Distinct linkage between post-translational processing and differential secretion of progastrin derivatives in endocrine cells

Prohormones often undergo extensive cellular processing prior to secretion. These post-translational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated in a pre-trans-Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C-terminal fragments derived from progastrin differ in characteristic manners in levels and patterns of carboxyamidation and tyrosine sulfation in accordance with the secretory pathway taken. Point mutations introduced into a sorting motif disrupt these patterns, suggesting that differences in post-translational modifications are attributable to differential intracellular sorting of precursors. The results suggest a two-step sorting mechanism for progastrin leading to differential secretion of processed fragments via different secretory pathways.     

Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing

Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg(94)Arg(95) and Lys(74)Lys(75) residues generating gastrin heptadecapeptide (G17-NH(2)). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH(2)) with little processing at Lys(74)Lys(75). Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH(2). To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys(74)Lys(75)) or mutant (Arg(74)Arg(75), Lys(74)Arg(75), and Arg(74)Lys(75)) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH(2) formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys(74)Lys(75) site within G34-NH(2) to Lys(74)Arg(75) resulted in the production of primarily G17-NH(2) rather than G34-NH(2), inhibition of PC1/PC3 did not significantly inhibit processing at the Lys(74)Arg(75) site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells.     

Alpha-carboxyamidation of antral progastrin. Relation to other post-translational modifications

Using radioimmunoassays for amidated and glycine-extended gastrin before and after trypsin-carboxypeptidase B cleavage and chromatography, alpha-carboxyamidation of porcine antral progastrin has been related to tyrosine-O-sulfation and proteolytic cleavages. Corresponding to the sequence at the proteolysis and amidation site, -Gly-Arg-Arg-, antrum contained three COOH-terminally extended precursor types. The glycine-extended gastrins were present in the highest concentrations (241 +/- 58 pmol/g). The degree of tyrosine-O-sulfation was identical for amidated and precursor gastrins irrespective of component size, whereas the component size differed for glycine-extended and amidated forms. For instance, gastrin-34-Gly constituted 54% of the glycine-extended gastrins, while gastrin-34 comprised 8% of the amidated gastrins. The results indicate that tyrosine-O-sulfation occurs prior to NH2-terminal cleavages, which again precede carboxyamidation; but a significant correlation between tyrosine-O-sulfation and proteolytic cleavages or alpha-carboxy-amidation of antral gastrin could not be demonstrated. Furthermore, our results suggest that the immediate precursor of the principal hormonal form, gastrin-17, is gastrin-17-Gly rather than gastrin-34 as previously believed.     

Identification and characterization of protein folding intermediates

In order to understand the mechanism by which a polypeptide chain folds into its functionally active native state it is necessary to characterize in detail all the species accumulated along the pathway. The elusive nature of protein folding intermediates poses their identification and characterization as an extremely difficult task in the protein folding field. In the case of small single domain proteins, the direct measurement of the thermodynamics and structural parameters of protein folding intermediates has provided new insights on the nature of the forces involved in the stabilization of nascent protein structures. Here we summarize some of the experimental approaches aimed at the detection and characterization of folding intermediates along with a discussion of some general structural features emerging from these studies.     

Identification of multiple post-translational modifications in the porcine brain specific p25alpha

P25α is a protein normally expressed in oligodendrocytes and subcellular relocalization of p25α occurs in multiple system atrophy, Parkinson's disease and Lewy body dementia along with ectopic expression in neurons. Moreover, it accumulates in Lewy body inclusions with aggregated α-synuclein and is a potent stimulator of α-synuclein aggregation. P25α is a phosphoprotein and post-translational modifications (PTMs) may play a role in its disease-related abnormalities. To investigate the spectrum of PTMs on p25α we cloned porcine p25α and isolated the protein from porcine brain. Using several complementary tandem mass spectrometry techniques for peptide mass analysis and amino acid sequencing, a comprehensive analysis of the PTMs on porcine p25α was performed. It was found that porcine p25α is heavily modified with a variety of modifications: phosphorylation, di- and trimethylation, citrullination and a HexNAc group. The modifications are localized within p25α's unfolded terminal domains and suggest that their functional states are regulated. This comprehensive mapping of p25α's PTMs will form the basis for future functional studies and investigations of p25α's potential role as a biomarker.     

