Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth.

Endoglin (CD105), a transmembrane protein of the transforming growth factor ß superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

Triad of polar residues implicated in pH specificity of acidic mammalian chitinase.

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X-ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the beta-anomer of the substrate. A triad of polar residues in the second-shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.

Regulation of secreted Frizzled-related protein-1 by heparin.

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition.

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.

Kinetic characterization of recombinant human acidic mammalian chitinase.

Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.

p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation.

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.

The Formin family protein, formin homolog overexpressed in spleen, interacts with the insulin-responsive aminopeptidase and profilin IIa.

Insulin stimulates translocation of glucose transporter isoform type 4 (GLUT4) and the insulin-responsive aminopeptidase (IRAP) from an intracellular storage pool to the plasma membrane in muscle and fat cells. A role for the cytoskeleton in insulin action has been postulated, and the insulin signaling pathway has been well investigated; however, the molecular mechanism by which GLUT4/IRAP-containing vesicles move from an interior location to the cell surface in response to insulin is incompletely understood. Here, we have screened for IRAP-binding proteins using a yeast two-hybrid system and have found that the C-terminal domain of FHOS (formin homolog overexpressed in spleen) interacts with the N-terminal cytoplasmic domain of IRAP. FHOS is a member of the Formin/Diaphanous family of proteins that is expressed most abundantly in skeletal muscle. In addition, there are two novel types of FHOS transcripts generated by alternative mRNA splicing. FHOS78 has a 78-bp insertion and it is expressed mainly in skeletal muscle where it may be the most abundant isoform in humans. The ubiquitously expressed FHOS24 has a 24-bp insertion encoding an in-frame stop codon that results in a truncated polypeptide. It is known that some formin family proteins interact with the actin-binding profilin proteins. Both FHOS and FHOS78 bound to profilin IIa via their formin homology 1 domains, but neither bound profilin I or IIb. Overexpression of FHOS and FHOS78 resulted in enhanced insulin-stimulated glucose uptake in L6 cells to similar levels. However, overexpression of FHOS24, lacking the IRAP-binding domain, did not affect insulin-stimulated glucose uptake. These findings suggest that FHOS mediates an interaction between GLUT4/IRAP-containing vesicles and the cytoskeleton and may participate in exocytosis and/or retention of this membrane compartment.