Interaction of the coiled‐coil domain with glycosaminoglycans protects angiopoietin‐like 4 from proteolysis and regulates its antiangiogenic activity

Abstract

Angiopoietin-like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled-coil domain (CCD) and fibrinogen-like domain (FLD) fragments. The full-length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM-bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure-function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full-length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule-like formation. In endothelial cells, ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor alpha1-PDX and abolished by the mutation of the (161)RRKR(164) cleavage site without modification of the ECM binding and release. These data suggest that the full-length form, which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.

Viral G protein-coupled receptor up-regulates Angiopoietin-like 4 promoting angiogenesis and vascular permeability in Kaposi's sarcoma

“…Although its secretion has been shown to modulate the disposition of circulating triglycerides by inhibiting lipoprotein lipase, its role in vascular biology is less clear. ANGPTL4 (or specific domains of the protein) has been shown to regulate endothelial cell adhesion (24) and to promote angiogenesis under some circumstances (21,22,35), but also to inhibit angiogenesis in other circumstances (23)(24)(25)(26). More recently, several studies have suggested similarly discordant functions for this factor in tumor dissemination: whereas systemic ANGPTL4 secretion in mice inhibited melanoma metastasis through inhibition of vascular permeability and tumor cell invasiveness (29), TGFβ-mediated induction of ANGPTL4 by breast cancer cells primed these cells for lung metastasis by increasing the permeability of lung capillaries (28).…”

“…In KS, this increased vessel permeability manifests with extravasated erythrocytes and inflammatory cells, whereas in other cancers, ANGPTL4 promotes tumor metastasis. Nonetheless, recent evidence also suggests that the biologic functions of ANGPTL4 may be highly dependent on its microenvironment (26). Indeed, the diverse and often contradictory functions of ANGPTL4 may be suggestive of a context or tissue-specific activity for this protein (19).…”

“…In this regard, ANGPTL proteins have been previously shown to regulate multiple aspects of endothelial cell function, including endothelial cell proliferation, migration, differentiation, and endothelial cell adhesion (19). However, conflicting data exists as to the pro-vs. antiangiogenic effects of ANGPTL4 (21)(22)(23)(24)(25)(26). To elucidate the contribution of ANGPTL4 to the angiogenic switch promoted by vGPCR, we used the directed in vivo angiogenesis assay (DIVAA), which examines the development of blood vessels within basement membrane extract (BME)-filled angioreactors implanted in immunosuppressed mice (27).…”

Proteolytic Processing of Angiopoietin-like Protein 4 by Proprotein Convertases Modulates Its Inhibitory Effects on Lipoprotein Lipase Activity

“…Early studies in endothelial and human embryonic kidney 293 (HEK293) cells showed that ANGPTL4 is cleaved after a conserved basic sequence (-RXKR-), and mutation of all four of these amino acids abolishes the cleavage of ANGPTL4 in vitro and in vivo (15,16). ␣1-Antitrypsin Portland variant (␣1-PDX), which was generated by mutating the reactive-site loop of ␣1-antitrypsin to contain the minimal consensus sequence for proprotein convertase (PC) cleavage (-RIPR-), acts as a competitive inhibitor of several PCs (16) and can partially block the processing of ANGPTL4 in human umbilical vein endothelial cells (HUVECs).…”

“…␣1-Antitrypsin Portland variant (␣1-PDX), which was generated by mutating the reactive-site loop of ␣1-antitrypsin to contain the minimal consensus sequence for proprotein convertase (PC) cleavage (-RIPR-), acts as a competitive inhibitor of several PCs (16) and can partially block the processing of ANGPTL4 in human umbilical vein endothelial cells (HUVECs). Thus, PCs are suspected to act as the enzymes that cleave ANGPTL4.…”