Purification and properties of different isoforms of bovine cathepsin B

Deval, Christiane; Béchet, Daniel; Obled, Alain; Ferrara, Marc

doi:10.1139/o90-121

Cited by 15 publications

(3 citation statements)

References 27 publications

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“…The 5 kDa light chain does not stain very strongly, and is easily distinguished on the gel. A similar result was obtained by Deval et at. (1990) who also isolated cathepsin B from bovine liver.…”

Section: Resultssupporting

confidence: 91%

Endolysosomal proteolysis and its regulation

Pillay

Elliott

Dennison

2002

Biochemical Journal

236

216

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The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H+-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

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Section: Resultssupporting

confidence: 91%

Endolysosomal proteolysis and its regulation

Pillay

Elliott

Dennison

2002

Biochemical Journal

236

216

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“…Different isoforms (charged forms) of cath B may therefore not be the only ones responsible for the change in substrate specificity. This is further supported by the findings of Deval et al (1990) who demonstrated two different purified isoforms of cath B with very similar pH optimum profiles, and that one single isolated isoform of cath B may display a pH profile with 'shoulders' in the acidic and in the slightly alkaline range. Hasnain et al (1992) found that British Journal of Cancer (1997) 75(8) Werle et al, 1995;Ledakis et al, 1996) (Sheahan et al, 1989), who found that the tumour cath B activity at pH 8.0 was more stable than in adjacent colon tissue.…”

Section: Discussionsupporting

confidence: 67%

Cathepsin B fraction active at physiological pH of 7.5 is of prognostic significance in squamous cell carcinoma of human lung

Werle¹,

Jülke²,

Lah³

et al. 1997

Br J Cancer

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Summary In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumours and normal lung tissue by means of fluorogenic assays with Z-Arg-Arg-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With respect to pH dependence of activity, we found a deviation from a normal-shaped pHactivity curve. Besides the typical activity peak at pH 6.0, there were shoulders at pH 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumour and normal tissue. To test the stability of cath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82-100% of residual cath B activity was found at pH 5.0-5.5, whereas activity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was still 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 is more abundant in tumour tissues compared with the normal counterparts, we determined this fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction active at pH 7.5 in tumour tissue, although this fraction represented only a small part (about 16%) of the native, acidic (pH 6.0) cath B activity. However, in contrast to native cath B at 6.0, the cath B fraction active at pH 7.5 was related to post-operative probability of survival in curatively operated patients, since activity values higher than 292 (,uEU mg-' protein) were significantly associated with poor prognosis in patients with squamous cell carcinomas (n = 33, P = 0.04). It is concluded that in lung tumour and in normal lung tissue, cath B activity can be divided into at least three fractions with stability optima at different pH values, indicating various forms of cath B. The cath B fraction active at pH 7.5 provides prognostic information in patients with squamous cell carcinoma.

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“…These substrates for Cathepsin B were not compared in controlled experiments before, though there was Cathepsin B activity reported on a variety of short peptides, with a typical k cat ranging from 1 to 364 s –1 and K M from 108 to 1190 μM, which vary by enzyme source and test conditions (Table S1). − These comparisons would help identify whether mAb carrier is a factor impacting drug release rate and, therefore, inform the design of ADC constructs.…”

Section: Introductionmentioning

confidence: 99%

Cathepsin B Cleavage of vcMMAE-Based Antibody–Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific

et al. 2016

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Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different sites is not a critical parameter to control in manufacturing of the vc-MMAE-based ADC conjugated at reduced disulfide bonds.

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Purification and properties of different isoforms of bovine cathepsin B

Cited by 15 publications

References 27 publications

Endolysosomal proteolysis and its regulation

Endolysosomal proteolysis and its regulation

Cathepsin B fraction active at physiological pH of 7.5 is of prognostic significance in squamous cell carcinoma of human lung

Cathepsin B Cleavage of vcMMAE-Based Antibody–Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific

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