Regulation of the gene that encodes the peptide core of heparin proteoglycan and other proteoglycans that are stored in the secretory granules of hematopoietic cells.
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Animals↗
Biomedical subjects
R L Stevens
Publications and source records attributed to R L Stevens.
GATA-binding transcription factors in mast cells regulate the promoter of the mast cell carboxypeptidase A gene.
The transcription factors GATA-1, GATA-2, and GATA-3 were found to be expressed in several mouse and rat mast cell lines that contain mast cell carboxypeptidase A (MC-CPA) and other proteases in their cytoplasmic granules. GATA-1 mRNA was not detected in P815 cells, an immature mouse mastocytoma-derived cell line that lacks electron-dense granules and has low levels of secretory granule proteases. Because the 5'-flanking regions of the mouse and human MC-CPA genes contained a conserved GATA-binding motif 51 base pairs upstream of their translation initiation sites, the ability of GATA-binding proteins to regulate the promoter activity of the MC-CPA gene was examined in rat basophilic leukemia cells, mouse P815 cells, and transfected mouse P815 cells that expressed GATA-1. In all three mast cell lines, the promoter activity of the MC-CPA gene depended on the GATA binding site. GATA-1, GATA-2, and GATA-3 are thus the first DNA-binding proteins identified in mast cells which regulate the promoter activity of a gene that encodes a secretory granule protease.
Animals↗
Molecular cloning of the mouse mast cell protease-5 gene. A novel secretory granule protease expressed early in the differentiation of serosal mast cells.
cDNAs were isolated that encode mouse mast cell protease-5 (MMCP-5), an approximately 30,000 Mr serine protease stored in the secretory granules of serosal mast cells (SMC) and Kirsten sarcoma virus-immortalized mast cells. Based on the deduced amino acid sequences of these cDNAs, MMCP-5 is synthesized as a 247-amino acid preproenzyme composed of a novel 19-residue hydrophobic signal peptide, a Gly-Glu activation peptide not present in other mast cell chymases, and a 226-amino acid protein that represents the mature enzyme. MMCP-5 possesses a unique Asn residue in the substrate binding cleft at residue 176 and is highly basically charged. The MMCP-5 gene was isolated, sequenced, and found to belong to a distinct subset of chymase genes. Allelic variations of the MMCP-5 gene were also detected. MMCP-5 is expressed in bone marrow-derived mast cells (BMMC), Kirsten sarcoma virus-immortalized mast cells, and SMC, but not in gastrointestinal mucosal mast cells of helminth-infected mice. The abundant levels of MMCP-5 mRNA in immature BMMC indicate that this chymase is expressed relatively early during the differentiation of mast cells. MMCP-5 is the first chymase to be molecularly cloned from progenitor mast cells and is also the first chymase shown to be expressed preferentially in the SMC subclass.
Amino Acid Sequence↗
Identification of aminopeptidase activity in the secretory granules of mouse mast cells.
Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme beta-hexosaminidase, thereby localizing approximately 60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 +/- 0.06 mM (mean +/- SD; n = 3) for leucine-beta-naphthylamide. When various amino acid beta-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala greater than Leu greater than Phe much greater than Arg much greater than Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of greater than 1 x 10(7) kDa as defined by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the amino-peptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
Aminopeptidases↗
Cytokine mRNA are preferentially increased relative to secretory granule protein mRNA in mouse bone marrow-derived mast cells that have undergone IgE-mediated activation and degranulation.
