Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 > 100 nm > 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the α(IV) and α2(IV) chains. Three peptides from the known sequence of the α1(IV) and α2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the α1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the α2(NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the α1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.
|Original language||English (US)|
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|State||Published - 1989|