Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is thought to be involved in the promotion of cell adhesion, the promotion of cell gro wth in the cancerous state, and the modulation of blood coagulation. These activities are consistent with the proposed function of HIP/L29 as a hepari n/heparan sulfate (Hp/HS) binding growth factor that has a preference for a nticoagulantly active Hp/HS. Previous studies showed that a peptide derived from the C terminus of human HIP/L29 (HIP peptide-1) can selectively bind anticoagulant Hp and support cell adhesion. However, a murine ortholog does not have an identical HIP peptide-1 sequence, yet still retains the abilit y to bind Hp, suggesting that there may be additional Hp/HS binding sites o utside of the HIP peptide-1 domain. To test this hypothesis, a systematic s tudy of the domains within human and murine HIP/L29 responsible for Hp/HS b inding activity was undertaken. Using deletion mutants, proteolytic fragmen ts, and protease protection of HIP/L29 by Hp, we demonstrate that multiple binding domains contribute to the overall Hp/HS binding activity of HIP/L29 proteins. Furthermore, a conformational change is induced in human HIP/L29 upon I-Ip binding as detected by circular dichroism spectroscopy. These st udies demonstrate the multiplicity of Hp/HS binding sequences within human and murine HIP/L29.