Constitutive and accelerated shedding of murine syndecan-1 is mediated by cleavage of its core protein at a specific juxtamembrane site

Abstract

Syndecan-1 is a developmentally regulated cell surface heparan sulfate proteoglycan (HSPG). It functions as a coreceptor for a variety of soluble and insoluble ligands and is implicated in several biological processes, including differentiation, cell migration, morphogenesis, and recently feeding behavior. The extracellular domain of syndecan-1 is proteolytically cleaved at a juxtamembrane site by tissue inhibitor of metalloprotease-3 (TIMP-3)-sensitive metalloproteinases in response to a variety of physiological stimulators and stress in a process known as shedding. Shedding converts syndecan-1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. We found that replacing syndecan-1 juxtamembrane amino acid residues A243-S-Q-S-L247 with human CD4 amino acid residues can completely block PMA-induced syndecan-1 ectodomain shedding. Furthermore, using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), we identified the proteolytic cleavage site of syndecan-1 as amino acids A243 and S244, generated by constitutive and PMA-induced shedding from murine NMuMG cells. Finally, we show that basal cleavage of syndecan-1 utilizes the same in vivo site as the in vitro site. Indeed, as predicted, transgenic mice expressing the syndecan-1/CD4 cDNA do not shed the syndecan-1 ectodomain in vivo. These results suggest that the same cleavage site is utilized for basal syndecan-1 ectodomain shedding both in vitro from NMuMG and CHO cells and in vivo.

Figure.1

PMA-accelerated shedding of syndecan-1 ectodomains results from cleavage at a juxtamembrane site in the core protein within amino acid residues A243SQSL247. CHO cells or COS-7 cells were transfected with wild type syndecan-1 (mSyn-1), Syn-1CD4, Syn-1D, Syn-1E, and Syn-1F cDNAs. PMA-accelerated shedding was analyzed by incubating the transfected cells in 6 well plates with or without PMA (1μM) in serum-free medium for 15 minutes. The soluble syndecan-1 ectodomain in the conditioned media was analyzed by a dot blot assay. The fold induction is presented as the ratio of the soluble syndecan-1 ectodomains in conditioned media from PMA stimulated cells compared to DMSO treated controls. The human CD4 amino acid residues are underlined.

Figure 2

Basal shedding of syndecan-1 ectodomain is reduced in mutants with domain swap containing amino acid residues A243SQSL247. CHO cells were transfected with wild type syndecan-1cDNA, syndecan-1CD cDNA or syndecan-1 mutant cDNAs, syn-1D, syn-1E, and syn-1F (cf Figure 1). Basal shedding was analyzed by incubating the transfected cells in 6 well plates in serum-free medium for 15 minutes. The soluble syndecan-1 ectodomain in the conditioned media was analyzed by a dot blot assay and normalized to the syndecan-1 content in total cell lysates. Expression of syndecan-1 in the total cell lysate was extracted was determined to be similar among all the cDNA constructs (data not shown). The relative shedding rate is expressed as the percentage of control (wild-type Syn-1) value. (Error bars = SD, n=3).

Figure 3

On-line LC-ESI-MS analysis of tryptic digests of PMA-stimulated soluble syndecan-1 ectodomain. A. Total ion current trace showing the distribution of tryptic peptides of PMA-stimulated syndecan-1 ectodomain. B. Collision induced decomposition spectrum of the singly charged ion of m/z = 1155.64 from PMA-stimulated syndecan-1 ectodomain.

Figure 3

On-line LC-ESI-MS analysis of tryptic digests of PMA-stimulated soluble syndecan-1 ectodomain. A. Total ion current trace showing the distribution of tryptic peptides of PMA-stimulated syndecan-1 ectodomain. B. Collision induced decomposition spectrum of the singly charged ion of m/z = 1155.64 from PMA-stimulated syndecan-1 ectodomain.

Figure 4

A. On-line LC-ESI-MS analysis of tryptic digests of basal constitutively shed syndecan-1 ectodomain. A. Total ion current trace showing the distribution of tryptic peptides of basal constitutively shed syndecan-1 ectodomain. B. Collision induced decomposition spectrum of the singly charged ion of m/z = 1155.55 from basal constitutively shed syndecan-1 ectodomain.

Figure 5

Generation of transgenic mouse lines carrying mutations in the juxtamembrane region of syndecan-1. a) DNA constructs used for the generation of transgenic mice. Synd/+ = mice overexpressing wild-type syndecan-1, UC/+ mice overexpressing an uncleavable syndecan-1 construct; CS/+ = mice overexpressing a syndecan-1 construct lacking the transmembrane domain (‘constitutively shed’); CMV= cytomegalovirus promoter, TM = transmembrane domain, HG-Poly A = Poly A tail. b) Transgenic mice expressing Syn-1CD4 cDNA do not shed syndecan-1 ectodomain into blood. The relative levels of circulating soluble syndecan-1 ectodomains in 10 μl of plasma were analyzed by a dot blot assay using a ¹²⁵I labeled 281−2 monoclonal antibody against murine syndecan-1 ectodomain. cf Fig 5 for genotype symbols. (Data are shown as mean ± SD).

Figure 6

On-line LC-ESI-MS analysis of tryptic digest of syndecan-1 ectodomain purified from transgenic mice expressing wild type syndecan-1 cDNA. Plasma from transgenic mice expressing the syndecan-1 cDNA was collected and pooled. Affinity purified soluble syndecan-1 ectodomain was subjected to tryptic digestion and LC-MS. A. Total ion current trace showing the distribution of tryptic peptides of syndecan-1 ectodomain purified from the plasma of mice. B. Collision induced decomposition spectrum of the singly charged ion of m/z = 1155.56 of syndecan-1 ectodomain purified from murine plasma.