RESUMO
BACKGROUND: Heparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin. RESULTS: Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities. CONCLUSION: Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.
Assuntos
Quimiocinas CXC/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Soroalbumina Bovina/metabolismo , Sulfotransferases/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/enzimologia , Quimiocina CCL21 , Quimiocina CXCL12 , Quimiocinas CC/metabolismo , Meios de Cultivo Condicionados/química , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Interleucina-8/metabolismo , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/fisiologia , Ligação Proteica , Proteínas Recombinantes de Fusão/fisiologia , Sulfatases , Sulfotransferases/genética , Sulfotransferases/isolamento & purificaçãoRESUMO
Sulf-2 is an endosulfatase with activity against glucosamine-6-sulfate modifications within subregions of intact heparin. The enzyme has the potential to modify the sulfation status of extracellular heparan sulfate proteoglycan (HSPG) glycosaminoglycan chains and thereby to regulate interactions with HSPG-binding proteins. In the present investigation, data mining from published studies was employed to establish Sulf-2 mRNA upregulation in human breast cancer. We further found that cultured breast carcinoma cells expressed Sulf-2 mRNA and released enzymatically active proteins into conditioned medium. In two mouse models of mammary carcinoma, Sulf-2 mRNA was upregulated in comparison to its expression in normal mammary gland. Although mRNA was present in normal tissues, Sulf-2 protein was undetectable; it was, however, detected in some premalignant lesions and in tumors. The protein was localized to the epithelial cells of the tumors. In support of the possible mechanistic relevance of Sulf-2 upregulation in tumors, purified recombinant Sulf-2 promoted angiogenesis in the chick chorioallantoic membrane assay.