Nonhuman cells correctly sort and process the human lysosomal enzyme cathepsin D

Conner, Gregory E.; Udey, Jenny A.; Pinto, Cristina Barroso; Sola, Juan E.

doi:10.1021/bi00434a057

Cited by 23 publications

(15 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have also confirmed the report that the polypeptide pattern of human cathepsin D is similar in human and in transfected heterologous cells [31]. Further, we have shown that the polypeptide backbones of the two subunits of mature cathepsin D obtained by partial deglycosylation are of a very similar size in the two cell types.…”

Section: Discussionsupporting

confidence: 90%

“…Our data extend previous reports on the formation of a partially processed human cathepsin D in Xenopus laevis oocytes micro-injected with human cathepsin D mRNA [30] and of a two-chain human cathepsin D in various heterologous cells transfected with human cathepsin D cDNA [31]. We have shown that the enzyme expressed in BHK cells is enzymically active and that its enzyme activity and antibody reactivity are similar to those of cathepsin D produced in human cells.…”

Section: Discussionsupporting

confidence: 89%

See 1 more Smart Citation

Expression and maturation of human cathepsin D in baby-hamster kidney cells

Horst

Hasilík

1991

Biochemical Journal

View full text Add to dashboard Cite

In medium and in homogenates from baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA, an elevated activity of cathepsin D was found as compared to non-transfected cells. The elevated activity was removed by titrating the homogenates with an anti-(human cathepsin D) antibody. Metabolic labelling and immunoprecipitation revealed that, in the transfected cells, human cathepsin D was synthesized as a 53-kDa precursor indistinguishable from that found in human cells. A portion of the precursor was secreted and the remainder was processed to intermediate and mature chains within a few hours of synthesis. The precursor that was released from the transfected cells had a slightly smaller apparent size than that from cultured human fibroblasts. This difference was abrogated when the precursors were treated with glycopeptidase F. In the intracellular small chain a difference was observed in the size of carbohydrate chains that were cleavable with endo-,l-N-acetylglucosaminidase H. Sequence analysis of the N-termini of mature intracellular cathepsin D indicated a N-terminal trimming in both large and small chains from both human and transfected hamster cells. The proteolytic maturation of human cathepsin D in BHK cells closely resembles that in human cells, whereas a portion of the carbohydrate side chains is processed differently. The trimmingof the N-termini in mature cathepsin D is proposed to be a part of the maturation and aging of this protein. INTRODUCTIONHuman cathepsin D is a lysosomal pepstatin-sensitive aspartic proteinase consisting of two polypeptides [1]. It is synthesized as a high-molecular-mass (53 kDa) precursor that is subject to maturation upon segregation from the secretory pathway [2]. In a cell-free system using porcine cathepsin D, cDNA synthesis of a preprocathepsin D with an N-terminal signal sequence has been demonstrated [3]. The amino acid sequence of human preprocathepsin D has been deduced from the nucleotide sequence of the corresponding cDNA [4][5][6]. Its sequence is similar

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 89%

Expression and maturation of human cathepsin D in baby-hamster kidney cells

Horst

Hasilík

1991

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…5. Sequences of normal cath-D cDNAs were longer in both humans (14,15) and rodents (16,17) compared with cDNAs isolated from estrogen-stimulated breast cancer cell lines (18,19) or from prion-infected cells (scrapie) (20). Moreover, the TATAA sequence was highly conserved at approximately the same position.…”

Section: Resultsmentioning

confidence: 98%

Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells.

Cavaillès

Augereau

Rochefort

1993

Proc. Natl. Acad. Sci. U.S.A.

139

View full text Add to dashboard Cite

The cathepsin D (cath-D) gene, coding for a ubiquitous Iysosomal aspartyl protease, is overexpressed in aggressive human breast cancers, and its transcription is induced by estrogens in hormone-responsive breast cancer cells. We have determined the structure and function of the proximal 5' upstream region of the human cath-D gene from MCF7 cells. We show that the promoter has a compound structure with features of both housekeeping genes (high G+C content and potential transcription factor Spl sites) and regulated genes (TATAA sequence). By RNase protection assay, we show that transcription is initiated at five major transcription sites (TSSI to -V) spanning 52 base pairs. In hormoneresponsive breast cancer cells, estradiol increased by 6-to 10-fold the level of RNAs initiated at TSSI, which is located about 28 base pairs downstream from the TATA box. The specific regulation by estradiol of transcription starting at site I exclusively was confimed by primer extension. Moreover, the same estradiol effect was observed in the ZR75-1 cell line and in MDA-Ml231 estrogen-resistant breast cancer cells stably transfecte4 with the estrogen receptor. Site-directed mutagenesis in,#1c!Ited that the TATA box is essential for initiation ofcatfll') gene transcription at TSSI. In breast cancer biopsy samples, high levels of TATA-dependent transcription were correlated with overexpression of cath-D mRNA. We conclude that cath-D behaves, depending on the conditions, as a housekeeping gene with multiple start sites or as a hormoneregulated gene that can be controlled from its TATA box.Steroid repeptors increase the initiation of transcription of specific geneX lay interacting with the transcriptional machinery at thy promoter level (1). Cathepsin D (cath-D), a lysosomal gspartyl protease, is induced by estrogens in human breast cancer cell lines (2) and is produced in excess in cancer cells both in vitro and in vivo, where its concentration in the primary tumor is correlated with increased risk of metastasis (for review see ref.3).In eukaryotes, gene expression is controlled by both proximal and distal elements, generally located in the 5' upstream region of the gene (4). Many class II gene promoters contain a TATA box, which binds the transcription factor IID and defines the transcription initiation site; genes with these promoters have been called facultative or regulated genes. In contrast, promoters of housekeeping genes, such as those coding for lysosomal enzymes (5), lack a recognizable TATA box but contain multiple GC boxes acting as putative binding sites for the transcription factor Spl (6).We have previously shown, in human breast cancer cell lines, that estrogens stimulate transcription of the cath-D gene by means of estrogen-responsive sequences located in the proximal region of the promoter (7). Here we show, using RNase protection and primer extension, that cath-D gene transcription in breast cancer cells is initiated at multiple sites by a mixed promoter having characteristics of the promoters of both houseke...

