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 Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane

1989 ◽  
Vol 264 (2) ◽  
pp. 457-465 ◽  
Author(s):  
L P W J van den Heuvel ◽  
J van den Born ◽  
T J A M van de Velden ◽  
J H Veerkamp ◽  
L A H Monnens ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Molecular Mass ◽  
Core Protein ◽  
Mammalian Species ◽  
Heparan Sulphate ◽  
Basement Membranes ◽  
Heparan Sulphate Proteoglycan ◽  
Average Molecular Mass ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.

scholarly journals Localization of heparan sulfate proteoglycan in basement membrane by side chain staining with cuprolinic blue as compared with core protein labeling with immunogold.

1992 ◽  
Vol 40 (10) ◽  
pp. 1559-1572 ◽  
Author(s):  
F L Chan ◽  
S Inoue ◽  
C P Leblond
Keyword(s):  
Basement Membrane ◽  
Core Protein ◽  
Immunogold Labeling ◽  
Basement Membranes ◽  
Side Chains ◽  
Sulfate Proteoglycan ◽  
Reaction Products ◽  
Lamina Densa ◽  
The Core ◽  
Cuprolinic Blue

We localized heparan sulfate proteoglycan (HSPG) in the basement membranes of ciliary epithelium and plantar epidermis, using Cuprolinic blue to stain its side chains and an immunogold procedure to detect its core protein. In accord with most of the literature, staining with Cuprolinic blue in glutaraldehyde fixative yielded three to five times as many reaction products along the two surfaces than along the center of the lamina densa, whereas immunogold labeling for the core protein after formaldehyde fixation yielded about twice as many gold particles over the center than along the surfaces of the lamina densa. It therefore appeared that HSPG side chains predominated outside, and the core protein within, the lamina densa. To find out whether the discrepancy was true or was an artifact caused by differences in processing, we attempted to combine the two approaches on the same material. This was found possible when Cuprolinic blue was used in formaldehyde fixative, embedding was in LR White, and immunogold labeling was performed on thin sections as usual. Under these conditions, both Cuprolinic blue reaction products and immunogold particles predominated over the lamina densa in the two basement membranes under study. Moreover, evidence was present that reaction products and immunogold particles either overlapped each other or were closely associated. The lens capsule (a thick basement membrane) also showed their co-localization. The discrepancy initially observed between side chains and core protein location was attributed to differences in processing, since Cuprolinic blue staining had been carried out in the course of glutaraldehyde fixation whereas immunogold labeling was done after formaldehyde fixation. The results lead to two conclusions. First, processing differences may alter the localization of HSPG and possibly other proteoglycans. Second, both HSPG side chains and core protein are localized in the same sites within basement membrane.

scholarly journals cDNA Cloning of the Basement Membrane Chondroitin Sulfate Proteoglycan Core Protein, Bamacan: A Five Domain Structure Including Coiled-Coil Motifs

1997 ◽  
Vol 136 (2) ◽  
pp. 433-444 ◽  
Author(s):  
Rong-Rong Wu ◽  
John R. Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Core Protein ◽  
Coiled Coil ◽  
Basement Membranes ◽  
Cdna Clones ◽  
Molecular Architecture ◽  
Unusual Structure ◽  
Extracellular Matrix Molecule

Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions. This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix. The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser-Gly dipeptides are unfavorable for substitution. Fusion protein antibodies stained basement membranes in a pattern commensurate with bamacan, and they also Western blotted bamacan core protein from rat L2 cell cultures. The antibodies could also specifically immunoprecipitate an in vitro transcription/translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models.

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 Modulation of aggrecan and link-protein synthesis in articular cartilage

1992 ◽  
Vol 288 (3) ◽  
pp. 721-726 ◽  
Author(s):  
A J Curtis ◽  
R J Devenish ◽  
C J Handley
Keyword(s):  
Articular Cartilage ◽  
Half Life ◽  
Core Protein ◽  
Calf Serum ◽  
Cartilage Explants ◽  
Dependent Manner ◽  
Link Protein ◽  
The Core ◽  
Explant Cultures ◽  
Igf I

The addition of serum or insulin-like growth factor-I (IGF-I) to the medium of explant cultures of bovine articular cartilage is known to stimulate the synthesis of aggrecan in a dose-dependent manner. The half-life of the pool of proteoglycan core protein was measured in adult articular cartilage cultured for 6 days in the presence and absence of 20 ng of IGF-I/ml and shown to be 24 min under both sets of conditions. The half-life of the mRNA pool coding for aggrecan was also determined and shown to be approx. 4 h in cartilage maintained in culture with or without IGF-I. The pool size of mRNA coding for aggrecan core protein increased 5-6-fold in cartilage explants maintained in culture in medium containing 20% (v/v) fetal-calf serum; however, in tissue maintained with medium containing IGF-I there was no increase in the cellular levels of this mRNA. This suggests that aggrecan synthesis is stimulated by IGF-I at the level of translation of mRNA coding for the core protein of this proteoglycan and that other growth factors are present in serum that stimulate aggrecan synthesis at the level of transcription of the core-protein gene. Inclusion of serum or IGF-I in the medium of cartilage explant cultures induced increases in the amounts of mRNA coding for type II collagen and link protein, whereas only serum enhanced the amount of mRNA for the core protein of decorin.

