Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity

Baechle, Daniel; Cansier, Alexander; Fischer, Rainer; Brandenburg, Jens; Burster, Timo; Driessen, Christoph; Kalbacher, Hubert

doi:10.1002/psc.607

Cited by 16 publications

(18 citation statements)

References 33 publications

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“…We did not observe any significant differences between male and female sweat. Next, we investigated if the ␤-chain of CatD is enzymatically active in sweat by performing in vitro digestion experiments using a specific fluorescent CatD substrate as we reported previously (30). The substrate was incubated for 30 min with purified CatD or pooled sweat, respectively.…”

Section: Identification Of Dominant Proteins In Human Eccrinementioning

confidence: 99%

“…Because no aminopeptidase activity was detectable using the specific substrate H-Leu-AMC (data not shown) this processing step is caused by an endoprotease and not by an aminopeptidase. The third processing step is the cleavage between Leu 29 and Glu 30 by CatD that obtains peptides LEK-26 and SSL-29, which are further processed to the proteolytical stable peptides LEK-24 and SSL-25 (Fig. 8C).…”

Section: Spectra)mentioning

confidence: 99%

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Cathepsin D Is Present in Human Eccrine Sweat and Involved in the Postsecretory Processing of the Antimicrobial Peptide DCD-1L

Baechle

Flad

Cansier

et al. 2006

Journal of Biological Chemistry

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The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu 44 and Asp 45 and between Leu 29 and Glu 30 almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatDprocessed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.

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Section: Identification Of Dominant Proteins In Human Eccrinementioning

confidence: 99%

Section: Spectra)mentioning

confidence: 99%

Cathepsin D Is Present in Human Eccrine Sweat and Involved in the Postsecretory Processing of the Antimicrobial Peptide DCD-1L

Baechle

Flad

Cansier

et al. 2006

Journal of Biological Chemistry

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“…Development of analytical methods involves a search for specific and sensitive substrates (beta-endorphin, synthetic fluorogenic peptides) and new analytical techniques: HPLC [145,220], capillary electrophoresis [221][222][223], fluorimetry in the near infrared region [224], flow cytofluorimetry [225][226][227][228][229], western blot [230][231][232], immunohistochemical techniques [168,169], fluorescence microscopy [233,234] and electron microscopy [235,236].…”

Section: Resultsmentioning

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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“…This peptide was characterized by the 'FF' cleavage site for Cat D recognition and by two biotin residues, which allowed for interaction with streptavidin [31,32].…”

Section: Enzyme-responsive Surfacesmentioning

confidence: 99%

“…The proposed structure was self-assembled onto glass substrates using the streptavidin-biotin interaction. The elements providing both sensing and actuation functions consisted of a biotinylated peptide containing the enzyme cleavage site [31,32].…”

Section: Introductionmentioning

confidence: 99%

Enzyme-responsive multifunctional surfaces for controlled uptake/release of (bio)molecules

Mortato

Argentiere

Gregorio

et al. 2014

Colloids and Surfaces B: Biointerfaces

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Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity

Cited by 16 publications

References 33 publications

Cathepsin D Is Present in Human Eccrine Sweat and Involved in the Postsecretory Processing of the Antimicrobial Peptide DCD-1L

Cathepsin D Is Present in Human Eccrine Sweat and Involved in the Postsecretory Processing of the Antimicrobial Peptide DCD-1L

Quantitative determination and localization of cathepsin D and its inhibitors.

Enzyme-responsive multifunctional surfaces for controlled uptake/release of (bio)molecules

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