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 Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan.

1989 ◽  
Vol 109 (6) ◽  
pp. 3187-3198 ◽  
Author(s):  
K J McCarthy ◽  
M A Accavitti ◽  
J R Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Core Protein ◽  
Rat Kidney ◽  
Basement Membranes ◽  
Chondroitin Sulfate Proteoglycan ◽  
In Vivo Model ◽  
Rat Tissues ◽  
Sulfate Proteoglycan ◽  
Reichert's Membrane

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.

scholarly journals Distinct effects of different matrix proteoglycans on collagen fibrillogenesis and cell-mediated collagen reorganization

2020 ◽  
Author(s):  
Dongning Chen ◽  
Lucas R. Smith ◽  
Gauri Khandekar ◽  
Pavan Patel ◽  
Christopher K. Yu ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Core Protein ◽  
Complex Mixture ◽  
List Type ◽  
Collagen Fibrillogenesis ◽  
Fibrous Network ◽  
Large Matrix ◽  
Carbohydrate Components ◽  
And Function

AbstractThe extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.HighlightsSmall leucine rich proteoglycans (SLRPs) and large chondroitin sulfate (CS) proteoglycans (PGs) have distinct effects on collagen fibrous network behavior.Unlike other matrix proteoglycans, versican promotes collagen fibrillogenesis in an in vitro spectrophotometric (turbidity) assay.The versican core protein has a larger impact on collagen behavior in a fibrillogenesis assay than its glycosaminoglycan chains do.Versican increases the diameter of collagen fibers and the porosity of collagen fibrous networks, unlike aggrecan and SLRPs.The addition of versican to collagen does not alter fibroblast contractility but leads to enhanced cell-mediated collagen reorganization and contraction.

scholarly journals Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues.

1990 ◽  
Vol 38 (10) ◽  
pp. 1479-1486 ◽  
Author(s):  
K J McCarthy ◽  
J R Couchman
Keyword(s):  
Chondroitin Sulfate ◽  
Dermatan Sulfate ◽  
Core Protein ◽  
Rat Kidney ◽  
Basement Membranes ◽  
Rat Tissues ◽  
Adult Rat ◽  
Unique Distribution ◽  
Reichert's Membrane

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.

scholarly journals The primary structure of NG2, a novel membrane-spanning proteoglycan.

1991 ◽  
Vol 114 (2) ◽  
pp. 359-371 ◽  
Author(s):  
A Nishiyama ◽  
K J Dahlin ◽  
J T Prince ◽  
S R Johnstone ◽  
W B Stallcup
Keyword(s):  
Amino Acids ◽  
Chondroitin Sulfate ◽  
Primary Structure ◽  
Disulfide Bonds ◽  
Transmembrane Domain ◽  
Core Protein ◽  
Extracellular Domain ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Extracellular Region

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.

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 Characterization of a novel heparan sulfate proteoglycan found in the extracellular matrix of liver sinusoids and basement membranes.

1991 ◽  
Vol 113 (5) ◽  
pp. 1231-1241 ◽  
Author(s):  
C J Soroka ◽  
M G Farquhar
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Rat Liver ◽  
Core Protein ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Sinusoidal Cell ◽  
Space Of Disse

A novel heparan sulfate proteoglycan (HSPG) present in the extracellular matrix of rat liver has been partially characterized. Proteoglycans were purified from a high salt extract of total microsomes from rat liver and found to consist predominantly (approximately 90%) of HSPG. A polyclonal antiserum raised against this fraction specifically recognized HSPG by immunoprecipitation and immunoblotting. The intact, fully glycosylated HSPG migrated as a broad smear (150-300 kD) by SDS-PAGE, but after deglycosylation with trifluoromethanesulfonic acid only a single approximately 40-kD band was seen. By immunocytochemistry this HSPG was localized in the perisinusoidal space of Disse associated with irregular clumps of basement membrane-like extracellular matrix material, some of which was closely associated with the hepatocyte sinusoidal cell surface. It was also localized in biosynthetic compartments (rough ER and Golgi cisternae) of hepatocytes, suggesting that this HSPG is synthesized and deposited in the space of Disse by the hepatocyte. The anti-liver HSPG IgG also stained basement membranes of hepatic blood vessels and bile ducts as well as those of kidney and several other organs (heart, pancreas, and intestine). An antibody that recognizes the basement membrane HSPG found in the rat glomerular basement membrane did not precipitate the 150-300-kD rat liver HSPG. We conclude that the liver sinusoidal space of Disse contains a novel population of HSPG that differs in its overall size, its distribution and in the size of its core protein from other HSPG (i.e., membrane-intercalated HSPG) previously described in rat liver. It also differs in its core protein size from HSPG purified from other extracellular matrix sources. This population of HSPG appears to be a member of the basement membrane HSPG family.

scholarly journals Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development.