The levels of mRNA that encode a number of cytokines have been reported by several laboratories to be increased in mouse mast cells after their IgE-bearing receptors have been cross-linked with Ag. In this study, we have compared the mRNA levels for Fc epsilon RI alpha, three cytokines (IL-6, granulocyte-macrophage CSF, and TNF-alpha), actin and three secretory granule-localized proteins (carboxypeptidase A, proteoglycan peptide core, and a generic serine protease) in mouse bone marrow-derived mast cells (BMMC) before and after IgE-mediated activation and degranulation to determine the kinetics and specificity of mRNA induction. An antigen concentration of approximately 10 ng/ml was optimal for the release of histamine from IgE-sensitized BMMC and for the generation and release of a cytokine that was functionally and immunochemically identical to TNF-alpha. In kinetic experiments, the levels of TNF-alpha, IL-6, and granulocyte-macrophage CSF mRNA increased greater than 23-fold 0.5 to 1 h after activation. As assessed by in situ hybridization, virtually all BMMC contained detectable proteoglycan peptide core mRNA before and after exposure to Ag, but only approximately one-half of the Ag-treated cells in the culture contained IL-6 mRNA 1 h after activation. There was a slight transient increase at 4 h in the level of proteoglycan peptide core mRNA, but no increase in the levels of those highly expressed mRNA that encode actin, Fc epsilon RI alpha, carboxypeptidase A, and serine protease. Thus, despite the remarkable increment in the levels of the transcripts that encode cytokines in BMMC after IgE-mediated, Ag-dependent activation, the levels of those transcripts that encode a plasma membrane-localized recognition receptor and several constituents of the secretory granule remain essentially unchanged. The failure to increase substantially the level of protease and proteoglycan peptide core mRNA in mast cells after the activation/secretion response suggests that regranulation of mast cells is a slow process.
Animals↗
Gene expression in different populations of mouse mast cells.
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Amino Acid Sequence↗
Characterization of the human gene that encodes the peptide core of secretory granule proteoglycans in promyelocytic leukemia HL-60 cells and analysis of the translated product.
Based upon the deduced amino acid sequence of a cDNA (cDNA-H4) that had been proposed to encode the peptide core of an eosinophil and a HL-60 cell secretory granule proteoglycan, a 16-amino acid peptide was synthesized. This peptide was then used to elicit rabbit antibodies for study of the translation and post-translational modification of this gene product in hematopoietic cells. When HL-60 cells were radiolabeled for 2 min with [35S]methionine, a protein that migrated in a sodium dodecyl sulfate-polyacrylamide electrophoresis gel with a Mr of 20,000 was immunoprecipitated with the IgG fraction of the anti-peptide serum. Kinetic experiments revealed that within 10 min this radiolabeled precursor protein was converted in HL-60 cells into an Mr approximately 150,000 chondroitin sulfate proteoglycan intermediate. After a 20-min to 1-h chase, this [35S]methionine- or [35S]sulfate-labeled proteoglycan intermediate lost its antigenicity, presumably due to proteolysis of its N terminus. A human genomic library was probed under conditions of high stringency with cDNA-H4 to isolate genomic clones that contain the gene that encodes this proteoglycan peptide core. This gene spans approximately 15 kilobases and consists of three exons. The first exon encodes the 5'-untranslated region of the mRNA transcript, as well as the entire 27-amino acid signal peptide of the translated molecule. The second exon encodes a 49-amino acid region of the peptide core, predicted to be the N terminus of the molecule after its proteolytic processing in the endoplasmic reticulum. The third exon encodes the remainder of the molecule, including its glycosaminoglycan attachment, serine-glycine repeat region. As assessed by S1 nuclease mapping and primer extension analysis, the transcription-initiation site in HL-60 cells for this gene resides 53 base pairs upstream from the translation-initiation site.
Amino Acid Sequence↗
Identification and molecular cloning of a novel mouse mucosal mast cell serine protease.
A novel 28,000 Mr serine protease, designated mouse mast cell protease-2 (MMCP-2), that is stored in the secretory granules of Kirsten sarcoma virus-immortalized mouse mast cells (KiSV-MC) has been identified and its NH2-terminal amino acid sequence has been determined. Analysis of a 953-base pair cDNA that encodes MMCP-2 revealed that this serine protease is a basically charged protein, possessing the histidine-aspartic acid-serine charge relay system that is characteristic of other serine proteases. DNA blot analysis using the full-length MMCP-2 cDNA indicated the existence of a family of highly related serine protease genes in the mouse genome. When the same DNA blot was probed with the 149-base pair KpnI----3' fragment of the cDNA, the probe hybridized to a single DNA fragment, thereby demonstrating that this 3' fragment could be used as a gene-specific probe. The presence of high levels of the MMCP-2 mRNA transcript in the intestines of nematode-infected mice, and its absence in mouse bone marrow-derived mast cells and peritoneal cavity-derived connective tissue mast cells, suggest that this member of the mouse mast cell protease family is preferentially expressed late in the differentiation of mucosal mast cells.
Amino Acid Sequence↗
The gene that encodes the peptide core of secretory granule proteoglycans of haematopoietic cells.
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Animals↗
Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.
Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine proteases comprise the major proteins of mouse SMC secretory granules. Each of the four SMC serine proteases was distinct from the two serine proteases present in mucosal mast cells in the intestines of helminth-infected mice. The secretory granules of a Kirsten sarcoma virus-immortalized mast cell line contained three of the SMC-derived serine proteases and one of the mucosal mast cell-derived serine proteases. Thus, the family of mouse mast cell secretory granule serine proteases has at least six distinct members that can be expressed in different combinations in different mast cell populations.
Amino Acid Sequence↗
The importance of mouse, rat, and dog mast cell lines.
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Animals↗
A morphometric study of normodense and hypodense human eosinophils that are derived in vivo and in vitro.
Hypodense eosinophils were obtained from two patients with the idiopathic hyperosinophilic syndrome (IHES), and hypodense eosinophils were derived by culturing normodense human eosinophils from control donors in the presence of endothelial cells alone, granulocyte/macrophage-colony-stimulating factor (GM-CSF) alone, or GM-CSF and fibroblasts. These eosinophils were examined ultrastructurally and stereologically for alterations in the volume density (Vv) of their electron-dense granules, the Vv of their lucent granules, the Vv of their lipid droplets, the numerical density of their granules with respect to cytoplasm (Nv), and the plasma membrane surface area-to-cell volume ratio (Sv) that might account for their decreased sedimentation density. The hypodense eosinophils that were obtained from the two patients with IHES exhibited a one-third reduction in granule Vv relative to normodense eosinophils from control donors, primarily because of a decrease in granule size. The culture-derived hypodense eosinophils exhibited 10% to 16% decreases in their granule Vv, significant increases in their lucent granules, and a approximately 7.5% decrease in their Sv. Calculation of the cell volume from cross-sectional area measurements showed that the eosinophils that had been cocultured with fibroblasts in the presence of GM-CSF increased their volume by approximately 15%. The eosinophils that had been cocultured with endothelial cells exocytosed some of their granules. In conclusion, a composite of factors including cell swelling, a decrease in the volume of the cytoplasm occupied by granules, and an increase in granule lucency contributes to the hypodense phenotype in vitro, but only cell swelling and hypogranulation are seen in cells from patients with IHES. The latter could reflect the response of 'primed' hypodense eosinophils in vivo to pertinent tissue ligands.
Adult↗
Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family.
Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.
Amino Acid Sequence↗
Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 fibroblasts.
A mouse liver genomic library was probed with a 450-base pair AccI----3' gene-specific fragment of a mouse bone marrow-derived mast cell proteoglycan cDNA to isolate 15-18-kilobase (kb) genomic clones containing the gene that encodes the peptide core of mouse secretory granule proteoglycans. Based on the nucleotide sequences of its 2.0-3.5-kb subcloned fragments, this mouse gene consists of three exons. The first exon contains 41 base pairs of untranslated nucleotides that are present in the 5' region of the transcript and also encodes the hydrophobic 25-amino acid signal peptide. The second exon encodes a 48-amino acid sequence that would be predicted to be the N terminus of the peptide core after the signal peptide has been removed in the endoplasmic reticulum. The third exon encodes a 79-amino acid sequence that includes the 15 amino acids immediately preceding an alternating serine-glycine 21-amino acid sequence for glycosaminoglycan attachment, and the subsequent C-terminal 43 amino acids; this exon also contains the 424 untranslated nucleotides present in the 3' region of the transcript. Primer extension and S1 nuclease protection analyses were performed to determine the transcription initiation site of the mouse gene. Rat-1 fibroblasts were cotransfected with the selectable marker pSV2 neo and a lambda clone (lambda MG-PG1) to obtain two rat-1 fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. RNA blot analysis of both cell lines revealed the presence of the 1.0-kb secretory granule proteoglycan peptide core mRNA transcript, indicating that lambda MG-PG1 contained the entire mouse secretory granule proteoglycan peptide core gene including some of the regulatory elements in its promoter region. The gene that encodes the peptide core of mouse secretory granule proteoglycans is the first proteoglycan gene to have its complete exon/intron organization determined and to be transfected and expressed in another cell type.
Amino Acid Sequence↗
IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity.
