scholarly journals Extracellular matrix microfibrils are composed of core proteins coated with fibronectin.

1985 ◽  
Vol 33 (4) ◽  
pp. 268-274 ◽  
Author(s):  
E Schwartz ◽  
S Goldfischer ◽  
B Coltoff-Schiller ◽  
O O Blumenfeld
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
Core Protein ◽  
Extracellular Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Core ◽  
Core Proteins ◽  
Double Label ◽  
Active Fragments ◽  
Partial Dissociation

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.

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 Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts

1985 ◽  
Vol 232 (1) ◽  
pp. 161-168 ◽  
Author(s):  
S Johansson ◽  
K Hedman ◽  
L Kjellén ◽  
J Christner ◽  
A Vaheri ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Human Articular Cartilage ◽  
Sulphate Proteoglycan ◽  
Fibroblast Cultures ◽  
Core Proteins ◽  
The Matrix

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called ‘link proteins’ were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.

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 Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin.

1987 ◽  
Vol 104 (6) ◽  
pp. 1683-1691 ◽  
Author(s):  
G Schmidt ◽  
H Robenek ◽  
B Harrach ◽  
J Glössl ◽  
V Nolte ◽  
...  
Keyword(s):  
Dermatan Sulfate ◽  
Core Protein ◽  
Human Fibroblasts ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sulfate Proteoglycan ◽  
Heparin Binding ◽  
Dermatan Sulfate Proteoglycan ◽  
Cultured Human Fibroblasts ◽  
Heparin Binding Domain ◽  
Double Label

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.

scholarly journals Pre-P Is a Secreted Glycoprotein Encoded as an N-Terminal Extension of the Duck Hepatitis B Virus Polymerase Gene

2008 ◽  
Vol 83 (3) ◽  
pp. 1368-1378 ◽  
Author(s):  
Feng Cao ◽  
Catherine A. Scougall ◽  
Allison R. Jilbert ◽  
John E. Tavis
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cultured Cells ◽  
Core Protein ◽  
Open Reading Frames ◽  
Duck Hepatitis B Virus ◽  
Reading Frame ◽  
The Core ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms “pre-P” by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.

scholarly journals Isolation and characterization of two sialoproteins present only in bone calcified matrix

1985 ◽  
Vol 232 (3) ◽  
pp. 715-724 ◽  
Author(s):  
A Franzén ◽  
D Heinegård
Keyword(s):  
Sialic Acid ◽  
Core Protein ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Dry Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Bone ◽  
Guanidinium Chloride ◽  
The Core ◽  
Isolation And Characterization

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

Alveolar type II cells synthesize hydrophobic cell-associated proteoglycans with multiple core proteins

1992 ◽  
Vol 263 (3) ◽  
pp. L348-L356 ◽  
Author(s):  
W. M. Maniscalco ◽  
M. H. Campbell
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Core Protein ◽  
Primary Cultures ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Core Proteins

Type II alveolar epithelial cells interact with the extracellular matrix via cell surface receptors for matrix ligands. Cell surface proteoglycans, which are hydrophobic due to their membrane insertion domains, are one of several classes of molecules that may be receptors for matrix ligands. To analyze the hydrophobic proteoglycans synthesized by adult alveolar type II cells, we labeled these cells with 35SO4 and [3H]leucine in short-term primary cultures. Cell-associated hydrophobic proteoglycans and culture medium-derived proteoglycans were purified and characterized. Both the hydrophobic proteoglycans and medium-derived proteoglycans, which were not hydrophobic, had mainly heparan sulfate glycosaminoglycans. Analysis of core proteins of the hydrophobic proteoglycans showed three proteins, 47, 65, and 90 kDa. The 47- and 65-kDa core proteins were substituted only with heparan sulfate chains. The 90-kDa core protein was seen only after digestion with both heparitinase and chondroitin ABC lyase, suggesting it was a hybrid having both heparan sulfate and chondroitin-dermatan sulfate chains. These findings were confirmed by iodination of the core proteins. The hydrophobic cell-associated proteoglycans inserted into artificial liposomes, whereas the medium-derived molecules did not. These data document heterogeneity in core protein and glycosaminoglycan chains among hydrophobic proteoglycans synthesized in vitro by adult alveolar type II cells. These molecules may have diverse functions in regulating type II cell interaction with the extracellular matrix.

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.

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