1993 ◽  
Vol 41 (3) ◽  
pp. 401-414 ◽  
Author(s):  
K J McCarthy ◽  
K Bynum ◽  
P L St John ◽  
D R Abrahamson ◽  
J R Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Basement Membranes ◽  
Chondroitin Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
Electron Microscopic ◽  
Ureteric Buds ◽  
Membrane Components ◽  
Almost All ◽  
Basement Membrane Components

We previously reported the presence of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG) in basement membranes of almost all adult tissues. However, an exception to this ubiquitous distribution was found in the kidney, where BM-CSPG was absent from the glomerular capillary basement membrane (GBM) but present in other basement membranes of the nephron, including collecting ducts, tubules, Bowman's capsule, and the glomerular mesangium. In light of this unique pattern of distribution and of the complex histoarchitectural reorganization occurring during nephrogenesis, the present study used light and electron microscopic immunohistochemistry to examine the distribution of BM-CSPG and basement membrane heparan sulfate proteoglycan (BM-HSPG) during prenatal and postnatal renal development in the rat. Our results show that the temporal and spatial pattern of expression of BM-CSPG during nephrogenesis is unlike that reported for other basement membrane components such as laminin, fibronectin, and BM-HSPG, all of which can be found in the earliest formed basement membranes of the vesicle-stage nephron. Although BM-CSPG is present in the basement membranes of the invading vasculature and ureteric buds, its first appearance in nephron basement membrane occurs during the late comma stage. In capillary loop-stage glomeruli of prenatal animals, BM-CSPG is present in the presumptive mesangial matrix but undetectable in the GBM. However, as postnatal glomerular maturation progresses BM-CSPG is also found in both the lamina rara interna and lamina densa of the GBM in progressively increasing amounts, being most evident in the GBM of 21-day-old animals. Micrographs of glomeruli from 42-day-old animals show that BM-CSPG gradually disappears from the GBM and, by 56 days after birth, appears to be completely absent from the GBM, its pattern of distribution resembling that of the adult animal. Our results show that BM-CSPG is not required for the initial assembly of basement membranes but may in fact serve to stabilize basement membrane structure after histoarchitectural reorganization is completed.

Alveolar epithelial cells in vitro produce gelatinases and tissue inhibitor of matrix metalloproteinase-2

1997 ◽  
Vol 273 (3) ◽  
pp. L663-L675 ◽  
Author(s):  
M. P. d'Ortho ◽  
C. Clerici ◽  
P. M. Yao ◽  
C. Delacourt ◽  
C. Delclaux ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Matrix Metalloproteinase ◽  
Primary Cultures ◽  
Basement Membranes ◽  
Alveolar Epithelial Cells ◽  
Matrix Metalloproteinase 2 ◽  
Type Ii ◽  
Matrix Metalloproteinase 1 ◽  
Type Ii Pneumocytes

Type II pneumocytes are key cells of the alveolar epithelium. They lie on the alveolar basement membrane, which influences their phenotype and functions. We hypothesized that type II pneumocytes degrade basement membrane components by producing gelatinases, members of the matrix metalloproteinase family. To investigate this hypothesis, we used primary cultures of rat type II pneumocytes and cultures of the human A549 cell line. We found by zymography that 70-kDa gelatinase was present in media conditioned by these cells. This 70-kDa gelatinase was identified as gelatinase A by a Western blot, and the presence of its mRNA was demonstrated by reverse transcription-polymerase chain reaction. A 95-kDa gelatinase could be induced under certain conditions. Production of gelatinases may take place during the turnover of basement membranes, in physiological and in pathophysiological processes. This was suggested by the increase in production of both gelatinases that we observed after in vitro exposure to LPS or interleukin-1. The presence of tissue inhibitors of matrix metalloproteinase-1 and -2 was also demonstrated, suggesting that degradation of extracellular matrix by type II pneumocytes is tightly regulated.

scholarly journals Formation of basement membranes in the embryonic kidney: an immunohistological study

1981 ◽  
Vol 91 (1) ◽  
pp. 1-10 ◽  
Author(s):  
P Ekblom
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Basement Membranes ◽  
Glomerular Endothelial Cell ◽  
Type Iv ◽  
Endothelial Cell Differentiation ◽  
Inductive Interaction ◽  
Immunohistological Study

Specific antibodies to laminin, type IV collagen, basement-membrane proteoglycan, and fibronectin have been used in immunofluorescence microscopy to study the development of basement membranes of the embryonic kidney. Kidney tubules are known to form from the nephrogenic mesenchyme as a result of an inductive tissue interaction. This involves a change in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses fibronectin but no detectable laminin, type IV collagen, or basement-membrane proteoglycan. During the inductive interaction, basement-membrane specific components (laminin, type IV collagen, basement membrane proteoglycan) become detectable in the induced area, whereas fibronectin is lost. While the differentiation to epithelial cells of the kidney requires an inductive interaction, the development of the vasculature seems to involve an ingrowth of cells which throughout development deposits basement-membrane specific components, as well as fibronectin. These cells form the endothelium and possibly also the mesangium of the glomerulus, and contribute to the formation of the glomerular basement membrane. An analysis of differentiation of the kidney mesenchyme in vitro in the absence of circulation supports these conclusions. Because a continuity with vasculature is required for glomerular endothelial cell differentiation, it is possible that these cells are derived from outside vasculature.

scholarly journals Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro.

1984 ◽  
Vol 99 (3) ◽  
pp. 947-961 ◽  
Author(s):  
D Gospodarowicz ◽  
J Lepine ◽  
S Massoglia ◽  
I Wood
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Cell Monolayer ◽  
Basement Membranes ◽  
Low Density ◽  
Kidney Tubule ◽  
Confluent Monolayer ◽  
Bovine Kidney ◽  
Fibroblastic Cells

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.

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