Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the nature of a protein domain that is shared among lysosomal hydrolases and is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues. Previously, elements of this recognition domain were identified using a chimeric protein approach. The combined substitution of two regions (amino acids 188-230, particularly lysine 203, and 265-292) from the carboxyl lobe of the lysosomal hydrolase cathepsin D into the homologous positions of the related secretory protein glycopepsinogen was sufficient to confer recognition by phosphotransferase and subsequent phosphorylation of the oligosaccharides when this chimeric protein was expressed in Xenopus oocytes. (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). The current study demonstrates that when these two regions are replaced in cathepsin D by the homologous glycopepsinogen amino acids, the resultant chimeric molecule is poorly phosphorylated. However, when either of these regions is substituted individually, the chimeric molecules are well phosphorylated. The phosphorylation of these latter chimeric proteins is dependent on the presence of procathepsin D amino lobe elements. By analyzing a series of chimeric proteins that contain all eight combinations of three consecutive segments of the entire amino lobe of procathepsin D, it was found that multiple regions of the amino lobe of cathepsin D enhance phosphorylation of the chimeric proteins. These elements may be part of an extended carboxyl lobe recognition domain or comprise a second independent recognition domain.
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PMID:Lysosomal enzyme phosphorylation. I. Protein recognition determinants in both lobes of procathepsin D mediate its interaction with UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. 133 Oct 81

The propeptides of lysosomal enzymes have been implicated in membrane association and mannose 6-phosphate-independent sorting to the lysosome (Rijnboutt, S., Aerts, H., Geuze, H. J., Tager, J. M., and Strous, G. J. (1991) J. Biol. Chem. 266, 4862-4868; McIntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445). In this report, the function of the propeptide of procathepsin D in sorting to the lysosome was directly assessed using a cathepsin D deletion mutant lacking the propeptide, and using a chimeric cDNA encoding the cathepsin D propeptide fused to the secretory protein alpha-lactalbumin. Proteins encoded by these cDNAs were expressed in mouse Ltk- cells and in human hepatoma Hep G2 cells, and then immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The deletion mutant was glycosylated but was rapidly degraded in a chloroquine-independent fashion and did not assume an active conformation. Thus the propeptide appeared to be necessary for correct folding. The chimeric protein was glycosylated and secreted. The coincidence of complex oligosaccharide modification and secretion of the chimeric protein suggested that it was slowly released from the endoplasmic reticulum and rapidly passed through the cell to the extracellular compartment. This was confirmed by immunofluorescent localization of the proteins. The data indicated that the propeptide appeared to be necessary for folding of cathepsin D but, unlike the yeast vacuolar propeptides, was not sufficient to direct a secretory protein to the lysosome in fibroblasts or in epithelial cells.
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PMID:The role of the cathepsin D propeptide in sorting to the lysosome. 140 Apr 84

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose-6-P residues. Previously, we generated a lysosomal enzyme recognition domain by substituting two regions (lysine 203 and amino acids 265-292) of the lysosomal hydrolase cathepsin D into a related secretory protein glycopepsinogen. When expressed in Xenopus oocytes, the oligosaccharides of the chimeric protein were efficiently phosphorylated (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). In the current study, incremental substitutions of cathepsin D residues into glycopepsinogen and alanine-scanning mutagenesis were utilized to define the recognition domain more precisely. A computer-generated model of the cathepsin D/pepsinogen chimeric molecule served as a guide for mutagenesis and for the interpretation of results. These studies indicate that the recognition domain is a surface patch that contains multiple interacting sites. There is a strict positional requirement for the lysine residue at position 203.
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PMID:Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting. 166 Apr 71

We have investigated the effect of a glycosylphosphatidylinositol anchor on the distribution of the soluble lysosomal enzyme cathepsin D. Only 10% of the chimeric protein (CD-GPI) could be detected on the plasma membrane after transfection in CHO cells. Similarly to endogenous cathepsin D, intracellular CD-GPI was detected in vesicular structures, suggesting that CD-GPI is targeted to lysosomes. CD-GPI is present as three forms with M(r) 55, 50 and 37 kD which could correspond to the precursor, intermediate and mature forms of cathepsin D, respectively. CD-GPI was shown to be GPI anchored by differential extractability with Triton X-114 before and after phosphatidylinositol phospholipase C hydrolysis. Intracellular CD-GPI is mainly substituted with oligosaccharides containing uncovered mannose 6-phosphate residues whereas these residues are covered in the cell surface precursor form of CD-GPI. Ammonium chloride treatment reduces the lysosomal delivery of CD-GPI and increases the cell surface expression of its precursor form.
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PMID:Localization and processing of glycosylphosphatidylinositol anchored cathepsin D. 759 25

