scholarly journals Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane

1988 ◽  
Vol 252 (1) ◽  
pp. 301-304 ◽  
Author(s):  
W H Baricos ◽  
Y Zhou ◽  
R W Mason ◽  
A J Barrett
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Kidney ◽  
Ph Optimum ◽  
Dose Dependent ◽  
Hydrolysis Of

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

scholarly journals Degradation of glomerular basement membrane by purified mammalian metalloproteinases

1988 ◽  
Vol 254 (2) ◽  
pp. 609-612 ◽  
Author(s):  
W H Baricos ◽  
G Murphy ◽  
Y W Zhou ◽  
H H Nguyen ◽  
S V Shah
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Degradation Products ◽  
Gel Chromatography ◽  
Collagen Types ◽  
Percentage Release ◽  
Dose Dependent

Neutral metalloproteinases degrade components of the extracellular matrix, including collagen types I-V, fibronectin, laminin and proteoglycan. However, their ability to degrade intact glomerular basement membrane (GBM) has not previously been investigated. Incubation of [3H]GBM (50,000 c.p.m.; pH 7.5; 24 h at 37 degrees C) with purified gelatinase or stromelysin (2 units) resulted in significant GBM degradation: gelatinase, 46 +/- 2.2; stromelysin, 59 +/- 5.8 (means +/- S.E.M.; percentage release of non-sedimentable radioactivity; n = 4). In contrast, 2 units of collagenase released only 5.6 +/- 0.52% (n = 3) of the [3H]GBM radioactivity compared with 2.0 +/- 0.15% (n = 7) released from [3H]GBM incubated alone. Sephadex G-200 gel chromatography of supernatants obtained from incubations of [3H]GBM with either gelatinase or stromelysin confirmed the ability of these enzymes to degrade GBM and revealed both high-(800,000) and relatively low-(less than 20,000) Mr degradation products for both enzymes. GBM degradation by gelatinase and stromelysin was dose-dependent (range 0.02-2.0 units), near maximal between pH 6.0 and 8.6, and was completely inhibited (greater than 95%) by 2 mM-o-phenanthroline. Collagenase (2 units) did not enhance the degradation of GBM by either gelatinase (0.02 or 0.2 unit) or stromelysin (0.02 or 0.2 unit). Our results indicate that metalloproteinase-mediated GBM degradation by neutrophils and glomeruli may be attributable to gelatinase (neutrophils) and/or stromelysin (glomeruli) and suggest an important role for these proteinases in glomerular pathophysiology.

Cathepsin L mediates intracellular ileal digestion of gastric intrinsic factor

1995 ◽  
Vol 268 (1) ◽  
pp. G33-G40 ◽  
Author(s):  
M. M. Gordon ◽  
T. Howard ◽  
M. J. Becich ◽  
D. H. Alpers
Keyword(s):  
Degradation Products ◽  
Cathepsin L ◽  
Intrinsic Factor ◽  
Recombinant Baculovirus ◽  
Human Kidney ◽  
Intracellular Degradation ◽  
Basolateral Side ◽  
Renal Tubule Cell

Although acidic proteases of lysosomal origin are implicated in the degradation of intrinsic factor (IF) during cobalamin (cbl) transport across enterocytes and proximal renal tubule cell lines, the enzyme(s) involved in this process is not known. Recombinant (baculovirus-produced) rat 125I-labeled IF (125I-rIF), 43 kDa, added in vivo to the lumen of rat ileum was converted intracellularly to peptides of 33 and 26 kDa. In vitro rat 125I-rIF was degraded to peptides of 33 and 31 kDa by addition of cathepsin L; this conversion was fully inhibited by leupeptin. Western blot analysis using antiserum against denatured native rat IF identified additional cathepsin L degradation products in the 17- to 23-kDa range. In vitro the binding of cobalamin partially inhibited cathepsin L degradation of IF. Rat rIF produced from either insect (Sf9) or mammalian (CHO) cells and native rat IF were all degraded by cathepsin L, although the prominence of the various products differed in the recombinant preparations, being 33 and 36 kDa, respectively. Native rat IF was most sensitive to proteolysis, and no degradation products were identified. Rat 125I-rIF was taken up by LLC-PK1 cells, and 125I from degraded IF appeared abundantly on the basolateral side of cell monolayers by 1 h. The intracellular products of rat rIF in LLC-PK1 cells were the same size as those produced in vitro by the action of cathepsin L. Antiserum against a human kidney cDNA cathepsin L fusion protein easily demonstrated the protease in rat intestinal mucosa, as well as in all other tissues tested. These data suggest that cathepsin L is the protease responsible for the leupeptin-sensitive intracellular degradation of IF.

