Vasoinhibin comprises a three-helix bundle and its antiangiogenic domain is located within the first 79 residues

Abstract

Vasoinhibin belongs to a family of angiogenesis inhibitors generated when the fourth α-helix (H4) of the hormone prolactin (PRL) is removed by specific proteolytic cleavage. The antiangiogenic properties are absent in uncleaved PRL, indicating that conformational changes create a new bioactive domain. However, the solution structure of vasoinhibin and the location of its bioactive domain are unknown. Molecular dynamic simulation (MD) showed that the loss of H4 exposes the hydrophobic nucleus of PRL and leads to the compression of the molecule into a three-helix bundle that buries the hydrophobic nucleus again. Compression occurs by the movement of loop 1 (L1) and its interaction with α-helix 1 (H1) generating a new L1 conformation with electrostatic and hydrophobic surfaces distinct from those of PRL, that may correspond to a bioactive domain. Consistent with this model, a recombinant protein containing the first 79 amino acids comprising H1 and L1 of human PRL inhibited the proliferation and migration of endothelial cells and upregulated the vasoinhibin target genes, IL1A and ICAM1. This bioactivity was comparable to that of a conventional vasoinhibin having the 123 residues encompassing H1, L1, Η2, L2, and Η3 of human PRL. These findings extend the vasoinhibin family to smaller proteins and provide important structural information, which will aid in antiangiogenic drug development.

Development of Vasoinhibin-Specific Monoclonal Antibodies

“…Here, microplate assays demonstrate specificity of the antibodies for vasoinhibin in the presence of PRL. An important precondition for the development of these antibodies was the recognition of the unique three-dimensional structure in the L1 region of vasoinhibin which does not exist in full-length PRL ( 23 ). The vasoinhibin antibodies are suitable for sandwich ELISA applications, and are therefore promising in terms of the development of an ELISA which could be used to quantitatively determine circulating vasoinhibin levels in patients.…”

“…Unlike for full-length PRL ( 20 , 21 ), an experimental three-dimensional structure for vasoinhibin is not available, also because aggregation of the vasoinhibin molecule prevented resolution of its structure by NMR ( 22 ). Information on potential epitopes unique to vasoinhibin emerged from the recently reported three-dimensional model of vasoinhibin, which revealed significant conformational differences with full-length PRL in the area within the loop 1 (L1) comprising amino acids 40 – 76 ( 23 ). This region appeared to be a new epitope created only after the loss of the 4 th helix that follows the proteolytic cleavage of PRL and, thereby, a suitable region for the generation of monoclonal anti-vasoinhibin antibodies that do not react with PRL ( Figure 1A ).…”

Short prolactin isoforms are expressed in photoreceptors of canine retinas undergoing retinal degeneration

“…Additionally, PRLΔE1 retains many of the potential cathepsin-D sites that are required to generate vasoinhibin fragments 7 . However, the first helix that has been shown to be required for the anti-angiogenic activity of vasoinhibins 62 is lacking in this isoform. Therefore, it is unlikely that the PRLΔE1 isoform generates bioactive vasoinhibin peptides.…”

Levosulpiride Increases the Levels of Prolactin and Antiangiogenic Vasoinhibin in the Vitreous of Patients with Proliferative Diabetic Retinopathy

“…Cells were allowed to proliferate in the presence of 10 µM of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) (Sigma-Aldrich), and the number of cells with newly synthesized DNA was evaluated after 24 hours by the click reaction as reported. 26 Images obtained in a fluorescence inverted microscope were quantified using CellProfiler software. 27 BUVECs were cultured and seeded in 10% FBS-F12K at 5000 cells/cm 2 , allowed to attach for 3 to 4 hours, and serum-starved (0.5% FBS) for 12 to 16 hours.…”