Ferric ions inhibit proteolytic processing of progastrin

The gastrointestinal hormone gastrin is generated from an 80 amino acid precursor (progastrin) by cleavage after dibasic residues by prohormone convertase 1. Phosphorylation of Ser₇₅ has previously been suggested, on the basis of indirect evidence, to inhibit cleavage of progastrin after Arg₇₃Arg₇₄. Gastrins bind two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. This study directly investigated the effect of iron binding and of serine phosphorylation on the cleavage of synthetic progastrin-derived peptides. The affinity of synthetic progastrin_55–80 for ferric ions, and the rate of cleavage by prohormone convertase 1, were not affected by phosphorylation of Ser₇₅. In contrast, in the presence of ferric ions the rate of cleavage of both progastrin_55–80 and phosphoSer₇₅progastrin_55–80 by prohormone convertase 1 was significantly reduced. Hence iron binding to progastrin may regulate processing and secretion in vivo, and regulation may be particularly important in diseases with altered iron homeostasis.     

36 Identification and characterization of small subunit ribosomal intermediates

Bacterial ribosome biogenesis is poorly understood especially in terms of the role of ribosomal RNA (rRNA) maturation in vivo. A major problem in addressing these questions are asynchronous biogenesis, a large population of mature particles and the lack of techniques to isolated in vivo formed ribosome biogenesis intermediates. Our group has taken multiple approaches to allow study of ribosome biogenesis in Escherichia coli. We have used genetic manipulation to discover that for specific biogenesis factors, there is a delicate balance that is necessary for viability. Additionally, we have pioneered an affinity purification approach to allow for isolation of in vivo formed intermediates. Data will be present on our findings for the role of rRNA maturation in biogenesis, subsequent ribosome function, and cell viability. Our findings may result in identification of novel targets for antimicrobial development.     

Tyrosine O-sulfation promotes proteolytic processing of progastrin.

Tyrosine O-sulfation is a common post-translational modification of secretory and membrane proteins. The biological function of sulfation is known in only a few proteins, where it appears to enhance protein-protein interactions. Based on known sequences around sulfated tyrosines, a consensus sequence for prediction of target tyrosines has been proposed. However, some proteins are tyrosine sulfated at sites that deviate from the proposed consensus. Among these is progastrin. It is possible that the deviation explains the incomplete sulfation characteristic for bioactive gastrin peptides. In order to test this hypothesis, we have performed site-directed mutagenesis of the gastrin gene followed by heterologous expression in an endocrine cell line. The results show that substitution of the alanyl residue immediately N-terminal to the sulfated tyrosine with an acidic amino acid promotes the sulfation of gastrin peptides. Hence, the study supports the proposed consensus sequence for tyrosine sulfation. Importantly, however, the results also reveal that complete sulfation increases the endoproteolytic maturation of progastrin. Thus, our study suggests an additional function for tyrosine sulfation of possible general significance.     

Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.     

Modifying secretion and post-translational processing in insect cells

The baculovirus-insect cell system is a valuable tool for the expression of heterologous proteins. Due to limitations in the intracellular processing environment, however, heterologous secreted and membrane proteins are often insoluble, poorly processed, or contain 'non-human' modifications. Recent attempts to modify the insect cell secretory pathway by overexpressing processing factors have demonstrated the potential to overcome these limitations.     

Intracellular topography of glycine-extended pro-gastrin-processing intermediates in human antral mucosa: an electron-microscopic immunocytochemical study

To identify and characterize the subcellular topography of glycine-extended pro-gastrin-processing intermediates (G-Gly) in human antral mucosa, we performed an electron microscopic immunocytochemical study using region-specific antisera generated against the synthetic peptide, Tyr-Gly-Trp-Met-Asp-Phe-Gly (GL7), and C-terminal-specific anti-gastrin antisera. As has been previously reported, G-cells contained both electron-dense and electron-lucent granules, with a range of intermediate forms. Gastrin immunoreactivity was demonstrated in almost all granules of each type, whereas anti-GL7 antisera immunostained chiefly electron-dense granules. The relative ratio of GL7/gastrin granules varied among different cells but was approximately 1:10 on average. Other cytoplasmic organelles were devoid of specific labeling for GL7 or gastrin. As we have assumed that G-Gly serves as the immediate precursor for each molecular form of gastrin, electron-dense granules with high labeling for GL7 are regarded as the principal site for conversion of G-Gly to gastrin. This speculation supports many previous reports that electron-dense granules are immature and that the granules become less electron-dense with maturation.     

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.     

Identification and nearly full-length genome characterization of novel porcine bocaviruses

The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7-56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A(2) (sPLA(2)) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca(2+)-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.     