In vivo biosynthesis and turnover of glomerular basement membrane in diabetic rats

1982 ◽  
Vol 242 (4) ◽  
pp. F385-F389
Author(s):  
M. P. Cohen ◽  
M. L. Surma ◽  
V. Y. Wu
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Diabetic Rats ◽  
Basement Membranes ◽  
Membrane Turnover ◽  
Proline Incorporation ◽  
Basement Membrane Collagen ◽  
Osmotic Lysis ◽  
Specific Activities

Glomerular basement membrane (GBM) was labeled in vivo by the injection of tracer amounts of tritiated proline into normal and streptozotocin-diabetic rats. Basement membrane biosynthesis and turnover were determined from the specific activities of proline and hydroxyproline in samples purified following osmotic lysis of glomeruli isolated 4 h to 12 days after injection. Peak radiolabeling of normal and diabetic GBM occurred within 24-48 h and 48-72 h, respectively, and, when corrected for differences in the serum proline specific activities, [3H]proline incorporation was greater in diabetic than in normal samples. In contrast to the subsequent time-dependent progressive decline in radiolabeling in basement membranes from normal animals, specific activities of proline and hydroxyproline in diabetic glomerular basement membrane did not change significantly over the same period of observation. Renal cortical mass and glomerular basement membrane collagen content were preserved in diabetic animals despite loss of body weight. The findings are compatible with prolongation of glomerular basement membrane turnover in experimental diabetes, and suggest that diminished degradation contributes to the accumulation of glomerular basement membrane that is characteristic of chronic diabetes.

scholarly journals Anti-glomerular basement membrane disease: an update on subgroups, pathogenesis and therapies

2018 ◽  
Vol 34 (11) ◽  
pp. 1826-1832 ◽  
Author(s):  
Mårten Segelmark ◽  
Thomas Hellmark
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
De Novo ◽  
Correct Diagnosis ◽  
Rapidly Progressive Glomerulonephritis ◽  
Human Leukocyte ◽  
Pulmonary Haemorrhage ◽  
Leukocyte Antigen ◽  
Type Iv

Abstract Most patients with anti-glomerular basement membrane (anti-GBM) disease present with rapidly progressive glomerulonephritis with or without pulmonary haemorrhage; however, there are several variants and vigilance is necessary to make a correct diagnosis. Such variants include overlap with anti-neutrophil cytoplasm antibodies-associated vasculitis and membranous nephropathy as well as anti-GBM occurring de novo after renal transplantation. Moreover, patients can present with isolated pulmonary haemorrhage as well as with negative tests for circulating anti-GBM. Virtually all patients with anti-GBM disease have autoantibodies that react with two discrete epitopes on the α3 chain of type IV collagen. Recent evidence suggests that healthy persons have low-affinity natural antibodies reacting with the same epitopes, but most people are protected from developing disease-causing high-affinity autoantibodies by human leukocyte antigen-dependent regulatory T-cells (Tregs). The α3 chain-derived peptides presented by the HLA-DR15 antigen lack the ability to promote the development of such Tregs. The detection of anti-GBM in circulation using the rapid assay test has led to early diagnosis and improved prognosis. However, our present tools to curb the inflammation and to eliminate the assaulting antibodies are insufficient. Only about one-third of all patients survive with functioning native kidneys. More effective therapies need to be developed; agents that inhibit neutrophil recruitment, deplete B cells and cleave immunoglobulin G (IgG) in vivo may become new weapons in the arsenal to combat anti-GBM disease.

scholarly journals Insight into the Effects of Sous Vide on Cathepsin B and L Activities, Protein Degradation and the Ultrastructure of Beef

