Fibromodulin and chondroadherin in connective tissue homeostasis

Sammanfattning: Connective tissue contains a prominent extracellular matrix (ECM), which provides for physiological and mechanical properties. Intra- and inter-molecular interactions between proteins in this ECM provide for the formation of molecular networks. These interactions determine the shape of the tissue as well as cell behavior. This thesis is focused on the leucine-rich repeat proteoglycans fibromodulin and chondroadherin and their interactions with other proteins in the ECM. The N-terminal domain of fibromodulin has a highly negative charge due to the presence of the posttranslational modification of O-sulfate on tyrosine residues. We hypothesized that this domain could mimic heparin binding to proteins containing domains with clusters of basic amino acids. Interactions to synthetic peptides with heparin binding properties, representing the basic domains of the ECM proteins chondroadherin and PRELP and the cytokine oncostatin M were identified. Additional interactions were found with the heparin binding proteins MMP-13, basic FGF, type IX collagen, thrombospondin-1 and interleukine-10. Fibromodulin can then function as a linker in the ECM, binding proteins containing basic domains through its N-terminal domain and other proteins, e.g. collagen. This may determine the appearance and properties of the collagen network. Fibromodulin may also function to sequester proteases and bioactive molecules in the matrix. Further, other functions of this domain were found. Presence of the domain enhanced the overall affinity of fulllength fibromodulin to collagen and it positively regulated collagen fibril formation in vitro. The regulatory role was evaluated by electron microscopy that showed involvement of the domain in the fine-tuning of collagen molecules assembly into highly organized fiber structures. The C-terminal domain of chondroadherin contains a cluster of basic amino acids, which besides binding fibromodulin, binds heparin. This thesis describes interaction of this domain with heparan sulfate chains attached to a cell surface proteoglycan. Upon binding this short peptide stimulated bound chondrocytes to spread and increased the attachment through integrins, which resulted in the formation of focal adhesions. Further, the short peptide elicited cell signaling via the MAPK/ERK signaling pathway. Chondroadherin exists in two forms, where one of the forms represents a cleavage product that lacks the last nine amino acids, i.e. including the heparin binding site. This cleavage may play an important role in tissue homeostasis through feedback regulation to the cell. Finally, the integrin binding sequence of chondroadherin was identified by protease digestion of the protein followed by mass spectrometry. The domain was shown to be located in one of the C-terminal disulfide loop structures. Cells adhered to a peptide containing the integrin binding site remain rounded, but yet rapidly induced MAPK/ERK signaling pathway. Thus, binding may be central for maintaining the chondrocyte phenotype and homeostasis of adult cartilage, e.g. through release of growth factors, proteases and molecules for matrix assembly. This thesis describes biologically active domains of the protein fibromodulin and chondroadherin. Binding of the described domains to other molecules is proposed to be important for the ECM assembly and how cells communicate with the surrounding extracellular matrix.