insertion and core glycosylation depended on formation of intrasubunit disulfide bonds and a conformational change. Formation of intrasubunit disulfide bonds was not sufficient for ligand-binding activity and is likely to precede the conformational change, which resulted in increased resistance toward trypsin, formation of highly antigenic epitopes, and association to dimers, concomitant with the acquisition of ligand-binding activity.

Animals↗
PubMed1989

Cloning and expression of human steroid-sulfatase. Membrane topology, glycosylation, and subcellular distribution in BHK-21 cells.

A 2.4-kilobase cDNA clone for human steroid-sulfatase (STS) was isolated and sequenced, which encoded an enzymatically active protein. The deduced amino acid sequence comprises 583 amino acids with an N-terminal signal peptide of 21 or 23 residues and four potential N-glycosylation sites. Two of the N-glycosylation sites are utilized and were localized to the asparagine residues 47 and 259. STS has the solubility properties of an integral membrane protein. The resistance of STS toward proteinase K after translocation into microsomes suggests that most, if not all, sequences of STS are exposed at the luminal side of microsomes. The deduced amino acid sequence predicts two membrane-spanning domains (amino acids 185-211 and 213-237) separated by a helix-breaking proline residue. We propose for STS a three-domain model. Two glycosylated luminally oriented domains of 161 and 346 residues are separated by a hydrophobic domain spanning the membrane twice in opposite directions. STS expressed in BHK-21 cells is located predominantly in the endoplasmic reticulum; smaller fractions are found in the Golgi, at the cell surface, multivesicular endosomes, as well as in lysosomes. The stability of STS in lysosomes may be related to the high homology of the two luminal domains of STS with the lysosomal sulfatases, arylsulfatase A, and arylsulfatase B. In spite of its similarity with these two lysosomal sulfatases, STS does not contain mannose 6-phosphate residues and is transported to lysosomes by a mannose 6-phosphate receptor-independent mechanism.

Amino Acid Sequence↗
PubMed1989

Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.

The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer.

Animals↗
PubMed1989

Immunohistochemical localization of secretogranin II in the rat cerebellum.

Secretogranin II (chromogranin C) is a peptide related to chromogranin A and secretogranin I (chromogranin B) which is secreted by a regulated pathway from both neurons and endocrine cells. In the present study we have determined by light microscopic immunocytochemistry its distribution in the cerebellum and in adjacent brain stem regions. Secretogranin II was found to be widely distributed throughout the gray matter of these regions. Highly immunoreactive structures in the cerebellar cortex included the majority of climbing fibers, a large number of mossy fibers, sparse varicose fibers in the molecular layer and a subpopulation of neuronal perikarya in the granule cell layer. The location and shape of these neurons are very similar to those of a novel type of cerebellar neurons which has been recently described. A moderate level of immunoreactivity was observed on fibers travelling among Purkinje cells and parallel to the pial surface in the Purkinje cell layer. A variable, but in general low, degree of immunoreactivity was also detectable in the perikarya of Purkinje cells. In the deep cerebellar nuclei a loose network of secretogranin II-positive fibers was visible. Neurons of the nuclei, however, were non-immunoreactive. A dense network of highly immunoreactive fibers was found throughout the brain stem regions adjacent to the cerebellum. Our results indicate that secretogranin II has in the cerebellum and adjacent regions a distribution more widespread than that of known regulatory peptides and suggest that the peptide-mediated signaling in the cerebellum plays a role more important that has been acknowledged so far.

Animals↗
PubMed1988

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.

Acid Phosphatase↗
PubMed1988

Identification of gastroenteropancreatic neuroendocrine cells in normal and neoplastic human tissue with antibodies against synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II.

Gastroenteropancreatic human neuroendocrine (NE) cells (normal and neoplastic) were investigated for the expression of the neuroendocrine-specific polypeptides synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II, using immunohistochemistry and immunoblotting. Monoclonal antibody against synaptophysin stained most, and possibly all, of the neuroendocrine cells in both normal and neoplastic tissue. Monoclonal antibody against chromogranin A also stained a high proportion of normal and neoplastic neuroendocrine cells. Immunostaining with polyclonal antisecretogranin I and antisecretogranin II antibodies was detectable in almost all of the normal and neoplastic tissue sections that were analyzed, and it was confined to a smaller population of neuroendocrine cells than that observed for synaptophysin and chromogranin A. Consistent with the immunohistochemical observations, immunoblotting revealed the presence of all four antigens in various tumors. The data show that synaptophysin and chromogranin A, for which monoclonal antibodies are commercially available, may be used as diagnostic markers for human gastroenteropancreatic tumors. Our results also suggest that the development of monoclonal antibodies against human secretogranins I and II will provide additional tools for a refined diagnosis of such tumors.

