INHIBITION OF ENDOTHELIAL-CELL PROLIFERATION BY SPARC IS MEDIATED THROUGH A CA2+-BINDING EF-HAND SEQUENCE

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM-40) is a metal-binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid-G(1), and that a synthetic peptide (amino acids 54-73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide-bonded region was in part responsible for the growth-suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648-2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C-terminal EF-hand sequence comprising a high-affinity Ca2+-binding site of SPARC and represented by a synthetic peptide (amino acids 254-273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790-25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [H-3]-thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019-0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose-responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti-SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell-surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration-dependent: peptide 4.2 K, containing an Asp(258) --> Lys substitution, and peptide 4.2 AA, in which the two disulfide-bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF-hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild-type 4.2 sequence, that was indicative of a compromised affinity for Ca2+. Moreover, ablation of the disulfide bond in peptide 4.2 AA potentially destabilized the Ca2+-binding loop structure, as assessed by fluorescence spectroscopy, such that the peptide competed poorly for the binding of [I-125]-peptide 4.2 to BAE cells. We conclude both that Ca2+-coordinating Asp at position 258 and the conformation of peptide 4.2 are necessary for the inhibition of DNA synthesis by SPARC in cultured endothelial cells. (C) 1995 Wiley-Liss, Inc.