The transmembrane domain of Golgi resident proteins such as beta-galactoside alpha 2,6-sialyltransferase (ST) and N-acetylglucosaminyltransferase 1 (NT) contain a Golgi retention signal which confers Golgi retention to reporter proteins appended to them in the appropriate context. Thus, chimeras of the cell surface protein dipeptidyl peptidase IV containing the transmembrane domain of ST and NT are retained in the Golgi apparatus in MDCK and COS cells, as assessed by indirect immunofluorescence microscopy. Transfection of these chimeric constructs into CHO cells, however, results in their transport to vesicular structures which do not colocalize with that of an endogenous Golgi marker, mannosidase II. Furthermore, the staining pattern of these structures are not affected by brefeldin A. Biochemical analysis of the transgene products in pulse-chase experiments revealed that the chimeric proteins eventually become resistant to endoglycosidase H, suggesting that they are transported beyond the medial Golgi and therefore the vesicular structures are likely to be post-Golgi. The vesicular structures colocalized well with a lysosomal marker, cathepsin D, and also with internalized FITC-dextran chased into the lysosomal compartment. Monitoring the cell surface appearance of the chimeric protein suggests that the majority is transported directly to the lysosomal compartment. Golgi retention can be completely restored for ST and improved for NT by the inclusion of sequences flanking the transmembrane domain. Our results reflect cell type differences in the interpretation of the transmembrane domain Golgi retention signal, established that general Golgi retention of type II glycosyltransferases requires the hydrophilic flanking sequence as well as the transmembrane domain, and demonstrate that proteins which escape Golgi retention may be channeled to the lysosomal pathway.
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PMID:Cell type differences in Golgi retention signals for transmembrane proteins. 765 2

Microsomal-type cytochrome P450s are integral membrane proteins bound to the membrane through their N-terminal transmembrane hydrophobic segment, the signal anchor sequence. To elucidate the determinants that enable the P450s to be located in the ER, we constructed cDNAs encoding chimeric proteins in which a secretory form of carboxyesterase, carboxyesterase Sec, was connected to the N-terminus of the full-length or truncated forms of a microsomal-type P450, P450(M1), and the constructed plasmids were expressed in COS cells. Since carboxyesterase Sec is an N-glycosylated secretory protein, endo H treatment could be used to determine whether these chimeric proteins were located in the ER or not. Carboxyesterase Sec with the N-terminal 20 amino acids, containing the transmembrane region, of P450(M1), was located in the ER, as determined from the endo H sensitivity of the expressed protein and immunofluorescence staining of the cells. As the expressed protein exhibited carboxyesterase activity, it was not retained in the ER through the BiP-dependent quality control system recognizing unfolded proteins. Another chimeric protein construct in which carboxyesterase Sec was connected to the C-terminal region of rat UDP-glucuronosyltransferase (UDP-GT), that contained a double-lysin ER retention motif, was also located in the ER, as determined from the endo H sensitivity and immunofluorescence staining. On the other hand, the sugar moiety of the carboxyesterase Sec connected to the transmembrane segment of UDP-GT, Sec/GTd, was partially resistant to the endo H treatment. From the results of immunofluorescent staining and cell fractionation, it was concluded that the Sec/GTd product was located in the Golgi apparatus. These observations indicated that the N-terminal hydrophobic segment of P450(M1) is sufficient for the ER membrane retention, whereas the transmembrane segment of UDP-GT is not. To determine whether microsomal P450s are recycled between the ER and Golgi compartments or not, a DNA construct encoding cathepsin D connected to the N-terminus of P450(M1) was prepared and expressed in COS cells. The fusion protein was phosphorylated, but the phosphorylation was sensitive to alkaline phosphatase. As a control, authentic cathepsin D was subjected to phosphorylation of its oligosaccharide chain that was resistant to the alkaline phosphatase treatment. Since GlcNAc-P-transferase, which forms the alkaline phosphatase-resistant phosphodiester in the sugar chains of lysosome-targeting proteins, is located in the Golgi apparatus, it was concluded that the oligosaccharide chain of the cathepsin D portion of the fusion protein was not phosphorylated, and that the chimeric protein did not go to the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal. 779 74

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.
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PMID:Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation. 822 84