scholarly journals Calreticulin Biosynthesis and Processing in Human Myeloid Cells: Demonstration of Signal Peptide Cleavage and N-Glycosylation

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 372-381 ◽  
Author(s):  
Gerene M. Denning ◽  
Kevin G. Leidal ◽  
Valerie A. Holst ◽  
Shankar S. Iyer ◽  
Doran W. Pearson ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Proteolytic Processing ◽  
Peptide Cleavage ◽  
Wide Range

Abstract Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

scholarly journals Calreticulin Biosynthesis and Processing in Human Myeloid Cells: Demonstration of Signal Peptide Cleavage and N-Glycosylation

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 372-381
Author(s):  
Gerene M. Denning ◽  
Kevin G. Leidal ◽  
Valerie A. Holst ◽  
Shankar S. Iyer ◽  
Doran W. Pearson ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Proteolytic Processing ◽  
Peptide Cleavage ◽  
Wide Range

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

scholarly journals Proteolytic Processing of Sapovirus ORF1 Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7283-7290 ◽  
Author(s):  
Tomoichiro Oka ◽  
Kazuhiko Katayama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
Tsutomu Kageyama ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Translation System ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Capsid Protein Vp1

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Abstract Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

scholarly journals Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation

2004 ◽  
Vol 78 (24) ◽  
pp. 13708-13716 ◽  
Author(s):  
Pornwaratt Niyomrattanakit ◽  
Pakorn Winoyanuwattikun ◽  
Santad Chanprapaph ◽  
Chanan Angsuthanasombat ◽  
Sakol Panyim ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Dimensional Structure ◽  
Wild Type ◽  
Structural Determinants ◽  
Protease Activation ◽  
Ns3 Protease

ABSTRACT Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Φx3Φ motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k cat/K m value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k cat values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.

Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis

1993 ◽  
Vol 13 (8) ◽  
pp. 4875-4883
Author(s):  
J Delic ◽  
M Morange ◽  
H Magdelenat
Keyword(s):  
Gene Expression ◽  
Gamma Irradiation ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mrna Level ◽  
Sodium Dodecyl ◽  
Nuclear Proteins ◽  
Classical Type ◽  
Apoptotic Death

Apoptosis (the classical type of programmed cell death) can be triggered in many cell types by widely diverse stimuli. gamma rays, at low doses, can induce apoptosis in vitro in interphase human lymphocytes. In this type of apoptosis induction, activated gene expression is necessary for the fulfillment of the death program. In this report, we present evidence for a relationship between ubiquitin gene expression or ubiquitination and gamma-irradiation-mediated apoptosis in normal circulating human lymphocytes. Using in vitro nuclear transcription assays (run-on), Northern (RNA) blot analysis, immunolocalization studies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation, we demonstrate that (i) the ubiquitin mRNA level is increased as a consequence of the activation of ubiquitin gene transcription 15 to 90 min after initiation of apoptosis; (ii) specifically in apoptotic cells, and not in all irradiated cells, nuclear proteins are highly ubiquitinated; and (iii) ubiquitin sequence-specific antisense oligonucleotide inhibition results in a decreased level of ubiquitinated nuclear proteins and considerably diminishes the proportion of cells exhibiting the apoptotic death pattern. Each of these results might be explained by different modifications occurring in irradiated cells. Their convergence strongly suggests that the ubiquitin gene is one of the genes with induced activity in the apoptotic death program and that ubiquitination of nuclear proteins might be involved in chromatin disorganization and oligonucleosomal fragmentation, which are among the key events occurring in apoptosis.

scholarly journals Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis.

1993 ◽  
Vol 13 (8) ◽  
pp. 4875-4883 ◽  
Author(s):  
J Delic ◽  
M Morange ◽  
H Magdelenat
Keyword(s):  
Gene Expression ◽  
Gamma Irradiation ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mrna Level ◽  
Sodium Dodecyl ◽  
Nuclear Proteins ◽  
Classical Type ◽  
Apoptotic Death

Apoptosis (the classical type of programmed cell death) can be triggered in many cell types by widely diverse stimuli. gamma rays, at low doses, can induce apoptosis in vitro in interphase human lymphocytes. In this type of apoptosis induction, activated gene expression is necessary for the fulfillment of the death program. In this report, we present evidence for a relationship between ubiquitin gene expression or ubiquitination and gamma-irradiation-mediated apoptosis in normal circulating human lymphocytes. Using in vitro nuclear transcription assays (run-on), Northern (RNA) blot analysis, immunolocalization studies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation, we demonstrate that (i) the ubiquitin mRNA level is increased as a consequence of the activation of ubiquitin gene transcription 15 to 90 min after initiation of apoptosis; (ii) specifically in apoptotic cells, and not in all irradiated cells, nuclear proteins are highly ubiquitinated; and (iii) ubiquitin sequence-specific antisense oligonucleotide inhibition results in a decreased level of ubiquitinated nuclear proteins and considerably diminishes the proportion of cells exhibiting the apoptotic death pattern. Each of these results might be explained by different modifications occurring in irradiated cells. Their convergence strongly suggests that the ubiquitin gene is one of the genes with induced activity in the apoptotic death program and that ubiquitination of nuclear proteins might be involved in chromatin disorganization and oligonucleosomal fragmentation, which are among the key events occurring in apoptosis.

scholarly journals Robust temporal map of human in vitro myelopoiesis using single-cell genomics

2021 ◽  
Author(s):  
Clara Alsinet ◽  
Maria Primo ◽  
Valentina Lorenzi ◽  
Andrew J Knights ◽  
Carmen Sancho-Serra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Regulatory Networks ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Wide Range

Myeloid cells have a central role in homeostasis and tissue defence. Characterising the current in vitro protocols of myelopoiesis is imperative for their use in research and immunotherapy as well as for understanding the early stages of myeloid differentiation in humans. Here, we profiled the transcriptome of more than 400k cells and generated a robust molecular map of the differentiation of human induced pluripotent stem cells (iPSC) into macrophages. By integrating our in vitro datasets with in vivo single-cell developmental atlases, we found that in vitro macrophage differentiation recapitulates features of in vivo yolk sac hematopoiesis, which happens prior to the appearance of definitive hematopoietic stem cells (HSC). During in vitro myelopoiesis, a wide range of myeloid cells are generated, including erythrocytes, mast cells and monocytes, suggesting that, during early human development, the HSC-independent immune wave gives rise to multiple myeloid cell lineages. We leveraged this model to characterize the transition of hemogenic endothelium into myeloid cells, uncovering poorly described myeloid progenitors and regulatory programs. Taking advantage of the variety of myeloid cells produced, we developed a new protocol to produce type 2 conventional dendritic cells (cDC2) in vitro. We found that the underlying regulatory networks coding for myeloid identity are conserved in vivo and in vitro. Using genetic engineering techniques, we validated the effects of key transcription factors important for cDC2 and macrophage identity and ontogeny. This roadmap of early myeloid differentiation will serve as an important resource for investigating the initial stages of hematopoiesis, which are largely unexplored in humans, and will open up new therapeutic opportunities.

Proteolytic processing of human coagulation factor IX by plasmin

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 943-951 ◽  
Author(s):  
John A. Samis ◽  
Gillian D. Ramsey ◽  
John B. Walker ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Proteolytic Processing ◽  
Factor Ix ◽  
Stable Complex ◽  
Factor X ◽  
Active Site Probe

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 μM) by 80% and the activity of F.IXa (4 μM) by 50% after 30 minutes at 37°C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated–F.IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator–catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.

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