Original article A fibrillin-1-fragment containing the elastin-binding-protein GxxPG consensus sequence upregulates matrix metalloproteinase-1: biochemical and computational analysis Patrick Booms a,1 , Andreas Ney a,1 , Frank Barthel b , Gautier Moroy c , Damian Counsell d , Christoph Gille e , Gao Guo a , Reinhard Pregla b , Stefan Mundlos a,f , Alain J.P. Alix c , Peter N. Robinson a,* a Institute of Medical Genetics, Charité University Hospital, Humboldt University, Augustenburger Platz, 13353 Berlin, Germany b German Heart Institute, Berlin, Germany c Laboratoire de Spectroscopies et Structures BioMoléculaires (LSSBM), IFR 53 Biomolécules, UFR Sciences Exactes et Naturelles, Université de Reims Champagne-Ardenne, Reims cedex 2, France d Rosalind Franklin Center for Genomic Research, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK e Institute for Biochemistry, Charité University Hospital, Humboldt University, Berlin, Germany f Max Planck Institute for Molecular Genetics, Berlin, Germany Received 13 July 2005; received in revised form 23 November 2005; accepted 29 November 2005 Abstract Mutations in the gene for fibrillin-1 cause Marfan syndrome (MFS), a common hereditary disorder of connective tissue. Recent findings suggest that proteolysis, increased matrix metalloproteinase activity, and fragmentation of fibrillin-rich microfibrils in tissues of persons with MFS contribute to the complex pathogenesis of this disorder. In this study we show that a fibrillin-1 fragment containing a EGFEPG sequence that conforms to a putative GxxPG elastin-binding protein (EBP) consensus sequence upregulates the expression and production of matrix metalloproteinase (MMP)-1 by up to ninefold in a cell culture system. A mutation of the GxxPG consensus sequence site abrogated the effects. This is the first demonstration of such an effect for ligands other than elastin fragments. Molecular dynamics analysis of oligopeptides with the wildtype and mutant sequence support our biochemical results by predicting significant alterations of structural characteristics such as the poten- tial for forming a type VIII β-turn that are thought to be important for binding to the EBP. These results suggest that fibrillin-1 fragments may regulate MMP-1 expression, and that the dysregulation of MMPs related to fragmentation of fibrillin might contribute to the development of MFS. Our Gene Ontology (GO) analysis of the human proteome shows that proteins with multiple GxxPG motifs are highly enriched for GO terms related to the extracellular matrix. Matrix proteins with multiple GxxPG sites include fibrillin-1, -2, and -3, elastin, fibronectin, laminin, and several tenascins and collagens. Some of these proteins have been associated with disorders involving alterations in MMP regulation, and the results of the present study suggest a potential mechanism for these observations. © 2006 Elsevier Ltd. All rights reserved. Keywords: Extracellular matrix; Fibrillin; Marfan syndrome; Matrix metalloproteinase; Elastin-binding protein; Molecular dynamics; Gene Ontology 1. Introduction The Marfan syndrome (MFS) is a relatively common her- editary disorder of connective tissue with prominent manifesta- tions in the cardiovascular, ocular, and skeletal systems [1,2]. The major cause of death in untreated persons with MFS is aortic dissection, which generally occurs following gradual di- latation of the aortic root over a period of many years. Muta- tions in the gene for fibrillin-1, FBN1, were shown to be the cause of MFS in 1991 [3]. However, the pathogenetic mechan- isms leading from a FBN1 mutation to clinical disease have not been completely elucidated. Several mechanisms have been proposed, ranging from a dominant negative effect of mutant monomers on the highly polymeric fibrillin-rich microfibrils [4], disturbances of tissue homeostasis [5,6], and alterations www.elsevier.com/locate/yjmcc Journal of Molecular and Cellular Cardiology 40 (2006) 234–246 * Corresponding author. E-mail address: peter.robinson@charite.de (P.N. Robinson). 1 The first two authors contributed equally to this work. 0022-2828/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.11.009