Identification of binding partners interacting with the α1-N-propeptide of type V collagen

The predominant form of type V collagen is the [alpha 1(V)](2)alpha 2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the alpha 1(V)- and alpha 2(V)-collagen chain, cause classic EDS (Ehlers-Danlos syndrome), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remain unexplained. Type V collagen controls collagen fibrillogenesis through its conserved alpha 1(V)-N-propeptide domain. To gain an insight into the role of this domain, a yeast two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the N-propeptide as a bait. We identified 12 interacting proteins, including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or co-immunoprecipitation: alpha 1(1)- and alpha 2(1)-collagen chains, alpha 1(VI)-, alpha 2(V1)- and alpha 3(VI)-collagen chains, tenascin-C, fibronectin, PCPE-1 (procollagen C-proteinase enhancer-1), TIMP-1 (tissue inhibitor of metalloproteinases-1), MMP-2 (matrix metalloproteinase 2) and TGF-beta 1 (transforming growth factor beta 1). Solid-phase binding assays confirmed the involvement of the alpha 1(V)-N-propeptide in the interaction between native type V collagen and type VI collagen, suggesting a bridging function of this protein complex in the cell-matrix environment. Enzymatic studies showed that processing of the alpha 1(V)-N-propeptide by BMP-1 (bone morphogenetic protein 1)/procollagen C-proteinase is enhanced by PCPE-1. These interactions are likely to be involved in extracellular matrix homoeostasis and their disruption could explain the pathogenetic mechanism in unresolved classic EDS cases.