Cathepsin D Is a Good Candidate for the Specific Release of a Stable Hemorphin from HemoglobinIn Vivo:VV-Hemorphin-7

“…Subsequently, the crystal structure of the peptidic inhibitor CH-66 complexed with mouse renin (1SMR) (13) was used to build homology models of octapeptide substrates. The molecular models were then used to examine the similarities and differences among known substrate cleavage sites in mammalian Hb reported previously for the cathepsin D of Schistosoma japonicum (4) and for human cathepsin D (11). The models revealed that the difference in cleavage sites was due, in general, to a single amino acid alteration in the cleavage site (P4-P4Ј) that either enhances or diminishes the enzymatic activity.…”

mentioning

confidence: 96%

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Brinkworth¹,

Prociv²,

Loukas³

et al. 2001

Journal of Biological Chemistry

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Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4 were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH 2 ]Leu-Tyr-Tyr-Ser-NH 2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4 residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.Blood flukes, hookworms, and the malaria parasites are among the most important pathogens of humans in terms of both numbers of people infected and the consequent morbidity and mortality (1). Although phylogenetically unrelated, these parasites all share the same food source; they are obligate blood feeders, or hematophages. Hb from ingested or parasitized erythrocytes is their major source of exogenous amino acids for growth, development, and reproduction; the Hb, a ϳ64-kDa tetrameric polypeptide, is comprehensively catabolized by parasite enzymes to free amino acids or small peptides. Intriguingly, all these parasites appear to employ cathepsin D-like aspartic proteases to make initial or early cleavages in the Hb substrate (2-4).The vertebrate endopeptidase, cathepsin D (EC 3.4.23.5), is a member of the aspartic protease category of hydrolases, which also includes renin, pepsin, chymosin, cathepsin E, HIV 1 protease, and several other enzymes (5, 6). Cathepsin D is expressed in a diverse range of mammalian cells and tissues and is located predominantly in lysosomes (6). The molecule comprises two rather similar lobes, each incorporating a homologous Asp-Thr-Gly catalytic site motif, with the substrate binding groove located between these lobes. In aspartic proteases generally, the nucleophile that attacks...

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“…A definite volume of the material is placed on plates with a micropipette or filter paper disc saturated with the solution studied. The plates are then incubated at 37°C and after 1-96 hours, depending on the activity of proteinases in the sample, the size of the digested protein field is read directly or after sprin- [65]. The values given are approximate, and are expressed in terms of the unit of assay I, which cerresponds to about 1,2 ?g human cathepsin D. The estimate of enzyme required per assay assumes that and unincubated blank is required for each chemical method, but is unnecesary in the radiochemical method.…”

Section: Native Denaturated and Labeled Proteinsmentioning

confidence: 99%

Quantitative determination and localization of cathepsin D and its inhibitors.

Minarowska

Karwowska²,

Gacko³

2009

Folia Histochem Cytobiol.

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Cathepsin D (EC 3.4.23.5) is a lysosomal aspartyl endopeptidase, localized in all cells and tissues, except for mature erythrocytes [1][2][3]. Methods used to determine the activity, concentration and cellular distribution of cathepsin D, but not its inhibitors, have previously been the subject of literature reports [4][5][6][7][8][9][10][11][12]. However, since the time of their publication a number of new substrates and analytical techniques have been implemented. Structure, specificity, mechanism of actionCathepsin D is synthesized in the rough endoplasmic reticulum as preprocathepsin D, built up of 412 amino acid residues [13][14][15]. As a result of cleavage of the 20-amino acid signal prepeptide, it is converted into procathepsin D which undergoes glycosylation and disulphide bridges are formed in its molecule. Procathepsin D is transported from cisterns of the rough endoplasmic reticulum to the Golgi apparatus, from which, with the involvement of mannoso-6-phosphate (M-P-6) receptors, it is transferred to primary lysosomes [16,17]. As the M-6-P receptors are known to occur in the primary lysosomes but not in the mature ones, they can be used to distinguish between these two types of lysosomes [18]. In the acidic environment of the lysosomes (pH 4.5-5.5), due to autocatalytic cleavage of the 44-amino acid propeptide from the N-terminal molecule, procathepsin D is converted into the active one-chain form. The actions of cysteine proteinase, aminopeptidases and carboxypeptidases lead to the formation of an active two-chain form of cathepsin D (Fig. 1). These chains are bound by hydrophobic bonds. The molecular weight of the ultimate mature form of cathepsin D is 48 (14+34) kDa. The proteolytic activities of the one-chain and two-chain forms are very similar [19,20]. Modification of the polypeptide chain, different oligosaccharide composition types and phosphorylation/dephosphorylation in the amino saccharide residues contribute to marked molecular heterogenicity of cathepsin D and cause differences in isoelectric points of the respective isoenzymes between pH 4.5 -6.5 [21,22].The use of peptides with the known primary structure allows identification of amino acid residues that form peptide bonds cleaved by cathepsin D. For this purpose, synthetic peptides [23] and chains A and B of bovine insulin can be used (Fig. 2). Cathepsin D cleaves the peptide bonds found within the polypeptide chain, formed by carboxyl groups of the hydrophobic amino acid residues: aromatic -trypto-

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Cathepsin D Is a Good Candidate for the Specific Release of a Stable Hemorphin from HemoglobinIn Vivo:VV-Hemorphin-7

Cited by 50 publications

References 25 publications

Human cathepsin D.

Human cathepsin D.

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Quantitative determination and localization of cathepsin D and its inhibitors.

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