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W B Huttner

Publications and source records attributed to W B Huttner.

At least 109 records · Page 6Linked to original sources
PubMed1990

Purification and characterization of tyrosylprotein sulfotransferase.

Tyrosylprotein sulfotransferase (TPST) is a Golgi membrane enzyme involved in the post-translational modification of secretory and membrane proteins. Here we describe the 140,000-fold purification of this enzyme from bovine adrenal medulla to apparent homogeneity and determine its substrate specificity. The key step in the purification was affinity chromatography on a substrate peptide to which the enzyme bound in the presence of nucleotide cosubstrate. TPST is a 54-50 kd integral membrane glycoprotein. The presence of sialic acid strongly suggests that within the Golgi complex, TPST is localized in the trans-most subcompartment. TPST was found to specifically sulfate tyrosine residues adjacent to acidic amino acids. These results define a major determinant for the specificity of protein sulfation in the trans Golgi.

Adrenal Medulla↗
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↗
PubMed1990

Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary.

We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.

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 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↗
PubMed1989

Synaptophysin and chromogranins/secretogranins--widespread constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis.

Normal and neoplastic neuroendocrine (NE) cells have been identified for many years by morphological criteria only. With the advent of immunocytochemistry, antibodies against NE-specific polypeptides have been used to identify NE cells that had been missed by conventional techniques, thus improving the diagnosis of NE cells. In this review article we discuss (i) the biochemical, cell biological and molecular biological data obtained so far for two major types of NE markers, synaptophysin, which is characteristic of the small "transparent-looking" neurosecretory vesicles, and the chromogranins/secretogranins, which are widespread constituents of the larger "dense-cored" secretory granules; (ii) the immunohistochemical data obtained for these marker proteins in normal and neoplastic human NE cells and tissues; and (iii) future possible developments involving these as well as other proteins that are associated with these two distinct secretory organelles of NE cells and may serve as potential markers in NE cell diagnosis.

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↗
PubMed1989

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Ammonium Chloride↗
PubMed1989

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry.

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.

Animals↗
PubMed1989

Morphological and biochemical evidence showing neuronal properties in AtT-20 cells and their growth cones.

AtT-20 cells, which were derived from a murine pituitary tumor and produce ACTH, have until now been considered to originate from pituitary corticotrophs. Here we show that AtT-20 cells constitutively express several neuronal features. First, AtT-20 cells develop cytoplasmic processes whose fine structure is essentially identical to that of neurites and neuronal growth cones. These growth cones (i) are characterized by an extensive membranous reticulum which is derived from the endoplasmic reticulum (ER) since it contains immunoglobulin heavy chain binding protein, protein disulfide isomerase and glucose-6-phosphatase; (ii) are a major site of endocytosis; (iii) form cell-to-cell contacts resembling immature synapses. Second, AtT-20 cells, in contrast to pituitary corticotrophs, contain neurofilaments and express all three neurofilament polypeptides. They also contain the high molecular weight form of microtubule-associated protein 2 and tau protein. Third, AtT-20 cells express the neuron-specific phosphoprotein synapsin I which accumulates in the growth cones prior to contacts forming between growth cones and cells. Our results show that AtT-20 cells exhibit several properties of peptidergic neuronal cells and that the constitutive expression of a variety of these properties is compatible with continuous cell division.

Animals↗
PubMed1988

Phosphorylation of an 85-kd membrane protein by a novel mechanism.

Protein phosphorylation has been recognized as a major mechanism for the regulation of cellular functions. The classical phosphate donor in protein phosphorylation reactions is ATP. Here we show that 3'-phosphoadenosine-5'-phosphosulphate (PAPS), a ubiquitous nucleotide so far known to have a central role in sulphate transfer, serves as phosphate donor for protein phosphorylation. In a very specific, rapid and probably autocatalytic reaction, the 3'-phosphate group of PAPS was found to be transferred to a serine residue of an 85-kd membrane protein (p85). ATP did not serve as phosphate donor in this reaction. Radioactive phosphate incorporated into p85 in a membrane fraction was rapidly lost by dephosphorylation after removal of PAPS or by exchange with unlabelled phosphate after addition of nonradioactive PAPS. PAPS-dependent phosphorylation of the 85-kd protein and other proteins was observed in all rat and bovine tissues examined, as well as in various mammalian cell lines. Our results indicate the existence of a novel widespread form of protein phosphorylation.

Adenosine Triphosphate↗
PubMed1988

Purification of yolk protein 2 of Drosophila melanogaster and identification of its site of tyrosine sulfation.

We have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 of Drosophila melanogaster. Yolk proteins were purified from [35S]sulfate-labeled flies, and yolk protein 2 was separated from yolk protein 1 and yolk protein 3 by preparative two-dimensional polyacrylamide gel electrophoresis. After digestion of yolk protein 2 with trypsin and reversed-phase high performance liquid chromatography, the sulfate label was recovered in two distinct sulfopeptides which, however, had identical NH2-terminal sequences and contained 3 tyrosine residues each. After chymotryptic digestion of the two tryptic sulfopeptides, the sulfate label was recovered in one sulfopeptide which contained a single tyrosine residue. NH2-terminal sequencing showed that this tyrosine residue corresponded to tyrosine 172 of the yolk protein 2 precursor (Hung, M.-C., and Wensink, P. C. (1983) J. Mol. Biol. 164, 487-492) in the sequence Glu-Thr-Thr-Asp-Tyr(S)-Ser-Asn-Glu-Glu. This insect tyrosine sulfation site is very similar to the known vertebrate tyrosine sulfation sites in terms of amino acid composition and secondary structure. In the accompanying paper (Friederich, E., Baeuerle, P. A., Garoff, H., Hovemann, B., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14930-14938), we report on the expression of Drosophila yolk protein 2 in mouse fibroblasts and show the in vivo sulfation of tyrosine 172 by the vertebrate tyrosylprotein sulfotransferase.

Amino Acid Sequence↗
PubMed1988

Expression, tyrosine sulfation, and secretion of yolk protein 2 of Drosophila melanogaster in mouse fibroblasts.

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14925-14929), we have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 (YP2) of Drosophila melanogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heterologous mammalian cell system. Mouse fibroblasts, transfected with Drosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies. YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was approximately 1 mol of sulfate/mol of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172. These results show that essential parameters of the tyrosine sulfation reaction are very similar in insects and mammals and thus highly conserved in evolution.

Animals↗
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

Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface.

The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.

Amino Acid Sequence↗
PubMed1988

Tyrosine sulfation and the secretory pathway.

Tyrosine sulfation is a widespread posttranslational modification. Most tyrosine-sulfated proteins identified so far are secretory, including several neuropeptides. Tyrosine sulfation occurs in the trans Golgi and is one of the last processing steps before proteins exit from the Golgi complex. The sulfation reaction is catalyzed by tyrosylprotein sulfotransferase, an integral membrane protein that recognizes tyrosine residues in exposed protein domains containing acidic amino acids. In the cases studied to date, tyrosine sulfation has been found to be irreversible, resulting in a life-long alteration in the phenotype of the secretory proteins. The biological role of tyrosine sulfation has so far been elucidated in only a few cases. The intracellular transport kinetics of a secretory protein and the biological activity of certain neuropeptides have been found to be affected by this modification. Future functional studies will be greatly facilitated by the use of chlorate, a sulfate analogue that has recently been found to be a potent and nontoxic inhibitor of sulfation in intact cells.

Animals↗