Collagen XXVII Organises the Pericellular Matrix in the Growth Plate Darren A. Plumb1,2¤a, Laila Ferrara1,2¤b, Tanja Torbica1,2, Lynnette Knowles2, Aleksandr Mironov, Jr.2, Karl E. Kadler1,2, Michael D. Briggs1,2, Raymond P. Boot-Handford1,2* 1 Wellcome Trust Centre for Cell-Matrix Research, The University of Manchester, Manchester, United Kingdom, 2 Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom Abstract In order to characterise the function of the novel fibrillar type XXVII collagen, a series of mice expressing mutant forms of the collagen were investigated. Mice harboring a glycine to cysteine substitution in the collagenous domain were phenotypically normal when heterozygote and displayed a mild disruption of growth plate architecture in the homozygous state. Mice expressing an 87 amino acid deletion in the collagenous domain of collagen XXVII were phenotypically normal as heterozygotes whereas homozygotes exhibited a severe chondrodysplasia and died perinatally from a lung defect. Animals expressing the 87 amino acid deletion targeted specifically to cartilage were viable but severely dwarfed. The pericellular matrix of proliferative chondrocytes was disrupted and the proliferative cells exhibited a decreased tendency to flatten and form vertical columns. Collagen XXVII plays an important structural role in the pericellular extracellular matrix of the growth plate and is required for the organisation of the proliferative zone. Citation: Plumb DA, Ferrara L, Torbica T, Knowles L, Mironov A Jr, et al. (2011) Collagen XXVII Organises the Pericellular Matrix in the Growth Plate. PLoS ONE 6(12): e29422. doi:10.1371/journal.pone.0029422 Editor: Nuno M. Neves, University of Minho, Portugal Received June 20, 2011; Accepted November 28, 2011; Published December 19, 2011 Copyright: 2011 Plumb et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors are grateful for the following support for this study: AR UK grant (17243 to RBH), National Institutes of Health grant (RO1 AR49547-01 to MDB, RBH and KEK), Eurogrow (LSHM-CT-2007-037471), MRC, and the Wellcome Trust (071161/Z/03/Z to MDB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ray.boot-handford@manchester.ac.uk ¤a Current address: Hospital for Special Surgery, New York, New York, United States of America ¤b Current address: Al-Fateh University, Tripoli, Libya Introduction The classical fibrillar collagens (types I, II, III, V and XI) comprise the major structural elements of the interstitial (ECM) matrix of vertebrates. These collagens share highly conserved C-terminal non- collagenous domains and uninterrupted major collagenous domains of 1011-1017 amino acid residues. At the N-terminus of the major triple helical domain is a short, non-collagenous telopeptide sequence followed by a second much shorter collagenous sequence termed the ‘minor’ helical domain. Finally at the N-terminus of each proa chain there is usually a von Willebrand factor C domain (type A clade genes) or a variable domain flanked by a thrombospondin motif (type B clade genes) (reviewed in [1]). These triple helical molecules co-polymerise to form the cross-striated fibrils apparent in connective tissues when negatively stained and viewed by electron microscopy [2,3]. Type XXVII collagen is a novel member of the fibrillar collagen gene family [4,5]. This homotrimeric collagen, together with the closely related type XXIV collagen [6], differ from the classical fibrillar collagens in several notable respects. The major triple helical domains of these novel members of the fibrillar collagen family are shorter than their classical counterparts being 991–997 amino acid residues in length. Type XXIV and XXVII collagens have two interruptions in the characteristic collagen Gly-X-Y repeat at conserved locations in their major helical domains. In addition, types XXIV and XXVII collagen lack the N-terminal telopeptide region and the N-terminal minor helical domain that characterise the classical fibrillar collagens. The N-terminus of both novel types of fibrillar collagen consists of a ‘variable’ domain and a thrombos- pondin domain similar to that of the type B clade genes. Phylogenetic analysis reveals that types XXVII and XXIV collagen form a distinct clade (named type C) within the fibrillar collagen family [4–6]. Type XXVII collagen is expressed in a variety of tissues during development including skin, stomach, gonad, lung, aorta and tooth but its most prominent expression is in cartilage [4,5,7]. Expression is particularly high in proliferative zone chondrocytes of the epiphyseal growth plate [7,8]. The SOX9 [9] and Lc-Maf [10] transcription factors have been shown to control chondrocyte expression of type XXVII collagen. Immunolocalisation of type XXVII collagen in the skeleton revealed weak pericellular staining around articular chondrocytes and in the growth plate, stronger staining in the matrix surrounding proliferative chondrocytes that became intense as the matrix around hypertrophic chondrocytes condensed [7,8]. Immuno-electron microscopy of cartilage extracts revealed that type XXVII collagen appears to form thin non-striated fibrils perhaps organised in a network but certainly distinct from the cross-striated fibrils formed by the classical fibrillar collagens [7,8]. In order to characterise further the function of type XXVII collagen, we generated a series of mice expressing mutant forms of type XXVII collagen. We decided to introduce mutations into the collagen XXVII gene rather than knock it out for the following PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29422