Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca2+-binding EF-hand sequence
| ISSN: | 1097-4644 0730-2312 |
|---|---|
| Přístupová URL adresa: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::528ddd70b7f7243e2477290bd1cdf3d7 https://doi.org/10.1002/jcb.240570113 |
| Rights: | CLOSED |
| Přírůstkové číslo: | edsair.doi.dedup.....528ddd70b7f7243e2477290bd1cdf3d7 |
| Autor: | E H Sage, M J Folkman, Timothy F. Lane, J C Yost, James A. Bassuk |
| Rok vydání: | 1995 |
| Předmět: |
Angiogenesis
Molecular Sequence Data Peptide Tritium Biochemistry Animals Osteonectin Amino Acid Sequence Cysteine Amino Acids Binding site Molecular Biology Peptide sequence Aorta Cells Cultured chemistry.chemical_classification Alanine Binding Sites Dose-Response Relationship Drug biology DNA synthesis Heparin DNA Cell Biology Molecular biology Capillaries Amino acid Endothelial stem cell chemistry Isotope Labeling biology.protein Calcium Cattle Fibroblast Growth Factor 2 Endothelium Vascular Cell Division Thymidine |
| Zdroj: | Journal of Cellular Biochemistry. 57:127-140 |
| ISSN: | 1097-4644 0730-2312 |
| Popis: | 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-G1, 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 Ca(2+)-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 [3H]-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 Asp258 --> 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(+).(ABSTRACT TRUNCATED AT 400 WORDS) |
| Databáze: | OpenAIRE |
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