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1441
Author(s):  
Yantao Yin ◽  
Jailson Pereira ◽  
Lei Zhou ◽  
Jose M. Lorenzo ◽  
Xiaona Tian ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Sarcomere Length ◽  
Cathepsin L ◽  
Myofibrillar Protein ◽  
Potential Mechanism ◽  
Beef Tenderness ◽  
Fragmentation Index ◽  
Sous Vide ◽  
Hydrolysis Of ◽  
Insight Into

This study aimed to evaluate the effects of sous vide cooking (SV) on beef tenderness and its underlying potential mechanism. Beef semimembranosus (SM) were subjected to SV treatments at 45 °C, 55 °C and 65 °C for 4 h. Compared with control samples (CK, cooked at 75 °C until a core temperature of 72 °C was attained), SV treatment significantly promoted the release of cathepsin B and cathepsin L from lysosomes and decreased the shear force of beef SM (p < 0.05). In comparison with CK, samples treated with SV had more hydrolysis of myosin heavy chain and obtained higher myofibrillar fragmentation index, collagen solubility as well as longer sarcomere length (p < 0.05). The current study showed that the proteolysis of myofibrillar protein and collagen induced by cathepsin B and cathepsin L, and the limited longitudinal shrinkage together contributed to the improvement of beef tenderness upon SV.

scholarly journals Porcine deltacoronavirus enters cells via two pathways: A protease-mediated one at the cell surface and another facilitated by cathepsins in the endosome

2019 ◽  
Vol 294 (25) ◽  
pp. 9830-9843 ◽  
Author(s):  
Jialin Zhang ◽  
Jianfei Chen ◽  
Da Shi ◽  
Hongyan Shi ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Cultures ◽  
Viral Entry ◽  
Cathepsin B ◽  
Cathepsin L ◽  
The United States ◽  
Protein Cleavage ◽  
S Protein ◽  
Endosomal Pathway

Porcine deltacoronavirus (PDCoV) is a pathogen belonging to the genus Deltacoronavirus that in 2014 caused outbreaks of piglet diarrhea in the United States. To identify suitable therapeutic targets, a more comprehensive understanding of the viral entry pathway is required, particularly of the role of proteases. Here, we identified the proteases that activate the viral spike (S) glycoprotein to initiate cell entry and also pinpointed the host-cellular pathways that PDCoV uses for entry. Our results revealed that cathepsin L (CTSL) and cathepsin B (CTSB) in lysosomes and extracellular trypsin in cell cultures independently activate the S protein for membrane fusion. Pretreating the cells with the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) completely inhibited PDCoV entry, and siRNA-mediated ablation of CTSL or CTSB expression significantly reduced viral infection, indicating that PDCoV uses an endosomal pathway for entry. Of note, trypsin treatment of cell cultures also activated PDCoV entry, even when the endosomal pathway was inhibited. This observation indicated that trypsin-induced S protein cleavage and activation in cell cultures enables viral entry directly from the cell surface. Our results provide critical insights into the PDCoV infection mechanism, uncovering two distinct viral entry pathways: one through cathepsin L and cathepsin B in the endosome and another via a protease at the cell surface. Because PDCoV infection sites represent a proteases-rich environment, these findings suggest that endosome inhibitor treatment alone is insufficient to block PDCoV entry into intestinal epithelial cells in vivo. Therefore, approaches that inhibit viral entry from the cell membrane should also be considered.

scholarly journals Localization of α-Dystroglycan on the Podocyte: from Top to Toe

2005 ◽  
Vol 53 (11) ◽  
pp. 1345-1353 ◽  
Author(s):  
Nils P.J. Vogtländer ◽  
Henry Dijkman ◽  
Marinka A.H. Bakker ◽  
Kevin P. Campbell ◽  
Johan van der Vlag ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Confocal Microscopy ◽  
Cell Membrane ◽  
Basement Membrane ◽  
Immunoelectron Microscopy ◽  
Glomerular Basement Membrane ◽  
Human Kidney ◽  
Charged Membrane ◽  
Apical Localization

α-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that α-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against α-DG. Therefore, we studied the exact localization of glomerular α-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of α-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that α-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that α-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.

scholarly journals The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B

1988 ◽  
Vol 253 (3) ◽  
pp. 751-758 ◽  
Author(s):  
C Crawford ◽  
R W Mason ◽  
P Wikstrom ◽  
E Shaw
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Inhibitory Effects ◽  
Enzyme Functions ◽  
Calpain Ii

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.

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