Self-association of N-Syndecan (Syndecan-3) Core Protein Is Mediated by a Novel Structural Motif in the Transmembrane Domain and Ectodomain Flanking Region

Abstract

We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of self-association and formed stable, noncovalent multimeric complexes. The formation of N-syndecan core protein complexes was also demonstrated in mammalian cells by in situ cross-linking. Identification of structural motifs in the core protein of N-syndecan responsible for the formation of these complexes was accomplished by analyzing a series of constructs comprising different regions of the protein as well as sitedirected mutants. Self-association was assayed by SDSpolyacrylamide gel electrophoresis, glutaraldehyde cross-linking, and size-exclusion high pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but not sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acids (ERKE) of the extracellular domain plus the transmembrane domain; (iii) point mutations that changed the basic residues in this sequence to alanine residues either partially or completely abolished the ability of the N-syndecan core protein to form complexes; and (iv) replacement of conserved glycine residues in the transmembrane domain with leucines abolished complex formation. This property is similar to the oligomerization activity of other transmembrane receptors and suggests that regulated self-association may be important for the biological activity of transmembrane proteoglycans.

Syndecans: multifunctional cell-surface co-receptors

“…To test this idea, mutant syndecan-3 core proteins were expressed in which two of the conserved glycine residues were changed to leucine residues. Consistently with the model, these syndecan-3 molecules failed to form stable dimers or other oligomers, presumably as a result of steric hindrance by the bulky leucine side chains [69]. There appear to be specific requirements for the structure of the ectodomain segment as well.…”

“…In syndecan-3 the tetrapeptide adjacent to the transmembrane domain is highly charged (GluArg-Lys-Glu). Mutation of either of the basic amino acids in this segment to alanine decreased the extent of dimer formation that was observed [69]. Together, these results provide strong evidence that syndecan oligomerization results from specific intermolecular interactions.…”

“…Recombinant syndecan-3 core proteins form tight, but non-covalent, dimers, tetramers, and higher-order oligomers that can be detected as SDS-resistant complexes after gel electrophoresis, by covalent cross-linking or by gel-permeation chromatography [69]. Analysis of recombinant core-protein domains and site-directed mutants revealed that the transmembrane domain of the core protein is required, but not sufficient, for oligomerization.…”

“…Syndecan-3 oligomerization can also be observed in membranes of living cells. Chemical cross-linking of syndecan-3 core proteins expressed on the surface of transfected cells reveals that most of the core proteins are present in oligomeric form [69].…”

Indian hedgehog and syndecans‐3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis

“…One explanation can be found in our recent study showing that syndecan-3 is present in dimeric and oligomeric forms on the surface of chondrocytes (Kirsch et al, 2002). Syndecan-3-syndecan-3 interactions had been invoked in previous studies to account for biological properties of the molecule (Asundi and Carey, 1995;Bernfield et al, 1999). We provided evidence that syndecan-3 dimers/oligomers are actually present on the chondrocyte cell surface and their as-sembly probably involves parallel pairing of ␤-sheet segments within the ectodomains of adjacent core proteins.…”