Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L.
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 CatD-processed 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.
Baechle
Daniel
D
Medical and Natural Sciences Research Center, University of Tübingen, Ob dem Himmelreich 7, 72074 Tübingen, Germany.
Flad
Thomas
T
Cansier
Alexander
A
Steffen
Heiko
H
Schittek
Birgit
B
Tolson
Jonathan
J
Herrmann
Timo
T
Dihazi
Hassan
H
Beck
Alexander
A
Mueller
Gerhard A
GA
Mueller
Margret
M
Stevanovic
Stefan
S
Garbe
Claus
C
Mueller
Claudia A
CA
Kalbacher
Hubert
H
D000595:Amino Acid Sequence; D002402:Cathepsin D / Q000378:metabolism*; D004439:Eccrine Glands / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013542:Sweat / Q000737:chemistry / Q000201:enzymology
D010455:Peptides; C442243:dermcidin; D002402:Cathepsin D