scholarly journals Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane.

1981 ◽  
Vol 90 (2) ◽  
pp. 527-532 ◽  
Author(s):  
Y S Kanwar ◽  
V C Hascall ◽  
M G Farquhar
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Density Gradient ◽  
Glomerular Basement Membrane ◽  
Chromatographic Behavior ◽  
Acid Oxidation ◽  
Sulfate Proteoglycan ◽  
Cartilage Proteoglycan ◽  
Core Proteins

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.

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.

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.

Decreased Glomerular Basement Membrane Heparan Sulfate Proteoglycan in Essential Hypertension

Hypertension ◽  
1995 ◽  
Vol 25 (3) ◽  
pp. 399-407 ◽  
Author(s):  
Bernhard Heintz ◽  
Georg Stöcker ◽  
Christian Mrowka ◽  
Uwe Rentz ◽  
Heinrich Melzer ◽  
...  
Keyword(s):  
Essential Hypertension ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan

scholarly journals Association between heparan sulfate proteoglycan excretion and proteinuria after renal transplantation.

1996 ◽  
Vol 7 (12) ◽  
pp. 2670-2676
Author(s):  
I Stefanidis ◽  
B Heintz ◽  
G Stöcker ◽  
C Mrowka ◽  
H G Sieberth ◽  
...  
Keyword(s):  
Renal Transplantation ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Excretion Rate ◽  
Heparan Sulfate Proteoglycan ◽  
Heparan Sulfate Proteoglycans ◽  
Second Phase ◽  
Sulfate Proteoglycan ◽  
Glomerular Proteinuria

The aim of the study presented here was to investigate whether, in patients showing immediate graft function after renal transplantation, cold-ischemia and reperfusion lead to damage of the glomerular basement membrane and consequently to a loss of heparan sulfate proteoglycans. Loss of these heparan sulfate proteoglycans is a major cause of proteinuria. Time-dependent changes in urinary excretion rates of heparan sulfate proteoglycans but also of total protein, albumin, low- and high-molecular-weight proteins were analyzed quantitatively and by polyacrylamid-gel-electrophoresis in eight patients. Immediately after renal transplantation, severe proteinuria with an excretion rate of up to 251 +/- 108 mg/min was apparent and rapidly declined within 24 h to 4.11 +/- 2.80 mg/min. The gel-electrophoretic pattern showed a nonselective glomerular and tubular proteinuria. The excretion rate of heparan sulfate proteoglycan was increased in this initial reperfusion phase (up to 7 h), most probably because of ischemia- and reperfusion-induced damage of the glomerular basement membrane. The initial nonselective glomerular proteinuria disappeared within 48 h, changing to a mild selective glomerular proteinuria. In this second phase (7 to 48 h), lower levels of heparan sulfate proteoglycan excretion were observed (0.54 +/- 0.54 microgram/min versus 1.66 +/- 1.93 micrograms/min, P < 0.05). However, during the repair process of the glomerular basement membrane, heparan sulfate proteoglycan is synthesized de novo, leading to an increasing heparan sulfate proteoglycan content of the glomerular basement membrane. This second phase is paralleled by the change from a nonselective to a selective glomerular proteinuria. In the third phase, when the heparan sulfate proteoglycan content of the glomerular basement membrane normalizes, glomerular proteinuria was abolished in most of the patients.

scholarly journals The Glomerular Basement Membrane as a Model System to Study the Bioactivity of Heparan Sulfate Glycosaminoglycans

2012 ◽  
Vol 18 (1) ◽  
pp. 3-21 ◽  
Author(s):  
Kevin J. McCarthy ◽  
Deborah J. Wassenhove-McCarthy
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Molecular Genetic ◽  
Research Area ◽  
Critical Elements ◽  
Core Proteins ◽  
Transmembrane Flux ◽  
Anionic Charge ◽  
A Charge

AbstractThe glomerular basement membrane and its associated cells are critical elements in the renal ultrafiltration process. Traditionally the anionic charge associated with several carbohydrate moieties in the glomerular basement membrane are thought to form a charge selective barrier that restricts the transmembrane flux of anionic proteins across the glomerular basement membrane into the urinary space. The charge selective function, along with the size selective component of the basement membrane, serves to limit the efflux of plasma proteins from the capillary lumen. Heparan sulfate glycosaminoglycans are anionically charged carbohydrate structures attached to proteoglycan core proteins and have a role in establishing the charge selective function of the glomerular basement membrane. Although there are a large number of studies in the literature that support this concept, the results of several recent studies using molecular genetic approaches to minimize the anionic charge of the glomerular basement membrane would suggest that the role of heparan sulfate glycosaminoglycans in the glomerular capillary wall are still not yet entirely resolved, suggesting that this research area still requires new and novel exploration.

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