Constitutive- and accelerated shedding of syndecan-1, a co-receptor of FGF-signalling, is mediated by cleavage of its core protein at a specific juxtamembrane site

Syndecan-1 (Sdc-1) is a developmentally regulated cell surface heparan sulfate proteoglycan. It functions as a coreceptor for a variety of soluble and insoluble ligands, including members of the FGF-family, and is implicated in several biological processes, including differentiation, cell migration, morphogenesis, and feeding behavior. The extracellular domain of Sdc-1 can be proteolytically cleaved at a juxtamembrane site by TIMP-3-sensitive metalloproteinases in response to a variety of physiological stimulators and stress in a process known as shedding. Shedding converts Sdc-1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands.

Objectives: The aim of this study was to identify the shedding site of murine Sdc-1.

Methods: Sdc-1 cDNA constructs with mutated juxtamembrane regions were expressed in cultured cells, transgenic mice were generated, and the amount of shed Sdc-1 was quantified. The shedding site of purified Sdc-1 was identified by Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-ESI-MS).

Results: siRNA-mediated knockdown of Sdc-1 expression in the human breast cancer cell line MCF-7 lead to a decrease in FGF-2-mediated MAP kinase signaling, demonstrating its co-receptor role in the context of malignant diseases. In addition, we found that replacing the Sdc-1 juxtamembrane amino acid residues A243SQSL247 with human CD4 amino acid residues can completely block PMA-induced Sdc-1 ectodomain shedding. Furthermore, using LC-ESI-MS, we identified the proteolytic cleavage site of Sdc-1 to amino acids A243 and S244, generated by constitutive and PMA-induced shedding from murine mammary gland (NMuMG) cells. Finally, we show that basal cleavage of Sdc-1 utilizes the same in vivo site as the in vitro site: Transgenic mice expressing the Sdc-1/CD4 cDNA do not shed the Sdc-1 ectodomain in vivo.

Conclusion: The same cleavage site is utilized for basal Sdc-1 ectodomain shedding both in vitro and in vivo. The identification of this site represents a first step towards the development of shedding inhibitors.