Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans

2001 ◽  
Vol 79 (2) ◽  
pp. 159-164 ◽  
Author(s):  
Tsuyoshi Higuchi ◽  
Shinri Tamura ◽  
Kanji Tanaka ◽  
Keiichi Takagaki ◽  
Yoshiharu Saito ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
High Performance ◽  
Core Protein ◽  
High Performance Liquid Chromatographic ◽  
Linkage Region ◽  
The Core ◽  
Core Proteins ◽  
Phosphatase Treatment ◽  
Cell Homogenate ◽  
Galactosyltransferase I

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-β-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 × g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100 000 × g centrifugation. When both fractions (the 100 000 × g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100 000 × g supernatant. In addition, the 100 000 × g precipitate, which was prepared from the 110 × g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100 000 × g supernatant activates galactosyltransferase-I activity.Key words: ATP, enzyme activator, galactosyltransferase-I, proteoglycan linkage region.

scholarly journals Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped

1999 ◽  
Vol 80 (10) ◽  
pp. 2647-2659 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Yong Shyang Yi ◽  
Szecheng J. Lo
Keyword(s):  
Hepatitis B ◽  
Kinase Activity ◽  
Core Protein ◽  
Surface Antigens ◽  
Human Hepatoma Cells ◽  
The Core ◽  
Core Proteins ◽  
B Virus ◽  
Transfection Medium ◽  
Cscl Gradient

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.

scholarly journals Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells

2006 ◽  
Vol 173 (2) ◽  
pp. 207-218 ◽  
Author(s):  
Xavier Darzacq ◽  
Nupur Kittur ◽  
Sujayita Roy ◽  
Yaron Shav-Tal ◽  
Robert H. Singer ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Ribosome Biogenesis ◽  
Core Protein ◽  
Single Cells ◽  
Telomere Maintenance ◽  
Rna Transcription ◽  
The Core ◽  
Core Proteins ◽  
Rna Accumulation

Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli.

scholarly journals The Vaccinia Virus I7L Gene Product Is The Core Protein Proteinase

2002 ◽  
Vol 76 (17) ◽  
pp. 8973-8976 ◽  
Author(s):  
Chelsea M. Byrd ◽  
Tove' C. Bolken ◽  
Dennis E. Hruby
Keyword(s):  
Vaccinia Virus ◽  
Gene Product ◽  
Transient Expression ◽  
Core Protein ◽  
Cysteine Proteinase ◽  
Gene Products ◽  
The Core ◽  
Core Proteins ◽  
Viral Core Protein ◽  
Cysteine Proteinase Activity

ABSTRACT Maturation of vaccinia virus (VV) core proteins is required for the production of infectious virions. The VV G1L and I7L gene products are the leading candidates for the viral core protein proteinase (vCPP). Using transient-expression assays, data were obtained to demonstrate that the I7L gene product and its encoded cysteine proteinase activity are responsible for vCPP activity.

Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus

1987 ◽  
Vol 7 (3) ◽  
pp. 1148-1155
Author(s):  
L D Fresco ◽  
M G Kurilla ◽  
J D Keene
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Binding ◽  
Core Protein ◽  
Protein A ◽  
Core Complex ◽  
The Core ◽  
Core Proteins ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Small Nuclear Ribonucleoproteins

After infection of baby hamster kidney cells with vesicular stomatitis virus (VSV), processing and assembly of small nuclear ribonucleoproteins (snRNP) were rapidly inhibited. The U1 and U2 snRNAs accumulated as precursor species approximately 3 and 10 nucleotides longer, respectively, than the mature RNAs. Alteration in snRNP assembly was noted because the precursor snRNAs were not associated with the U-series RNA-core protein complex in infected cells. However, antibodies specific for the U2 RNA-binding protein, A', were able to precipitate pre-U2 RNAs from VSV-infected cells. These results indicated that precursors to U2 RNA were bound to A' and remained bound during virus infection. Analysis of the synthesis of proteins normally associated with U1 and U2 RNAs indicated that synthesis was unaffected at times when snRNP assembly with core proteins was blocked by the VSV. These findings suggested that the core proteins associate with one another in the absence of the snRNAs in VSV-infected cells. They further suggest a correlation between the inability of the core complex to bind the U-series snRNPs and the failure to process the 3' ends of U1 and U2 RNAs in VSV-infected cells. These effects of VSV on snRNP assembly may be related to the shutoff of host-cell macromolecular synthesis.

scholarly journals Antibodies to basement membrane heparan sulfate proteoglycans bind to the laminae rarae of the glomerular basement membrane (GBM) and induce subepithelial GBM thickening.

