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And N-Procollagen Proteinases Important for Collagen Assembly and Tensile Strength

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And N-Procollagen Proteinases Important for Collagen Assembly and Tensile Strength Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength Claudia Brodera, Philipp Arnoldb, Sandrine Vadon-Le Goffc, Moritz A. Konerdingd, Kerstin Bahrd, Stefan Müllere, Christopher M. Overallf, Judith S. Bondg, Tomas Koudelkah, Andreas Tholeyh, David J. S. Hulmesc, Catherine Moalic, and Christoph Becker-Paulya,1 aUnit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany; bInstitute of Zoology, Johannes Gutenberg University, 55128 Mainz, Germany; cTissue Biology and Therapeutic Engineering Unit, Centre National de la Recherche Scientifique/University of Lyon, Unité Mixte de Recherche 5305, Unité Mixte de Service 3444 Biosciences Gerland-Lyon Sud, 69367 Lyon Cedex 7, France; dInstitute of Functional and Clinical Anatomy, University Medical Center, Johannes Gutenberg University, 55128 Mainz, Germany; eDepartment of Gastroenterology, University of Bern, CH-3010 Bern, Switzerland; fCentre for Blood Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z3; gDepartment of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033; and hInstitute of Experimental Medicine, University of Kiel, 24118 Kiel, Germany Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved July 9, 2013 (received for review March 22, 2013) Type I fibrillar collagen is the most abundant protein in the human formation (22). A tight balance between synthesis and break- body, crucial for the formation and strength of bones, skin, and down of ECM is required for the function of all tissues, and tendon. Proteolytic enzymes are essential for initiation of the dysregulation leads to pathophysiological events, such as arthri- assembly of collagen fibrils by cleaving off the propeptides. We tis, cancer, atherosclerosis, aneurysms, and fibrosis (23–34). −/− − − report that Mep1a and Mep1b / mice revealed lower amounts We previously reported that both meprin α and meprin β are fi of mature collagen I compared with WT mice and exhibited signif- expressed in human dermal broblasts and are overexpressed in fi icantly reduced collagen deposition in skin, along with markedly skin brosis, such as keloids (35). In addition, meprins are known decreased tissue tensile strength. While exploring the mechanism to release C- and N-propeptides from type III procollagen in vitro, where the C-propeptide is cleaved at exactly the same site and even of this phenotype, we found that cleavage of full-length human fi procollagen I heterotrimers by either meprin α or meprin β led to more ef ciently than the procollagen C-proteinase (PCP) BMP-1. Here we report that meprin α and meprin β are both C- and N- the generation of mature collagen molecules that spontaneously proteinases for type I procollageninvitroandinvivoandare assembled into collagen fibrils. Thus, meprin α and meprin β are important for the maintenance of connective tissue integrity in skin. unique in their ability to process and release both C- and N-propep- tides from type I procollagen in vitro and in vivo and contribute to Results the integrity of connective tissue in skin, with consequent implica- Meprin α and Meprin β Release C- and N-Propeptides from tions for inherited connective tissue disorders. Heterotrimeric Human Procollagen I and Miniprocollagen α1(I) BIOCHEMISTRY Homotrimers in Vitro. We measured the procollagen proteinase proteolysis | Ehlers–Danlos syndrome | proteomics | fibrosis | activity of meprin α and meprin β using human recombinant connective tissue procollagen I heterotrimer as the substrate (Fig. 1A). Procollagen was incubated with meprin α or meprin β, and time-dependent ollagens are the major components of the extracellular ma- processing was analyzed by Western blotting with specific Ctrix (ECM) responsible for tensile strength and stability of antibodies raised against procollagen α1(I) C-propeptide, connective tissues. Fibril-forming collagens, such as types I, II, N-propeptide, or C-telopeptide. As a control, procollagen I was and III, are secreted into the ECM as precursor molecules, also processed with recombinant BMP-1 in the presence of pro- known as procollagens, that require enzymatic removal of the C- collagen C-proteinase enhancer (PCPE)-1, which is known to and N-propeptides, an essential step in collagen fibril assembly stimulate BMP-1 proteinase activity on fibrillar procollagen (9). (1–6). The N-propeptides of procollagen I are cleaved by a dis- As expected, BMP-1 generated pN-collagen by removing the integrin and metalloproteinase with thrombospondin motifs C-propeptides but not the N-propeptides, corresponding to its (ADAMTS)-2, -3, and -14, whereas excision of the C-propeptide PCP activity (Fig. 1B). Interestingly, meprin α and meprin β were is performed by bone morphogenetic protein-1 (BMP-1) and able to remove both the C-propeptides and the