Mapping of macrophage elastase cleavage sites in insoluble human skin elastin Samuel Taddese a , Anthony S. Weiss b , Reinhard H.H. Neubert a , Christian E.H. Schmelzer a, ⁎ a Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany b School of Molecular and Microbial Biosciences, University of Sydney, Sydney, Australia Received 24 December 2007; received in revised form 3 February 2008; accepted 4 February 2008 Abstract Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28–31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed. © 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: MMP-12; Insoluble elastin; Matrix metalloproteinases; Elastolysis; Mass spectrometry; Cleavage site specificity 1. Introduction MMPs are members of a tightly regulated zinc- and calcium- dependent family of enzymes that are capable of degrading numerous pericellular substrates and thus play an important role in extracellular matrix turnover (Kähäri and Saarialho-Kere, 1997; Sternlicht and Werb, 2001). They are synthesized as prepro- enzymes and secreted in most cases as inactive pro-MMPs. Their activation essentially requires the disruption of a specific Cys–Zn 2+ interaction (cysteine switch), and the removal of the propeptide which often proceeds in a stepwise manner (Nagase, 1997). In vivo, most pro-MMPs are likely to be activated by tissue or plasma proteinases or opportunistic bacterial proteinases (Nagase and Woessner, 1999). The macrophages, which are intimately associated with many inflammatory diseases, are rich sources of MMPs, from which MMP-12 is one of the most active against elastin (Shapiro, 1999; Chung et al., 2002; Filippov et al., 2003). Its abnormal expression in tissues is associated with many pathological conditions, including chronic obstructive pulmonary disease (Molet et al., 2005; Nenan et al., 2005; Demedts et al., 2006), abdominal aortic aneurysms (Campa et al., 1987; Longo et al., 2005), and atherosclerosis (Matsumoto et al., 1998). The unusual expression of MMPs in the skin is also associated with many skin disorders, including cutaneous wound healing, bullous diseases, granulomas, tumors, carcinoma, melanoma, and dermal photoaging (Kähäri and Saarialho-Kere, 1997; Okada et al., 1997; Saarialho-Kere et al., 1999; Vaalamo et al., 1999; Herouy, 2001). MMP-12 plays an important role in many of these skin conditions and is especially associated with granulomatous skin disorders and photoaging (Chung et al., 2002). Although elastin comprises only 2 to 4% of the total protein content of the skin (Rosenbloom et al., 1993) it is an essential component of the skin that provides recoil and resilience. Its degradation is responsible for many problems including an aged Available online at www.sciencedirect.com Matrix Biology 27 (2008) 420 – 428 www.elsevier.com/locate/matbio ⁎ Corresponding author. Tel.: +49 345 5525215; fax: +49 345 5527292. E-mail address: schmelzer@pharmazie.uni-halle.de (C.E.H. Schmelzer). 0945-053X/$ - see front matter © 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2008.02.001