show abstract

“…In order to facilitate this development, we are working to define the subtle differences in ligand/enzyme interactions that exist between cathepsin D and other aspartic proteinases. Previously, in order to examine active site requirements, human fibroblast procathepsin D has been overexpressed in Escherichia coli (17), refolded from solubilized inclusion bodies (18), and purified using pepstatinyl affinity chromatography (19). The resulting recombinant protein is active, nonglycosylated, not fully processed to the mature amino terminus, and retains 18 residues (27p-44p) of the pro-segment.…”

mentioning

confidence: 99%

Self-activation of Recombinant Human Lysosomal Procathepsin D at a Newly Engineered Cleavage Junction, “Short” Pseudocathepsin D

Beyer

Dunn

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

To obtain a recombinant model of human cathepsin D with kinetic properties that are identical with native human liver enzyme, we have addressed the significant differences in structure and catalytic function between naturally occurring enzyme and bacterially derived pseudocathepsin D. Human procathepsin D was expressed in a baculovirus system to obtain correctly folded, glycosylated enzyme that upon acidification completely converts to the active intermediate, pseudocathepsin D. The oligosaccharide moieties of this recombinant enzyme contributed to about 5% of the apparent molecular mass of the enzyme, and the carbohydrate composition was quite similar to the native material. However, specificity constants (k cat /K m ) of this glycosylated pseudoform for several synthetic chromogenic substrates were considerably less (33%-50%) than those for the native enzyme and were virtually identical with those observed with nonglycosylated pseudocathepsin D.A cleavable junction suitable for self-processing at the normal maturation point of human cathepsin D was engineered into procathepsin D according to known specificity requirements of this enzyme, and the construct was expressed using baculovirus. Following experiments that demonstrated that the new proenzyme failed to process to the expected point, the new cleavage junction was moved 6 residues toward the amino terminus of procathepsin D and expressed in Escherichia coli. After refolding, the protein containing the newly engineered junction self-processed, generating a shortened mutant form of pseudocathepsin D that is 6 residues longer at the amino terminus than the native material. The kinetic properties of this newly engineered pseudoform proved to be identical with those of the native enzyme, thus establishing an improved recombinant model for this important aspartic proteinase.The major intracellular aspartic proteinase cathepsin D (EC 3.4.23.5) is normally found in the endosomes and lysosomes of higher eukaryotes (1). This abundant endopeptidase comprises approximately 10% of total protein in the lysosome and is expressed in all mammalian tissues with the exception of erythrocytes (2). In the lysosome, cathepsin D has been shown to be involved in protein turnover and has been postulated to play a role in antigen and prohormone processing (3, 4). Interest in cathepsin D as a target for drug design results from its correlation with several biological processes of pathological significance. Cathepsin D overexpression and activity have been implicated with breast cancer progression (5-7) and the release of ␤-amyloid peptides from the amyloid precursor in Alzheimer's disease (8, 9).Cathepsin D is synthesized on the rough endoplasmic reticulum as a preproenzyme that undergoes several proteolytic cleavages during biosynthesis to produce the mature form (10). Following the initial co-translational removal of the signal peptide to yield procathepsin D, sugars are attached at two N-linked glycosylation sites and the proenzyme is transported to the Golgi stacks. Procathepsi...

show abstract

Nonhuman cells correctly sort and process the human lysosomal enzyme cathepsin D

Cited by 23 publications

References 24 publications

Expression and maturation of human cathepsin D in baby-hamster kidney cells

Expression and maturation of human cathepsin D in baby-hamster kidney cells

Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells.

Self-activation of Recombinant Human Lysosomal Procathepsin D at a Newly Engineered Cleavage Junction, “Short” Pseudocathepsin D

Contact Info

Product

Resources

About