scholarly journals The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures.

1989 ◽  
Vol 108 (4) ◽  
pp. 1567-1574 ◽  
Author(s):  
D S Grant ◽  
C P Leblond ◽  
H K Kleinman ◽  
S Inoue ◽  
J R Hassell
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Three Dimensional ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Lamina Densa ◽  
Dimensional Network ◽  
Type C

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.

scholarly journals Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

1988 ◽  
Vol 36 (3) ◽  
pp. 271-283 ◽  
Author(s):  
D S Grant ◽  
C P Leblond
Keyword(s):  
Heparan Sulfate ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Enamel Organ ◽  
Sulfate Proteoglycan ◽  
Lamina Densa ◽  
Type Iv ◽  
Reichert's Membrane

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

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 Association of malachite green-positive material with heparan sulfate proteoglycan double tracks in basement membrane of mouse kidney tubules.

1995 ◽  
Vol 43 (3) ◽  
pp. 293-297
Author(s):  
S Inoue
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Malachite Green ◽  
Heparan Sulfate Proteoglycan ◽  
Mouse Kidney ◽  
Basement Membranes ◽  
Kidney Cortex ◽  
Sulfate Proteoglycan ◽  
Kidney Tubules ◽  
Thin Sections

The presence of lipids in the basement membrane of the mouse kidney tubules was examined by histochemical staining with malachite green. Pieces of mouse kidney cortex were immersed in a fixative containing 3% glutaraldehyde and 0.1% malachite green in 0.067 M sodium cacodylate buffer, pH 6.8, for 18 hr at 4 degrees C. Control tissue was fixed in the same way except that no malachite green was added to the fixative. The tissue pieces were cryoprotected, frozen in Freon 22, and subjected to freeze-substitution in dry acetone containing 1% OsO4. Thin sections of Epon-embedded specimens were observed by electron microscopy at first without uranyl-lead counterstaining. The basement membrane of mouse kidney tubules was positively stained in a pattern composed of an irregular assembly of 5-8-nm wide strands. The nature of these malachite green-positive strands was further examined by counterstaining thin sections with uranyl-lead, and they were identified as 4.5-5-nm wide ribbon-like "double tracks" previously characterized as the form taken by heparan sulfate proteoglycan in basement membranes. It is concluded that lipids are present in the basement membrane of mouse kidney tubules in association with heparan sulfate proteoglycan.

scholarly journals Immunoelectron microscopy of endothelial cells in rat incisor suggests that most basement membrane components are produced by young cells, whereas heparan sulfate proteoglycan is produced by both young and old cells.

1988 ◽  
Vol 36 (7) ◽  
pp. 763-773 ◽  
Author(s):  
I C Murray ◽  
C P Leblond
Keyword(s):  
Endothelial Cells ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
Incisor Tooth ◽  
Rat Incisor ◽  
Membrane Components ◽  
Basement Membrane Components

When periodontal capillaries of rat incisor tooth were immunostained for four basement membrane components (laminin, collagen IV, fibronectin, heparan sulfate proteoglycan), all four were detected in the secretory organelles of endothelial cells located within 3 mm of the tooth's proximal end, but only the proteoglycan was observed in cells located 4 mm away and beyond (Experiment I). [3H]-Thymidine autoradiography revealed that the endothelial cells located at the tooth's proximal end were young and actively dividing, whereas those located 4 mm or more away were older and generally quiescent (Experiment II). Since immunostaining of a cell's secretory organelles for a given substance indicates production of this substance, the first experiment shows that endothelial cells at the proximal end produce the four basement membrane components. The second experiment discloses that these cells are young. As for the endothelial cells located 4 mm or more beyond the proximal end, the first experiment reveals that they produce only heparan sulfate proteoglycan, while the second shows that they are relatively old. Production of laminin, collagen IV, and fibronectin only by young cells implies that these substances are long-lived and stable components of basement membrane, whereas production of the proteoglycan by both young and old cells implies that it is labile and continually replaced.

scholarly journals Agrin Is a Major Heparan Sulfate Proteoglycan in the Human Glomerular Basement Membrane

1998 ◽  
Vol 46 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Alexander J. Groffen ◽  
Markus A. Ruegg ◽  
Henri Dijkman ◽  
Thea J. van de Velden ◽  
Carin A. Buskens ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Linear Distribution ◽  
Cell Matrix ◽  
Sds Page ◽  
Strong Staining

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactiv-ity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200–210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction.

scholarly journals Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells.

1983 ◽  
Vol 31 (1) ◽  
pp. 35-45 ◽  
Author(s):  
I Leivo
Keyword(s):  
Cell Membrane ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Type Iv Collagen ◽  
Heparan Sulfate Proteoglycan ◽  
Ruthenium Red ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Type Iv ◽  
The Matrix

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.

scholarly journals High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations.

1988 ◽  
Vol 107 (4) ◽  
pp. 1599-1610 ◽  
Author(s):  
J C Schittny ◽  
R Timpl ◽  
J Engel
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycan ◽  
Low Density ◽  
Sulfate Proteoglycan ◽  
Lamina Densa ◽  
Epithelial Basement Membrane ◽  
Domain 3 ◽  
Epithelial Basement ◽  
Domain 4

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

scholarly journals Heparan sulfate proteoglycan is present in basement membrane as a double-tracked structure.

1989 ◽  
Vol 37 (5) ◽  
pp. 597-602 ◽  
Author(s):  
S Inoue ◽  
D Grant ◽  
C P Leblond
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Core Protein ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Parallel Lines ◽  
Reichert's Membrane

Basement membranes contain 4.5-nm wide sets of two parallel lines, along which short prongs called "spikes" occur at regular intervals. The nature of this structure, referred to as "double tracks," was investigated in Lowicryl sections of mouse kidney and rat Reichert's membrane immunolabeled for basement membrane components using secondary antibodies conjugated to 5-nm gold particles. When the mouse glomerular basement membrane and rat Reichert's membrane were exposed to antibodies directed to the core protein of heparan sulfate proteoglycan, 95% or more of the gold particles were over double tracks, whereas after exposure of Reichert's membrane to antisera against laminin, collagen IV, or entactin, labeling of the double tracks remained at the random level. When heparan sulfate proteoglycan was incubated in Tris buffer, pH 7.4, at 35 degrees C for 1 hr, a precipitate resulted which, on electron microscopic examination, was found to consist of 5- to 6-nm wide sets of two parallel lines along which densities were observed. Immunolabeling confirmed the presence of the proteoglycan's core protein in the sets. Since double tracks were closely similar to this structure and were labeled with the same antibodies, they were likely to be also composed of heparan sulfate proteoglycan.

scholarly journals Localization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin to the basal lamina of basement membranes.

1982 ◽  
Vol 95 (1) ◽  
pp. 340-344 ◽  
Author(s):  
G W Laurie ◽  
C P Leblond ◽  
G R Martin
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Basal Lamina ◽  
Type Iv Collagen ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Enamel Organ ◽  
Sulfate Proteoglycan ◽  
Type Iv ◽  
Membrane Region

Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-microns thick slices of formaldehyde-fixed tissues. In the basement-membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries. The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.

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