scholarly journals Tensin2 is important for podocyte-glomerular basement membrane interaction and integrity of the glomerular filtration barrier

2020 ◽  
Vol 318 (6) ◽  
pp. F1520-F1530
Author(s):  
Kozue Uchio-Yamada ◽  
Keiko Yasuda ◽  
Yoko Monobe ◽  
Ken-ichi Akagi ◽  
Osamu Suzuki ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Glomerular Filtration ◽  
Glomerular Basement Membrane ◽  
Dependent Manner ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Gbm Thickening ◽  
Strain Dependent ◽  
Deficient Mice

Tensin2 (Tns2), an integrin-linked protein, is enriched in podocytes within the glomerulus. Previous studies have revealed that Tns2-deficient mice exhibit defects of the glomerular basement membrane (GBM) soon after birth in a strain-dependent manner. However, the mechanisms for the onset of defects caused by Tns2 deficiency remains unidentified. Here, we aimed to determine the role of Tns2 using newborn Tns2-deficient mice and murine primary podocytes. Ultrastructural analysis revealed that developing glomeruli during postnatal nephrogenesis exhibited abnormal GBM processing due to ectopic laminin-α2 accumulation followed by GBM thickening. In addition, analysis of primary podocytes revealed that Tns2 deficiency led to impaired podocyte-GBM interaction and massive expression of laminin-α2 in podocytes. Our study suggests that weakened podocyte-GBM interaction due to Tns2 deficiency causes increased mechanical stress on podocytes by continuous daily filtration after birth, resulting in stressed podocytes ectopically producing laminin-α2, which interrupts GBM processing. We conclude that Tns2 plays important roles in the podocyte-GBM interaction and maintenance of the glomerular filtration barrier.

scholarly journals Endothelial cells interact with the core protein of basement membrane perlecan through beta 1 and beta 3 integrins: an adhesion modulated by glycosaminoglycan.

1992 ◽  
Vol 119 (4) ◽  
pp. 945-959 ◽  
Author(s):  
K Hayashi ◽  
J A Madri ◽  
P D Yurchenco
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Basement Membrane ◽  
Core Protein ◽  
Affinity Column ◽  
Heparin Binding ◽  
The Core ◽  
Heparan Sulfates ◽  
Domain Iii ◽  
Endothelial Basement Membrane

Aortic endothelial cells adhere to the core protein of murine perlecan, a heparan sulfate proteoglycan present in endothelial basement membrane. We found that cell adhesion was partially inhibited by beta 1 integrin-specific mAb and almost completely blocked by a mixture of beta 1 and alpha v beta 3 antibodies. Furthermore, adhesion was partially inhibited by a synthetic peptide containing the perlecan domain III sequence LPASFRGDKVTSY (c-RGD) as well as by GRGDSP, but not by GRGESP. Both antibodies contributed to the inhibition of cell adhesion to immobilized c-RGD whereas only beta 1-specific antibody blocked residual cell adhesion to proteoglycan core in the presence of maximally inhibiting concentrations of soluble RGD peptide. A fraction of endothelial surface-labeled detergent lysate bound to a core affinity column and 147-, 116-, and 85-kD proteins were eluted with NaCl and EDTA. Polyclonal anti-beta 1 and anti-beta 3 integrin antibodies immunoprecipitated 116/147 and 85/147 kD surface-labeled complexes, respectively. Cell adhesion to perlecan was low compared to perlecan core, and cell adhesion to core, but not to immobilized c-RGD, was selectively inhibited by soluble heparin and heparan sulfates. This inhibition by heparin was also observed with laminin and fibronectin and, in the case of perlecan, was found to be independent of heparin binding to substrate. These data support the hypothesis that endothelial cells interact with the core protein of perlecan through beta 1 and beta 3 integrins, that this binding is partially RGD-independent, and that this interaction is selectively sensitive to a cell-mediated effect of heparin/heparan sulfates which may act as regulatory ligands.

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 Glomerular basement membrane proteoglycans are derived from a large precursor.

1988 ◽  
Vol 106 (3) ◽  
pp. 963-970 ◽  
Author(s):  
D J Klein ◽  
D M Brown ◽  
T R Oegema ◽  
P E Brenchley ◽  
J C Anderson ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Core Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Immunological Methods ◽  
Core Proteins ◽  
Single Precursor ◽  
Species Being

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.

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