Angiopoietins 3 and 4: diverging gene counterparts in mice and humans

Abstract

The angiopoietins have recently joined the members of the vascular endothelial growth factor family as the only known growth factors largely specific for vascular endothelium. The angiopoietins include a naturally occurring agonist, angiopoietin-1, as well as a naturally occurring antagonist, angiopoietin-2, both of which act by means of the Tie2 receptor. We now report our attempts to use homology-based cloning approaches to identify new members of the angiopoietin family. These efforts have led to the identification of two new angiopoietins, angiopoietin-3 in mouse and angiopoietin-4 in human; we have also identified several more distantly related sequences that do not seem to be true angiopoietins, in that they do not bind to the Tie receptors. Although angiopoietin-3 and angiopoietin-4 are strikingly more structurally diverged from each other than are the mouse and human versions of angiopoietin-1 and angiopoietin-2, they appear to represent the mouse and human counterparts of the same gene locus, as revealed in our chromosomal localization studies of all of the angiopoietins in mouse and human. The structural divergence of angiopoietin-3 and angiopoietin-4 appears to underlie diverging functions of these counterparts. Angiopoietin-3 and angiopoietin-4 have very different distributions in their respective species, and angiopoietin-3 appears to act as an antagonist, whereas angiopoietin-4 appears to function as an agonist.

Figure 1

Amino acid sequences and evolutionary relationships of the angiopoietins and their distant relatives. (A) Full-length sequences of the definitive angiopoietins as aligned by the clustal method by using the megalign program from DNAstar; arrowhead marks the predicted signal peptide cleavage site, bent arrows indicate the limits of the coiled–coil and fibrinogen-like domains, closed circles denote conserved cysteines, and the open circle marks a cysteine present in Ang1 but absent in all other angiopoietins. (B) Alignment of the conserved fibrinogen-like domain of all definitive members of the angiopoietin family with the fibrinogen-like domains of the more distant angiopoietin homologs described here (AngX and AngY), as well as of previously described members of the fibrinogen superfamily (i.e., ficolin-a, ficolin-b, hFrep, hpt49, and human fibrinogen γ); dots mark the three distinctive cysteines that are present in the angiopoietin family but absent from AngX and AngY, as well as all other members of the fibrinogen superfamily. For ease of alignment, some amino acids were deleted in the alignments: in PT49 a “g” was deleted after the boldfaced “n,” in FREP the sequence “dslagnf” was deleted before the boldfaced “h,” and in fibrinogen the sequence “sdkfftshng” was deleted after the boldfaced “p.” (C) Cladogram (built by the clustal method by using the megalign program from DNAstar) comparing the evolutionary relationships between members of the angiopoietin family and the distant relatives depicted in B; also indicated are the percent amino acid identity between paired orthologs or between the distant relatives and their closest angiopoietin homolog (only within the fibrinogen-like domain) as well as the chromosomal localizations of the paired ortholog in both mouse and human.

Figure 2

BIAcore assay for evaluation of angiopoietin-3 and -4 binding to the Tie1 and Tie2 receptors. BIAcore binding assays of the indicated angiopoietins or chimeric angiopoietins (having the fibrinogen-like domains of angiopoietin-3 or -4; as noted, binding of chimeric angiopoietins is determined by the fibrinogen-like domain) to BIAcore chips coated with immobilized Tie1 or Tie2 receptors was assessed in the presence of competition with excess amounts of either an irrelevant soluble receptor (TrkB) or soluble Tie1 or Tie2 receptors (6). For all the angiopoietins, binding is noted only to the Tie2 surface and not the Tie1 surface; consistent with this observation, only soluble Tie2 receptors compete for binding to the surface.

Figure 3

Human and mouse chromosomal mapping of the angiopoietin gene family. (A) Fluorescent in situ hybridization images of digoxigenin-labeled probes for human angiopoietin-1, human angiopoietin-2, and human angiopoietin-4 to human chromosomes in metaphase cells from phytohemagglutinin-stimulated human peripheral blood lymphocytes. The designated name and hybridization loci are denoted underneath each panel. (B and C) Mouse chromosome linkage maps for murine angiopoietin-1, angiopoietin-2, and angiopoietin-3.

Figure 4

Differing tissue distributions of angiopoietin-3 in mice and angiopoietin-4 in human. Depicted are Northern blots containing Poly(A)⁺ RNA from mouse tissues hybridized to a mouse angiopoietin-3 probe (Left) or from human tissues hybridized to a human angiopoietin-4 probe (Right).

Figure 5

Differing biological activities of mouse angiopoietin-3 as compared with human angiopoietin-4. Angiopoietin chimeras containing the angiopoietin-4 fibrinogen-like domain activate Tie2 receptors in either human (A) or mouse (B) endothelial cells as well as in mouse fibroblasts (C), whereas angiopoietin chimeras containing the angiopoietin-3 fibrinogen-like domain cannot activate Tie2 receptors in either human (A) or mouse (B) endothelial cells, although they can in mouse fibroblasts (C). Furthermore, excess amounts of chimeras containing the angiopoietin-3 fibrinogen-like domain can blunt the ability of angiopoietin-1 to activate the Tie2 receptor (D). Thus, whereas angiopoietin-4 derivatives act as agonists (like angiopoietin-1), angiopoietin-3 derivatives act as context-dependent antagonists (like angiopoietin-2).