A membrane topology model for human interferon inducible transmembrane protein 1.

InterFeron Inducible TransMembrane proteins 1-3 (IFITM1, IFITM2 and IFITM3) are a family of proteins capable of inhibiting the cellular entry of numerous human and animal viruses. IFITM1-3 are unique amongst the currently described viral restriction factors in their apparent ability to block viral entry. This restrictive property is dependant on the localisation of the proteins to plasma and endosomal membranes, which constitute the main portals of viral entry into cells. The topology of the IFITM proteins within cell membranes is an unresolved aspect of their biology. Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular. Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins. This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

Studying the mechanism of CD47-SIRPα interactions on red blood cells by single molecule force spectroscopy.

The interaction forces and binding kinetics between SIRPα and CD47 were investigated by single-molecule force spectroscopy (SMFS) on both fresh and experimentally aged human red blood cells (hRBCs). We found that CD47 experienced a conformation change after oxidation, which influenced the interaction force and the position of the energy barrier between SIRPα and CD47. Our results are significant for understanding the mechanism of phagocytosis of red blood cells at the single molecule level.

NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains.

Galectin-3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal domain that includes several repeats of a proline-tyrosine-glycine-rich motif. Earlier work based on a crystal structure of human galectin-3 CRD, and modeling and mutagenesis studies of the closely homologous hamster galectin-3, suggested that N-terminal tail residues immediately preceding the CRD might interfere with the canonical subunit interaction site of dimeric galectin-1 and -2, explaining the monomeric status of galectin-3 in solution. Here we describe high-resolution NMR studies of hamster galectin-3 (residues 1--245) and several of its fragments. The results indicate that the recombinant N-terminal fragment Delta 126--245 (residues 1--125) is an unfolded, extended structure. However, in the intact galectin-3 and fragment Delta 1--93 (residues 94--245), N-terminal domain residues lying between positions 94 and 113 have significantly reduced mobility values compared with those expected for bulk N-terminal tail residues, consistent with an interaction of this segment with the CRD domain. In contrast to the monomeric status of galectin-3 (and fragment Delta 1--93) in solution, electron microscopy of negatively stained and rotary shadowed samples of hamster galectin-3 as well as the CRD fragment Delta 1--103 (residues 104--245) show the presence of a significant proportion (up to 30%) of oligomers. Similar imaging of the N-terminal tail fragment Delta 126--245 reveals the presence of fibrils formed by intermolecular interactions between extended polypeptide subunits. Oligomerization of substratum-adsorbed galectin-3, through N- and C-terminal domain interactions, could be relevant to the positive cooperativity observed in binding of the lectin to immobilized multiglycosylated proteins such as laminin.

CD38 functions are regulated through an internalization step.

The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines. CD38 internalization was followed using radioiodinated Abs in an acidic elution endocytosis assay to monitor the effects of cross-linking on internalization processes and to quantify the ratio of the internalized molecule. Second, conventional, confocal, and electron microscopies were used to evaluate the morphologic effects induced by ligation of the molecule with Abs mimicking the natural ligand(s). The results demonstrated that internalization is a reproducible phenomenon following CD38 ligation with both agonistic and nonagonistic specific Abs and involving only a fraction of the entire amount of the surface molecule. It is independent from signal transduction as can be inferred by the observation that 1) both agonistic and non agonistic Abs are effective and 2) the dynamic of internalization is much slower than that of cellular signaling. Morphologic studies demonstrated that endocytosis induced as a result of CD38 ligation presents a very specific pathway consisting of subcellular organelles fundamental to the processing of the complex. Our data indicate that down-regulation by endocytosis may be, in parallel with shedding, a regulatory element in activation and adhesion processes mediated by CD38. However, internalization seems not to be a key step in triggering intracellular signaling; more likely, it is a negative feedback control mechanism which interrupts signal transduction or cell-cell cross-talks mediated by membrane CD38.

Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro.

All blood vessels are lined by endothelium and, except for the capillaries, surrounded by one or more layers of smooth muscle cells. The origin of the embryonic vascular smooth muscle cell has until now been described from neural crest and locally differentiating mesenchyme. In this study, we have substantial evidence that quail embryonic endothelial cells are competent in the dorsal aorta of the embryo to transdifferentiate into subendothelial mesenchymal cells expressing smooth muscle actins in vivo. At the onset of smooth muscle cell differentiation, QH1-positive endothelial cells were experimentally labeled with a wheat germ agglutinin-colloidal gold marker (WGA-Au). No labeled subendothelial cells were observed at this time. However, 19 hours after the endothelial cells had endocytosed, the WGA-Au-labeled subendothelial mesenchymal cells were observed in the aortic wall. Similarly, during the same time period, subendothelial cells that coexpressed the QH1 endothelial marker and a mesenchymal marker, alpha-smooth muscle actin, were present. In such cells, QH1 expression was reduced to a cell membrane localization. A similar antigen switch was also observed during endocardial-mesenchymal transformation in vitro. Our results are the first direct in vivo evidence that embryonic endothelial cells may transdifferentiate into candidate vascular smooth muscle cells. These data arouse new interpretations of the origin and differentiation of the cells of the vascular wall in normal and diseased vessels.

Self-aggregation of the transmembrane glycoprotein CD38 purified from human erythrocytes.

The 46 kDa human transmembrane glycoprotein CD38 is a multicatalytic enzyme exhibiting ADPribosyl cyclase, cyclic ADPribose hydrolase and NAD(+)-glycohydrolase activities at its extracellular domain. When CD38, purified to homogeneity from human erythrocyte membranes, was incubated with NAD+ or beta-mercaptoethanol, extensive aggregation took place. Addition of both compounds to CD38 led to the formation of still larger aggregates (over 300 nm), which were resistant to TCA precipitation. Extensive and stable CD38 self-aggregation was shown by, i) SDS-PAGE and autoradiography of the [32P]NAD(+)-incubated CD38, ii) SDS-PAGE followed by immunochemical detection of CD38 on the transblots, iii) direct electron microscopy on negatively stained CD38 samples. Self-aggregation of CD38 might be correlated with its putative function as a transducer of activation and proliferation signals in a number of hematopoietic cells.

[Fc gamma receptors: structure, function, and clinical significance]. / Fc gamma-Rezeptoren: Struktur, Funktion und klinische Bedeutung.

Fc gamma receptors are a group of three different receptors with several subtypes. They are widely distributed on many cells of the immune system and contribute to the pathogenesis of immune complex- and autoantibody-mediated diseases such as vasculitis, rheumatoid arthritis, idiopathic thrombocytopenic purpura or autoimmune neutropenia. This review focuses on the structure, distribution and function in Fc gamma receptors and their subtypes.