EMILIN-3, a peculiar member of the EMILIN/Multimerin protein family, has a distinct expression pattern, forms oligomeric assemblies and serves as a pro TGF-β antagonist.

EMILIN-3 is a glycoprotein of the extracellular matrix belonging to a family that contains a characteristic N-terminal cysteine-rich EMI domain. Currently, EMILIN-3 is the least characterized member of the EMILIN/Multimerin family. Using RNA, immunohistochemical and protein chemistry approaches, we carried out a detailed characterization of the expression and of the biochemical properties of EMILIN-3 in mouse. During embryonic and postnatal development, EMILIN-3 showed a peculiar and dynamic pattern of gene expression and protein distribution. EMILIN-3 mRNA was first detected at E8.5-E9.5 in the tail bud and in the primitive gut, and at later stages it became abundant in the developing gonads and in the osteogenic mesenchyme. Interestingly, and in contrast to other EMILIN/Multimerin genes, EMILIN-3 was not found in the cardiovascular system. Despite the absence of the globular C1q domain, immunoprecipitation and western blot analyses demonstrated that EMILIN-3 forms disulfide-bonded homotrimers and higher-order oligomers. Circular dichroism spectroscopy indicated that the most C-terminal part of EMILIN-3 has a substantial α-helical content and forms coiled-coil structures, involved in EMILIN-3 homo-oligomerization. Transfection experiments with recombinant constructs showed that the EMI domain contributes to the higher-order self-assembly but was dispensable for homotrimer formation. EMILIN-3 was found to bind heparin with high affinity, a property mediated by the EMI domain, thus revealing a new function for this domain that may contribute to the interaction of EMILIN-3 with other ECM and/or cell surface molecules. Finally, in vitro experiments showed that EMILIN-3 is able to function as an extracellular regulator of the activity of TGF-β ligands.