Human peripheral blood-derived eosinophils were assessed for their viability, density, and functional properties after 7 days of culture with purified mouse IL-5 and mouse 3T3 fibroblasts. Whereas none of the eosinophils remained viable after 7 days of culture in the absence of IL-5, 38 +/- 12% and 61 +/- 14% (n = 6, mean +/- SD) of the eosinophils survived in the presence of 1 pM IL-5 alone or 1 pM IL-5 in the presence of 3T3 fibroblasts, respectively (p less than 0.05). Direct contact between the fibroblasts and the eosinophils was not needed for this enhanced IL-5-dependent viability. After 7 days, 66 +/- 7% (n = 6) of the cocultured eosinophils were viable when the two cell types were separated by a 0.4-microns filter. As assessed by density-gradient centrifugation after 7 days of IL-5 exposure, all of the original normodense eosinophils became hypodense. The time course of this conversion was accelerated by the presence of 3T3 fibroblasts. Enhanced helminthic cytotoxicity was maintained by the 7-day cultured eosinophils only if they had been cocultured with fibroblasts. Eosinophils killed 10 +/- 11% (n = 5), 48 +/- 17%, and 31 +/- 15% of the larvae when they were cultured for 7 days in IL-5 alone, in IL-5 in direct contact with 3T3 fibroblasts, or in IL-5 with filter separation of the fibroblasts and the eosinophils, respectively. The ability of IL-5 to induce progenitor cells to differentiate selectively into eosinophils, and of 3T3 fibroblasts to facilitate the IL-5-mediated conversion of normodense eosinophils to hypodense eosinophils with increased viability and antibody-dependent cytotoxicity suggests a role for both hematopoietic and tissue factors in determining the presence and pathobiologic function of activated hypodense eosinophils in patients with hypereosinophilic conditions.
Animals↗
Interleukin 5 and phenotypically altered eosinophils in the blood of patients with the idiopathic hypereosinophilic syndrome.
We report that the hypodense eosinophil population in three patients with corticosteroid-unresponsive IHES was uniquely long lived ex vivo in the absence of exogenous cytokines. Serum or plasma from these patients conferred prolonged viability ex vivo to normodense eosinophils from reference donors and converted them to a functionally activated hypodense phenotype. In that antibody against IL-5 neutralized this activity in IHES serum, excessive quantities of this cytokine may account for the characteristic eosinophilia and long-lived, functionally augmented eosinophil phenotype in this disorder.
Adult↗
Regulation of the growth rate of mouse fibroblasts by IL-3-activated mouse bone marrow-derived mast cells.
When mouse bone marrow-derived mast cells (BMMC) are cocultured with a confluent layer of mouse 3T3 fibroblasts in the presence of WEHI-3-conditioned medium, the mast cells undergo a phenotypic change toward that of a connective tissue mast cell, and the fibroblasts increase their synthesis of globopentaosylceramide. We now demonstrate that fibroblasts lose their contact inhibition and multiply such that by the 2nd and the 4th wk of coculture there are, respectively, approximately four-fold and six-fold more fibroblasts than in the cultures that are not exposed to BMMC. This in vitro increase in the number of fibroblasts is dependent on the number of mast cells (over the range of 6 x 10(4) to 1 x 10(6) BMMC/culture) initially seeded with the fibroblasts and on the concentration of WEHI-3-conditioned medium present during the coculture. That the fibroblasts also multiply in BMMC/fibroblast cocultures exposed to synthetic IL-3 or to purified IL-3 indicates that IL-3 is a component in WEHI-3-conditioned medium that induces mast cells to produce the fibroblast growth factor. The number of fibroblasts does not increase if fibroblasts are exposed to lysates of BMMC, or to BMMC-derived conditioned medium, or if the two cell types are separated from one another during the coculture with a 3-microns filter or a 0.4-microns filter. Thus, IL-3-activated BMMC must be in proximity to fibroblasts to induce them to multiply. Because of their increased numbers per culture dish, total fibroblasts that were cocultured with mast cells synthesized approximately two-fold more 35S-labeled proteoglycans, incorporated approximately 3-fold more [3H] proline into collagenase-sensitive proteins, and had substantially more alpha 2(I) collagen mRNA than fibroblasts that were maintained in the absence of mast cells. These is vitro studies reveal a sequence by which IL-3-activated mast cells may play a role in the induction of fibrosis.
Animals↗
Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells.
Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.
Carboxypeptidases↗