Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V

The C-terminal perlecan domain V of about 90 kDa consists of laminin-type G domain modules (LG) (25 kDa) and epidermal growth factor-like modules (EG) (4 kDa) in the tandem arrangement LG1-EG1-EG2-LG2-EG3-EG4-LG3. Several sho rter fragments have been prepared by recombinant production in mammalian ce lls and used to map the single glycosaminoglycan (GAG) substitution site an d the binding of several carbohydrate and protein ligands. This identified a Ser3511 residue located in a short link region between EG4 and LG3 as bei ng involved in GAG attachment. :Electron microscopy provided evidence that the same substitution exists in tissue forms of perlecan. Heparan sulphate attached to this site was shown to bind to the alpha 1LG4 module of laminin -l, indicating a role in basement membrane assembly and cell-matrix interac tions. This site is also close to an Asn-Asp bond which is readily cleaved by an endogenous protease that depends on the presence of Asp and the LG2 m odule. A weak:heparin binding site was shown to include the EG2 module, whi ch contains five basic residues. Binding to sulphatides and the alpha-dystr oglycan receptor was much stronger and required at least two LG modules. Ho wever, single LG modules appear to be sufficient for the interaction with t he laminin-nidogen complex, while EG3-4 and some flanking regions are appar ently involved in fibulin-2 binding. These observations indicate that a com plex modular structure is required for domain V in order to provide a rich repertoire of potential biological functions. (C) 1999 Academic Press.