Identification and characterization of genes required for post-translational modification of Campylobacter coli VC167 flagellin

Two genes have been identified in Campylobacter coli VC167 which are required for the biosynthesis of post-translational modifications on flagellin proteins. The ptmA gene encodes a protein of predicted M _r 28 486 which shows significant homology to a family of alcohol dehydrogenases from a variety of bacteria. The ptmB gene encodes a protein of predicted M _r 26 598 with significant homology to CMP- N -acetylneuraminic acid synthetase enyzmes involved in sialic acid capsular biosynthesis in Neisseria meninigitidis and Escherichia coli K1. Site-specific mutation of either ptmA or ptmB caused loss of reactivity with antisera specific to the post-translational modifications and a change in the isoelectric focusing fingerprints relative to the parent strains. Mutation of ptmB , but not of ptmA , caused a change in apparent M _r of the flagellin subunit in SDS–PAGE gels. The ptmA and ptmB genes are present in other strains of Campylobacter . In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild-type strain. This suggests that the surface-exposed post-translational modifications may play a significant role in the protective immune response.     

Identification of two intermediates during processing of profilaggrin to filaggrin in neonatal mouse epidermis

A major event in the keratinization of epidermis is the production of the histidine-rich protein filaggrin (26,000 mol wt) from its high molecular weight (greater than 350,000) phosphorylated precursor (profilaggrin). We have identified two nonphosphorylated intermediates (60,000 and 90,000 mol wt) in NaSCN extracts of epidermis from C57/Bl6 mice by in vivo pulse-chase studies. Results of peptide mapping using a two-dimensional technique suggest that these intermediates consist of either two or three copies of filaggrin domains. Each of the intermediates has been purified. The ratios of amino acids in the purified components are unusual and essentially identical. The data are discussed in terms of a precursor containing tandem repeats of similar domains. In vivo pulse-chase experiments demonstrate that the processing of the high molecular weight phosphorylated precursor involves dephosphorylation and proteolytic steps through three-domain and two-domain intermediates to filaggrin. These processing steps appear to occur as the cell goes through the transition cell stage to form a cornified cell.     

Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser(3), was isolated from rat stomach and found to be an endogenous ligand for the growth-hormone secretagogue receptor (GHS-R). This octanoyl modification is essential for ghrelin-induced GH release. We report here the purification and identification of human ghrelin from the stomach, as well as structural analysis of the human ghrelin gene and quantitation of changes in plasma ghrelin concentration before and after gastrectomy. Human ghrelin was purified from the stomach by gel filtration and high performance liquid chromatography, using a ghrelin-specific radioimmunoassay and an intracellular calcium influx assay on a stable cell line expressing GHS-R to test the fractions. In the course of purification, we isolated human ghrelin of the expected size, as well as several other ghrelin-derived molecules. Classified into four groups by the type of acylation observed at Ser(3); these peptides were found to be non-acylated, octanoylated (C8:0), decanoylated (C10:0), and possibly decenoylated (C10:1). All peptides found were either 27 or 28 amino acids in length, the former lacking the C-terminal Arg(28), and are derived from the same ghrelin precursor through two alternative pathways. The major active form of human ghrelin is a 28-amino acid peptide octanoylated at Ser(3), as was found for rat ghrelin. Synthetic octanoylated and decanoylated ghrelins produce intracellular calcium increases in GHS-R-expressing cells and stimulate GH release in rats to a similar degree. Both ghrelin and the ghrelin-derived molecules were found to be present in plasma as well as stomach tissue. Plasma levels of immunoreactive ghrelin after total gastrectomy in three patients were reduced to approximately half of their pre-gastrectomy values, after which they gradually increased. This suggests that the stomach is the major source of circulating ghrelin and that other tissues compensate for the loss of ghrelin production after gastrectomy.     

Expression and post-translational processing of gastrin in heterologous endocrine cells

The biosynthesis of gastrin involves a complex series of post-translational processing reactions that result in the formation of a biologically active secretory product. To study the mechanisms for two specific reactions in gastrin processing, namely dibasic cleavage and amidation, we infected AtT-20, GH3, and Rin5-f cells with the retroviral expression vector, pZip-NeoSV(X), containing human gastrin cDNA. We detected gastrin and its glycine extended post-translational processing intermediates (G-gly) in the media and cell extracts of successfully infected cells. Characterization of the molecular forms of gastrin in these cell lines revealed that GH3 and Rin5-f processed gastrin in a manner similar to antral G-cells but the cleavage of the Lys74-Lys75 bond that converts G34 to G17 appeared to be suppressed in AtT-20 cells. Even after conversion of this site to Arg74-Arg75 via site-directed mutagenesis, the At-20 cells synthesized G34 predominantly. All of the infected cells amidated gastrin but the gastrin/G-gly ratio, a reflection of amidation within the cells, was enhanced in GH3 and Rin5-f cells but diminished in AtT-20 cells upon treatment with dexamethasone (10(-4) M) for 3 days. The dibasic cleavage of gastrin was uneffected by dexamethasone. Our data suggest that the activities of post-translational processing reactions responsible for the synthesis of biologically active gastrin exhibit considerable tissue and substrate specificity.