Antibodies, Neoplasm↗
PubMed1988

Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors.

The distribution of chromogranin A and secretogranin I (chromogranin B) in normal and neoplastic human endocrine tissues was analyzed with two human monoclonal antibodies against chromogranin A, anti-bovine antiserum against chromogranin A, and an anti-rat antiserum against secretogranin I. Western blotting analyses showed both chromogranin A and secretogranin I in normal adrenals, pheochromocytomas, a pituitary adenoma, and in normal pituitary glands, but not in a bladder carcinoma. Rat adrenal medullary and anterior pituitary tissues reacted with the polyclonal chromogranin A and secretogranin I antisera, but not with the two monoclonal chromogranin A antibodies. All antibodies reacted with most of the neuroendocrine cells and tumors examined. Pituitary prolactinomas contained immunoreactive secretogranin I, but not chromogranin A. Analysis of the distribution of chromogranin A and secretogranin I in pancreatic islet cells showed that chromogranin A was found predominantly in the glucagon-producing A cells, whereas secretogranin I was present in less than 5% of islet cells. These results indicate that chromogranin A and secretogranin I are both useful in the characterization of some neuroendocrine cells and neoplasms.

Adrenal Medulla↗
PubMed1987

Sulfated oligosaccharides in human lysosomal enzymes.

Cathepsin D, arylsulfatase A and the alpha-chain of beta-hexosaminidase are synthesized in human fibroblasts as sulfated polypeptides. The sulfate is added posttranslationally. Its half-life is less than one-tenth of that of the respective polypeptide chains. The sulfate residues were found on asparagine-linked oligosaccharides sensitive to endoglycosidase F and peptide: N-glycosidase F and resistant to endoglycosidase H. Inhibition of formation of complex type oligosaccharides by 1-deoxy-manno-nojirimycin prevented sulfation, indicating that the sulfate residues were added to complex type oligosaccharides.

Acetylglucosaminidase↗
PubMed1987

The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region.

We have determined and analyzed the primary structure of human secretogranin I (chromogranin B), a tyrosine-sulfated secretory protein found in a wide variety of peptidergic endocrine cells. A 2.5-kb cDNA clone, hybridizing to an mRNA of similar length, was isolated from a cDNA library of human pheochromocytoma. The identity of the clone was established by comparison of its deduced amino acid sequence with N-terminal and several internal secretogranin I sequences as well as by immunoprecipitation of the protein produced by in vitro transcription-translation of the cloned cDNA. Secretogranin I is a 657 amino acid long polypeptide of 76 kd and is preceded by a cleaved N-terminal signal peptide of 20 residues. Comparison of the predicted amino acid sequence of human secretogranin I with that of bovine chromogranin A reveals significant homologies near the N termini and at the C termini. The N-terminal homologous domains contain the only two cysteine residues of both proteins and form disulfide-stabilized loop structures. The sequences between the homologous terminal domains in both proteins differ but are characterized by a remarkable hydrophilicity, an abundance of acidic amino acids and potential dibasic cleavage sites for the generation of smaller, perhaps hormone-like, peptides.

Adrenal Gland Neoplasms↗
PubMed1985

Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway.

We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pI, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules.

Adrenal Gland Neoplasms↗
PubMed1984

Tyrosine sulfation: a post-translational modification of proteins destined for secretion?

Protein sulfation was studied in germ-free rats by prolonged in vivo labeling with [35S]sulfate. Specific sets of sulfated proteins were observed in all tissues examined, in leucocytes, and in blood plasma. No protein sulfation was detected in erythrocytes. Analysis of the type of sulfate linkage showed that sulfated proteins secreted into the plasma contained predominantly tyrosine sulfate, whereas sulfated proteins found in tissues contained largely carbohydrate sulfate. This implies some kind of selection concerning the intracellular processing, secretion, turnover or re-uptake of sulfated proteins which is responsible for the enrichment of tyrosine-sulfated proteins in the plasma.

Animals↗