scholarly journals Proteolytic processing and glycosylation of cathepsin B. The role of the primary structure of the latent precursor and of the carbohydrate moiety for cell-type-specific molecular forms of the enzyme

1992 ◽  
Vol 282 (2) ◽  
pp. 577-582 ◽  
Author(s):  
L Mach ◽  
K Stüwe ◽  
A Hagen ◽  
C Ballaun ◽  
J Glössl
Keyword(s):  
Heavy Chain ◽  
Cathepsin B ◽  
Hepg2 Cells ◽  
Cysteine Proteinase ◽  
Carbohydrate Moiety ◽  
Molecular Forms ◽  
Cell Type ◽  
Single Chain ◽  
Cell Type Specific ◽  
Chain Form

The lysosomal cysteine proteinase cathepsin B is synthesized in cultured human hepatoma HepG2 cells as an inactive 44 kDa precursor and subsequently processed to the mature single-chain enzyme with a molecular mass of 33 kDa. Intralysosomal conversion into the two-chain form results in subunits of 27 kDa, 24 kDa (heavy chain) and 5 kDa (light chain). Enzymic deglycosylation reveals that the 27 kDa polypeptide is the glycosylated variant of the carbohydrate-free 24 kDa heavy-chain form. The intracellular transport to the lysosomes is dependent upon mannose 6-phosphate-containing N-linked oligosaccharides. Receptor-mediated endocytosis of human skin-fibroblast-derived procathepsin B by HepG2 cells resulted in processed molecular forms that are not distinguishable from endogenous cathepsin B, thus favouring rather a cell-type-specific processing than structural differences due to the source of the proenzyme. The conversion step of single-chain catehpsin B into the two-chain enzyme is inhibited in vivo by the irreversible cysteine-proteinase inhibitors Z-Phe-Ala-CHN2 and, albeit weaker, Z-Phe-Phe-CHN2. Both substances have no effect on the activation of procathepsin B to the mature enzyme. The carbohydrate moiety of cathepsin B exerts no significant influence on the stability and the enzymatic activity of the enzyme.

scholarly journals Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself

1993 ◽  
Vol 293 (2) ◽  
pp. 437-442 ◽  
Author(s):  
L Mach ◽  
H Schwihla ◽  
K Stüwe ◽  
A D Rowan ◽  
J S Mort ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Mammalian Cells ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Human Hepatoma ◽  
Single Chain ◽  
Human Hepatoma Cells ◽  
Chain Form

In order to elucidate the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent autoprocessing. When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed. Inhibitors of metallo-, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro. However, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074. Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B. On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself.

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

scholarly journals Human tumour cathepsin B. Comparison with normal liver cathepsin B

1992 ◽  
Vol 285 (2) ◽  
pp. 427-434 ◽  
Author(s):  
K Moin ◽  
N A Day ◽  
M Sameni ◽  
S Hasnain ◽  
T Hirama ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Human Liver ◽  
Normal Liver ◽  
Polyacrylamide Gels ◽  
Single Chain ◽  
Human Tumour ◽  
Double Chain ◽  
Normal Human Liver ◽  
Human Cathepsin ◽  
Chain Form

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.

Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies

2003 ◽  
Vol 81 (4) ◽  
pp. 317-326 ◽  
Author(s):  
M A Sentandreu ◽  
L Aubry ◽  
A Ouali
Keyword(s):  
Cathepsin B ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Peptide Sequence ◽  
Size Exclusion ◽  
Single Chain ◽  
Double Chain ◽  
Bovine Kidney ◽  
Chain Form

Cathepsin B (EC 3.4.22.1) has been highly purified (14 225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl–Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate – polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS–PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.Key words: cathepsin B, bovine kidney, MALDI-TOF, cathepsin B isoforms, antibodies.

Cell-type-specific synthesis of murine immunoglobulin mu RNA from an adenovirus vector

1986 ◽  
Vol 6 (1) ◽  
pp. 123-133
Author(s):  
J E Ruether ◽  
A Maderious ◽  
D Lavery ◽  
J Logan ◽  
S M Fu ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Adenovirus Vector ◽  
Immunoglobulin Heavy Chain ◽  
Adenovirus Type ◽  
Cell Types ◽  
Myeloma Cell ◽  
Host Protein ◽  
Cell Type ◽  
Region Gene ◽  
Cell Type Specific

The mouse immunoglobulin heavy-chain mu constant region gene was cloned into the early region 1B of an adenovirus type 5 vector to allow reproducible kinetics of expression of the mu gene in the presence of continuous host protein synthesis after infection by the recombinant. The immunoglobulin-adenovirus recombinant is helper independent in infecting human fibroblastic and B- and T-cell lines and expresses mu in a cell-type-specific manner. By Northern blot analysis, correctly polyadenylated and spliced E1B-mu S and E1B-mu m mRNAs are found to be equally abundant at steady state in fibroblasts. In contrast, and appropriately, only E1B-mu S mRNAs accumulate in a lambda light-chain-secreting myeloma cell line. Analysis of nascent transcripts pulse labeled in isolated nuclei demonstrates equimolar polymerase loading throughout the mu region in all cell types infected by mu-Ad. Thus, correct polyadenylation and splicing of E1B-mu S and E1B-mu m in fibroblasts does not require transcription termination in the region separating the mu S and mu m polyadenylation sites. Furthermore, differential expression of mu transcripts in the background of myeloma cells is regulated at the level of RNA processing and does not require the presence of the immunoglobulin heavy-chain enhancer or promoter element.

scholarly journals Cell-Type-Specific Gene Transfer into Human Cells with Retroviral Vectors That Display Single-Chain Antibodies

1998 ◽  
Vol 72 (12) ◽  
pp. 10148-10156 ◽  
Author(s):  
An Jiang ◽  
Te-Hua T. Chu ◽  
F. Nocken ◽  
Klaus Cichutek ◽  
Ralph Dornburg
Keyword(s):  
Gene Delivery ◽  
Cell Surface ◽  
Retroviral Vectors ◽  
Human Cells ◽  
Specific Gene ◽  
Cell Type ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Cell Type Specific ◽  
Vector Particles

ABSTRACT The successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from spleen necrosis virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 105 CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.

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