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

Abstract

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.

Antimicrobial Peptides, Skin Infections, and Atopic Dermatitis

“…86 Dermcidin is secreted as a proprotein, and post secretory processing by cathepsin D cleaves the peptide from the C terminus of the proprotein thus allowing for the dermcidin peptides to be distributed to the skin surface in sweat. 87 Dermcidin is an anionic peptide whose specific method of killing of microbes is still unknown, however they have been shown to have potent antimicrobial activity against Staphylococcus aureus, Escherichia coli and candida. 88…”

Cathepsin D—Many functions of one aspartic protease

“…In some physiological and pathological conditions, pCD/CD escapes normal targeting mechanism and is secreted from the cells. pCD was found in human, bovine and rat milk [20][21][22], serum [23] and the presence of both pCD and 34 kDa CD was demonstrated in human eccrine sweat and urine [23,24]. pCD is a major secreted protein of numerous types of cancer cells [25].…”

A Cleavage-potentiated Fragment of Tear Lacritin Is Bactericidal

“…1A), which flows onto the eye during basal and reflex tearing (21,31). Lacritin is 21% identical to dermcidin, whose proteolytically processed C terminus contributes to the bactericidal activity of human sweat (26,27,32). We first sought to validate whether basal human tears (33)(34)(35) (Fig.…”

“…1A and 2E), whose proteolytically processed C terminus contributes to the bactericidal activity of human sweat (26,27,31) and is in tears (13). Tear bactericidal activity has been the subject of much curiosity for over a century (7), including the original discovery of lysozyme (15) and a variety of other tear antimicrobial factors, particularly lactoferrin, lipocalin 2, immunoglobulin A antibodies (IgA), secretory phospholipase A 2 , mucins, ␣-and ␤-defensins (20), histatins, and cathelicidin LL-37 (7,19,37), and recently cytokeratin fragments (59).…”

“…Lacritin is 21% identical (25) with dermcidin, whose cathepsin D-releasable C-terminal domain is processed (26) to the potently bactericidal SSL-25 peptide, the main bactericidal activity in sweat (27). These observations were the rationale for challenging cultures of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis with lacritin and lacritin truncation mutants, peptides, and fragments.…”