Endoglin (CD105), a transmembrane protein of the transforming growth factor ␤ 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. Endoglin (CD105) is a homodimeric glycosylated cell-surface protein of 180 kDa previously identified as a co-receptor belonging to the TGF␤ superfamily (1). Several lines of evidence support an important role of endoglin in cardiovascular development and vascular remodeling (2). Loss-of-function mutations in endoglin are implicated in the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1), 3 which is a bleeding disorder characterized by arteriovenous malformations in the brain, lungs, and liver and is attributed to haploinsufficiency (3-5). Homozygous endoglin knock-out mice die during early gestation due to the lack of development of normal mature blood vessels (6). Adult endoglin heterozygous mice (7-8) or mice with a conditional mutation in the endoglin gene (9) exhibited similar angiogenic abnormalities and were used as animal models for HHT1. Mutations in the endoglin gene found in numerous HHT1 patients are localized most exclusively in the extracellular domain (4). The specific role of endoglin in the vascular dysplasia observed in HHT patients is not known, but it is likely to be related to the role of TGF␤ family signaling in angiogenesis (2, 10). Interestingly, another form of HHT, known as HHT2, which is also characterized by the presence of telangiectases as well as arteriovenous malformations in brain, lungs and liver, results from the loss of TGF␤ type I receptor ALK1 (11), which suggests an interrelatedness between endoglin and ALK1 and possibly involvement of the same ligand(s) in the mechanism of action of both molecules.A soluble form of endoglin has been observed in the serum of patients with different types of solid malignancies (12) and of pregnant women suffering from preeclampsia, a disease leading to vascular permeability (13), hypertension, and placental abruption (14). This soluble form, which reportedly results from partial shedding of the membrane-bound form of endoglin by the matrix metalloproteinase 14 (MT1-MMP) (15), a phenomenon also observed for the other type III receptor betaglycan (16), has been proposed to act as a scavenger or trap for circulating TGF␤ family liga...

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 Å 2 ) 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.Injured neurons in mature organisms are unable to effectively regrow their axons after central nervous system damage. One of the many factors restricting axonal regeneration after injury is the growth-inhibiting components associated with damaged myelin. At least three of these components, Nogo-66, myelin-associated glycoprotein (MAG), 3 and oligodendrocyte myelin glycoprotein, either individually or collectively, have been shown to be potent inhibitors of neurite outgrowth (1, 2). All three signal inhibition through the Nogo receptor complex, composed of the ligand-binding Nogo-66 receptor (NgR) and two complementary co-receptors p75 and Lingo-1 that act as a signal-transducing pair on an axon's cell membrane (3, 4). Although both NgR and the p75 nerve growth factor receptor have well documented roles in the context of myelin inhibition, reports exploring the role of Lingo-1 are more recent.Human Lingo-1 is a central nervous system-specific transmembrane glycoprotein (Fig. 1) also known as LERN-1, which belongs to a larger family of LRR-Ig-containing proteins involved in central nervous system development and axonal growth (5). Its large extracellular or ectodomain is thought to be of functional importance in protein-protein recognition and is characterized by a tandem array of multiple LRRs and one Iglike domain. The first studies examining the role of Lingo-1 demonstrated that in cultured neurons Lingo-1 directly associates with NgR and p75 and that whenever myelin-NgR/p75-mediated growth inhibition is observe...

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. INTRODUCTIONInsulin normalizes blood glucose levels by mobilizing the muscle and adipocyte glucose transporter isoform, GLUT4, from intracellular storage vesicles and moving it to the plasma membrane (Simpson et al., 2001;Bryant et al., 2002). Various models of GLUT4 trafficking suggest that GLUT4 must exist in more than one intracellular compartment and the major insulin sensitive pool is localized to a compartment that is distinct from endosomal markers and is commonly referred to as glucose transporter storage vesicles (GSVs), or insulin responsive vesicle (IRVs). Despite the critical function of glucose transport in glucose homeostasis, many of the details by which adipocytes and muscle form a pathway of insulin-sensitive GLUT4 trafficking remain unknown. However, it is virtually certain that the major cargo proteins of GSVs must interact with a number of cytosolic and membrane proteins, such as adaptors and tethers, in order to be properly sorted and regulated by insulin.Insulin-responsive aminopeptidase (IRAP) was identified as an abundant cargo protein associated with GLUT4 vesicles that translocates in response to insulin in a manner seemingly identical to GLUT4 Mastick et al., 1994;Keller et al., 1995;Malide et al., 1997;Martin et al., 1997;Ross et al., 1997). In fact, it is more abundantly expressed in vesicles than the transporter (Kupriyanova et al., 2002). When the cytoplasmic N-terminus of IRAP was microinjected into 3T3-L1 adipocytes, GLUT4 was localized on the plasma membrane even in the basal state (Waters et al., 1997), suggesting IRAP can play a role in GSV movement/targeting. A chimeric protein containing the intracellular domain of IRAP and ...

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.

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 b-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.