1986 ◽  
Vol 163 (5) ◽  
pp. 1064-1084 ◽  
Author(s):  
A Miettinen ◽  
J L Stow ◽  
S Mentone ◽  
M G Farquhar
Keyword(s):  
Basement Membrane ◽  
Core Protein ◽  
Basement Membranes ◽  
Immunoperoxidase Staining ◽  
Dependent Manner ◽  
The Core ◽  
Core Proteins ◽  
Rabbit Igg ◽  
Gbm Thickening ◽  
Glomerular Capillaries

Antibodies specific for the core protein of basement membrane HSPG (Mr = 130,000) were administered to rats by intravenous injection, and the pathologic consequences on the kidney were determined at 3 min to 2 mo postinjection. Controls were given antibodies against gp330 (the pathogenic antigen of Heymann nephritis) or normal rabbit IgG. The injected anti-HSPG(GBM) IgG disappeared rapidly (by 1 d) from the circulation. The urinary excretion of albumin increased in a dose-dependent manner during the first 4 d, was increased 10-fold at 1-2 mo, but remained moderate (mean = 12 mg/24 h). By immunofluorescence the anti-HSPG(GBM) was seen to bind rapidly (by 3 min) to all glomerular capillaries, and by immunoperoxidase staining the anti-HSPG was seen to bind exclusively to the laminae rarae of the GBM where it remained during the entire 2-mo observation period. C3 was detected in glomeruli immediately after the injection (3 min), where it bound exclusively to the lamina rara interna; the amount of C3 bound increased up to 2 h but decreased rapidly thereafter, and was not detectable after 4 d. Mononuclear and PMN leukocytes accumulated in glomerular capillaries, adhered to the capillary wall, and extended pseudopodia through the endothelial fenestrae to contact in the LRI of the GBM where the immune deposits and C3 were located. At 1 wk postinjection, staining for C3 reappeared in the glomeruli of some of the rats, and by this time most of the rats, including controls injected with normal rabbit IgG, had circulating anti-rabbit IgG (by ELISA) and linear deposits of rat IgG along the GBM (by immunofluorescence). Beginning at 9 d, there was progressive subepithelial thickening of the GBM which in some places was two to three times its normal width. This thickening was due to the laying down of a new layer of basement membrane-like material on the epithelial side of the GBM, which gradually displaced the old basement membrane layers toward the endothelium. The results show that the core proteins of this population of basement membrane HSPG (Mr = 130,000), which are ubiquitous components of basement membranes, are exposed to the circulation and can bind anti-HSPG(GBM) IgG in the laminae rarae of the GBM. Binding of these antibodies to the GBM leads to changes (C3 deposition, leukocyte adherence, moderate proteinuria, GBM thickening) considered typical of the acute phase of anti-GBM glomerulonephritis. Antibody binding interferes with the normal turnover of the GBM, presumably by affecting the biosynthesis and/or degradation of basement membrane components.

scholarly journals Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions

2009 ◽  
Vol 83 (19) ◽  
pp. 9923-9939 ◽  
Author(s):  
Li-Shuang Ai ◽  
Yu-Wen Lee ◽  
Steve S.-L. Chen
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Complex Formation ◽  
Signal Peptide ◽  
Core Protein ◽  
The Core ◽  
Hcv Core Protein ◽  
Core Proteins ◽  
Multimeric Complex ◽  
Protein Multimerization

ABSTRACT The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis.

Exploration of Human Xylosyltransferase for Chemoenzymatic Synthesis of Proteoglycan Linkage Region

2021 ◽  
Author(s):  
Jia Gao ◽  
Po-han Lin ◽  
Setare Tahmasebi Nick ◽  
Kunli Liu ◽  
Kefei Yu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Growth Factor ◽  
Tumor Progression ◽  
Chemoenzymatic Synthesis ◽  
Biological Processes ◽  
Linkage Region ◽  
The Core ◽  
Core Proteins ◽  
Regulation Of Growth

Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature,...

Sign in / Sign up

Export Citation Format

Share Document