Figure 2.

Cleavage of the extracellular Ca²⁺ binding domain of human SPARC by human cath-D at acidic pH. (A) Time-course of cath-D-induced SPARC cleavage. Recombinant human FL SPARC was incubated with recombinant human auto-activated pseudo-cath-D (51-kDa) in cleavage buffer at pH 5.9 with or without pepstatin A (Pepst.) at 37 °C for the indicated times. SPARC cleavage was analyzed by 13.5% SDS-PAGE and immunoblotting with an anti-SPARC antibody (clone AON-5031). (B) pH dependence of cath-D-induced SPARC cleavage. Recombinant human FL SPARC was incubated with recombinant human auto-activated pseudo-cath-D (51-kDa) in cleavage buffer with or without pepstatin A (Pepst.) at the indicated pH at 37 °C overnight. SPARC cleavage was analyzed as in (A). (C) Detection of the cath-D-induced SPARC fragments by silver staining. Recombinant SPARC was incubated with recombinant auto-activated pseudo-cath-D (51-kDa) or fully-mature cath-D (34 + 14-kDa) at pH 5.9 for the indicated times. SPARC cleavage was analyzed by 17% SDS-PAGE and silver staining. (D) Cath-D cleavage sites in SPARC extracellular Ca²⁺ binding domain. The entire C-terminal extracellular Ca²⁺ binding domain of human SPARC (amino acids 154-303) is shown. SPARC cleaved peptides generated in the extracellular Ca²⁺ binding domain by auto-activated pseudo-cath-D (51-kDa) and fully-mature (34 + 14-kDa) cath-D at pH 5.9 were resolved by iTRAQ-ATOMS. Arrows, cleavage sites. (E) Schematic representation of the SPARC fragments generated by cath-D according to (C) and (D).