N-propeptides, related tolloid-like proteinases (1, 7–11). thereby releasing mature collagen I (Fig. 1B). Clearly, the mat- As deduced from its known substrates, BMP-1 prefers an uration of procollagen I is more efficient by meprin β compared aspartic acid residue at the P1′ position (1, 8, 12). Notably, this with meprin α, resulting in full conversion of procollagen after 30 specificity for negatively charged amino acid residues in the P1′ min. Incubation with meprin α not only resulted in release of the site is also characteristic of two other members of the astacin full C-propeptides, but also indicated a second cleavage event family, meprin α and meprin β (13). Meprin proteases are further C-terminal within the globular propetide. Similarly, this in expressed in a range of tissues, where they activate or release vitro procollagen C- and N-proteinase activity of meprin α and growth factors and other biologically active peptides, and are involved in immunological and neuropathological processes, angiogenesis, and matrix remodeling (14–20). Author contributions: C.B., T.K., A.T., D.J.S.H., C.M., and C.B.-P. designed research; C.B., P.A., Regulation of ECM remodeling relies on complex molecular S.V.-L.G., K.B., T.K., D.J.S.H., and C.M. performed research; M.A.K., S.M., C.M.O., J.S.B., A.T., interactions and is a key event in many physiological conditions. D.J.S.H., C.M., and C.B.-P. contributed new reagents/analytic tools; C.B., P.A., K.B., T.K., A.T., Defects in the processing of type I procollagen into its mature, and C.B.-P. analyzed data; and C.B. and C.B.-P. wrote the paper. fibril-forming form result in such disorders as Ehlers–Danlos The authors declare no conflict of interest. syndrome type VII (21). This inherited connective tissue disorder This article is a PNAS Direct Submission. involves mutations that prevent removal of the N-propeptide of Freely available online through the PNAS open access option. type I procollagen. Retention of the amino-terminal propeptides 1To whom correspondence should be addressed. E-mail: [email protected]. fi leads to incorporation into the bril of the partially processed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. form pN-collagen, resulting in abnormal connective tissue 1073/pnas.1305464110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305464110 PNAS | August 27, 2013 | vol. 110 | no. 35 | 14219–14224 Downloaded by guest on October 1, 2021 C-propeptide from homotrimeric miniprocollagen α1(I) released after incubation with meprin β confirmed the cleavage site at po- sition Arg1227/Asp1228 (Fig. S1A). We further identified the meprin α and meprin β cleavage sites in the N-propeptides of the procollagen α1(I) and α2(I) chains at positions Tyr166/Asp167 and Tyr81/Asp82, respectively (Fig. 1C and Fig. S2). Meprins Trigger Collagen Fibril Formation in Vitro. Removal of the globular C- and N-propeptides is the key step in the formation of collagen fibrils. Because meprin α and meprin β show both procollagen C- and N-proteinase activity in vitro, we further investigated whether they were able to trigger the self-assembly of mature collagen into fibrils. By electron microscopy after negative staining, de novo collagen fibril formation could be observed when heterotrimeric full-length procollagen I was in- cubated with meprin α or meprin β (Fig. 2). After 60 min, in- cubation with either meprin α or meprin β led to the assembly of fibrils, particularly with meprin β, where the characteristic D- periodicity was apparent (Fig. 2A), consistent with the faster maturation observed by Western blot analysis (Fig. 1B). As expected, BMP-1 was not able to trigger the self-assembly of collagen fibrils; it removed the C-propeptides, but not the N- propeptides, of procollagen I (Figs. 1 and 2 and Fig. S1). Maturation of Type I Procollagen Is Reduced in Skin and Primary −/− − − Fibroblasts from Mep1a and Mep1b / Mice. To determine whether meprins are procollagen proteinases in vivo, skin biopsy specimens as well as cultured primary fibroblasts isolated from −/− −/− WT, Mep1a , and Mep1b mice were analyzed by Western blotting using antibodies against the collagen α1(I) C-propeptide or C-telopeptide. Interestingly, procollagen I processing was −/− − − decreased in the skin of Mep1a and Mep1b / mice (Fig. 3 A and B), as well as in the corresponding primary fibroblasts (Fig. Fig. 1. Cleavage of type I procollagen by meprin α and meprin β.(A) S3A). The activity of both procollagen C- and N-proteinase was −/− − − Schematic of the type I procollagen molecule consisting of an N-terminal affected. Extracts of skin from Mep1a and Mep1b / mice propeptide (NP I), the N-telopeptide (N-telo), the triple helical region, the C- contained lower amounts of mature collagen I compared with telopeptide (C-telo), and the C-terminal propeptide (CP I). (B) Cleavage of the WT skin (Fig. 3A). In addition, significantly decreased − − recombinant procollagen heterotrimer by BMP-1, meprin α, and meprin β.
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