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W B Huttner

Publications and source records attributed to W B Huttner.

At least 127 records · Page 7Linked to original sources
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

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↗
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↗
PubMed1987

Tyrosine sulfation is a trans-Golgi-specific protein modification.

The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.

Animals↗
PubMed1986

Chlorate--a potent inhibitor of protein sulfation in intact cells.

Chlorate is known to be an in vitro inhibitor of ATP-sulfurylase, the first enzyme in the biosynthesis of PAPS which is the ubiquitous co-substrate for sulfation. Here, the effect of chlorate on protein sulfation in intact cells was investigated. Treatment of various cell cultures with 1 mM sodium chlorate in a medium low in sulfate and sulfur-containing amino acids resulted in an inhibition of protein sulfation greater than 95%. Tyrosine as well as carbohydrate sulfation was blocked. Chlorate did not inhibit protein synthesis and did not exhibit any other toxic effects, even after prolonged treatment of cell cultures. Thus, chlorate treatment provides a powerful tool for studying the biological significance of protein sulfation.

Animals↗
PubMed1986

The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells.

We have determined the primary structure of bovine chromogranin A as a first step in the elucidation of the function of this widespread protein. After oligonucleotide screening of a cDNA library of bovine adrenal medulla, a clone (insert length 1.9 kb) containing the entire coding region for chromogranin A was isolated and sequenced. The authenticity of the sequence was verified by comparison with N-terminal, several internal, and C-terminal amino acid sequences as well as the amino acid composition of chromogranin A. The cDNA clone hybridized to an mRNA of 2.1 kb and, after in vitro transcription-translation, yielded a polypeptide with a similar electrophoretic mobility in SDS gels to chromogranin A. The polypeptide chain of chromogranin A comprises 431 amino acid residues, corresponding to an unmodified protein of 48 kd, and is preceded by a cleaved signal peptide of 18 amino acid residues. Interesting features of the chromogranin A structure include repeated clusters of glutamic acid residues, the occurrence of eight potential dibasic cleavage sites, six of which are located in the C-terminal domain, and the presence, in the N-terminal domain, of -Arg-Gly-Asp- (RGD), a three amino acid sequence involved in the binding of several constitutively secreted proteins to cell membranes.

Adrenal Medulla↗
PubMed1986

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.

Adenosine↗
PubMed1985

Cell-free sulfation of the contact site A glycoprotein of Dictyostelium discoideum and of a partially glycosylated precursor.

An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrate Metabolism↗
PubMed1985

Nature of sulphated macromolecules in mouse Reichert's membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins.

Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan.

Animals↗
PubMed1985

Tyrosine sulfation of yolk proteins 1, 2, and 3 in Drosophila melanogaster.

Protein sulfation was studied in Drosophila melanogaster after in vivo labeling of flies with inorganic [35S]sulfate. After separation of total fly protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins with sulfated carbohydrates and proteins containing tyrosine sulfate were found in all the molecular weight ranges analyzed. When female and male fly proteins were compared with each other, the electrophoretic patterns of protein-bound carbohydrate sulfate were found to be similar, whereas those of protein-bound tyrosine sulfate were distinct. The most prominent difference was the exclusive presence in female flies of three major tyrosine-sulfated proteins with apparent molecular masses between 48 and 45 kDa. Radioimmunolabeling after two-dimensional polyacrylamide gel electrophoresis was used to identify these proteins as yolk proteins 1, 2, and 3. Each of the three yolk proteins existed in several isoelectric forms, all of which were sulfated. Since the number of tyrosine residues in the yolk proteins is known, the stoichiometry of tyrosine sulfation could be determined by a novel method and was found to be 2.2, 0.9, and 1.2 mol of tyrosine sulfate per mol of yolk protein 1, 2, and 3, respectively. The present results, together with the recently reported molecular cloning of the yolk protein genes, make the yolk proteins suitable objects for genetic approaches to investigate the biological role(s) of tyrosine sulfation of secretory proteins.

Animals↗
PubMed1985

(Glu62, Ala30, Tyr8)n serves as high-affinity substrate for tyrosylprotein sulfotransferase: a Golgi enzyme.

Tyrosylprotein sulfotransferase, the enzyme catalyzing the sulfation of proteins on tyrosine residues, was characterized by using the acidic polymer containing tyrosine (Glu62, Ala30, Tyr8)n (referred to as Glu,Ala,Tyr) as exogenous "protein" substrate. After subcellular fractionation of a bovine adrenal medulla homogenate, tyrosylprotein sulfotransferase activity was found to be highest in fractions enriched in Golgi membrane vesicles. Tyrosylprotein sulfotransferase required the presence of a nonionic detergent for sulfation of exogenous Glu,Ala,Tyr, indicating an orientation of the catalytic site of the enzyme toward the Golgi lumen. Tyrosylprotein sulfotransferase was solubilized by Triton X-100, suggesting that the enzyme was tightly associated with the Golgi membrane, possibly as an integral membrane protein. The apparent Golgi localization of tyrosylprotein sulfotransferase was supported by the observation that tyrosine sulfation of proteins in intact cells was blocked by monensin and was in line with previous observations that all tyrosine-sulfated proteins known so far are secretory. Glu,Ala,Tyr was found to have a very high affinity for tyrosylprotein sulfotransferase (apparent Km, 300 nM), similar to that reported for certain tyrosylprotein kinases. While this may suggest some similarity between these enzymes, the Golgi localization of tyrosylprotein sulfotransferase segregates tyrosine sulfation from the sites of tyrosine phosphorylation of proteins in the intact cell. If, however, tyrosylprotein sulfotransferase was allowed to react with cytoplasmic proteins by using a nonionic detergent, tyrosine sulfation of tubulin was observed.

Adrenal Medulla↗
PubMed1985

The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs.

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine-sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.

Animals↗
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↗
PubMed1984

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction.

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.

Adenine Nucleotides↗
PubMed1984

A Mr 70,000 phosphoprotein of sympathetic neurons regulated by nerve growth factor and by depolarization.

The effect of nerve growth factor (NGF) on protein phosphorylation was investigated in cultures of dissociated, purified chick sympathetic neurons labeled with inorganic [32P]phosphate or [35S]methionine. For at least 90 min after dissociation and purification of the neurons, overall protein phosphorylation was similar in the absence and presence of added NGF, indicating that the neurons were not unspecifically affected by this period of NGF deprivation. Addition of NGF resulted in a marked decrease in the phosphorylation of a 70,000-dalton protein, designated p70 . p70 existed in five isoelectric variants, referred to as p70 /1-5. p70 /1 was unphosphorylated and was the least acidic variant. p70 /2-5 contained progressively more phosphate and they were increasingly acidic. NGF, via dephosphorylation (or via a highly specific and very limited proteolysis), induced the conversion of p70 /5 and p70 /4 to p70 /2. The effect of NGF on p70 involved phosphothreonine residues to a greater extent than phosphoserine residues and occurred rapidly, being detectable after 5 min and complete after 15 min. 8-Br-cAMP did not mimic the effect of NGF on p70 . Depolarization of the neurons with high K+ and addition of the calcium ionophore A23187 produced effects on the phosphorylated p70 variants similar to those induced by NGF. The response of p70 to two distinct "survival factors," NGF and depolarization, may suggest a role for this phosphoprotein in the survival of sympathetic neurons.

8-Bromo Cyclic Adenosine Monophosphate↗