Manduca sexta serpin-7, a putative regulator of hemolymph prophenoloxidase activation.

Serpins regulate various physiological reactions in humans and insects, including certain immune responses, primarily through inhibition of serine proteases. Six serpins have previously been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a full-length cDNA sequence of another Manduca serpin, named serpin-7. The open reading frame of serpin-7 encodes a polypeptide of 400 amino acid residues with a predicted signal peptide of the first 15 residues. Multiple protein sequence alignment of the reactive center loop region of the M. sexta serpins indicated that serpin-7 contains Arg-Ile at the position of the predicted scissile bond cleaved by protease in the serpin inhibition mechanism. The same residues occur in the scissile bond of the reactive center loop in M. sexta serpin-4 and serpin-5, which are protease inhibitors that can block prophenoloxidase activation in plasma. Serpin-7 transcript was detected in hemocytes and fat body, and its expression increased in fat body after injection of larvae with Micrococcus luteus. Recombinant serpin-7 added to larval plasma inhibited spontaneous melanization and decreased prophenoloxidase activation stimulated by bacteria. Serpin-7 inhibited prophenoloxidase-activating protease-3 (PAP3), forming a stable serpin-protease complex. Considering that serpin-3 and serpin-6 are also efficient inhibitors of PAP3, it appears that multiple serpins present in plasma may have redundant or overlapping functions. We conclude that serpin-7 has serine protease inhibitory activity and is likely involved in regulation of proPO activation or other protease-mediated aspects of innate immunity in M. sexta.

Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes.

The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Herein, we demonstrate that their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the ;garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function.

The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts.

The body wall muscle of a Drosophila larva is generated by fusion between founder cells and fusion-competent myoblasts (FCMs). Initially, a founder cell recognizes and fuses with one or two FCMs to form a muscle precursor, then the developing syncitia fuses with additional FCMs to form a muscle fiber. These interactions require members of the immunoglobulin superfamily (IgSF), with Kin-of-IrreC (Kirre) and Roughest (Rst) functioning redundantly in the founder cell and Sticks-and-stones (Sns) serving as their ligand in the FCMs. Previous studies have not resolved the role of Hibris (Hbs), a paralog of Sns, suggesting that it functions as a positive regulator of myoblast fusion and as a negative regulator that antagonizes the activity of Sns. The results herein resolve this issue, demonstrating that sns and hbs function redundantly in the formation of several muscle precursors, and that loss of one copy of sns enhances the myoblast fusion phenotype of hbs mutants. We further show that excess Hbs rescues some fusion in sns mutant embryos beyond precursor formation, consistent with its ability to drive myoblast fusion, but show using chimeric molecules that Hbs functions less efficiently than Sns. In conjunction with a physical association between Hbs and SNS in cis, these data account for the previously observed UAS-hbs overexpression phenotypes. Lastly, we demonstrate that either an Hbs or Sns cytodomain is essential for muscle precursor formation, and signaling from IgSF members found exclusively in the founder cells is not sufficient to direct precursor formation.

Myoblast fusion in Drosophila.

Myogenic differentiation in Drosophila melanogaster, as in many other organisms, involves the generation of multinucleate muscle fibers through the fusion of myoblasts. Prior to fusion, the myoblasts become specified as one of two distinct cell types. They then become competent to fuse and express genes associated with cell recognition and adhesion. Initially, cell-type- specific adhesion molecules mediate recognition and fusion between these two distinct populations of myoblasts. Intracellular proteins that are essential for the fusion process are then recruited to points of cell-cell contact at the membrane, where the cell surface molecules have become localized. Many of these cytosolic proteins contribute to reorganization of the cytoskeleton through activation of small guanosine triphosphatases and recruitment of actin nucleating proteins. Following the initial fusion event, the ultimate size of the syncytia is achieved through multiple rounds of fusion between the developing syncytia and mononucleate myoblasts. Ultrastructural changes associated with cell fusion include recruitment of electron-dense vesicles to points of cell-cell contact, resolution of these vesicles into fusion plaques, fusion pore formation, and membrane vesiculation. This chapter reviews our current understanding of the genes, pathways, and ultrastructural events associated with fusion in the Drosophila embryo, giving rise to multinucleate syncytia that will be used throughout larval life.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

An integrin-tetraspanin interaction required for cellular innate immune responses of an insect, Manduca sexta.

In their encounters with foreign intruders, the cells of the insect innate immune system, like those of the mammalian immune system, exhibit both humoral and cell-mediated responses. Some intruders can be dispatched by the humoral immune system alone, but many must be phagocytosed by individual hemocytes or encapsulated by interacting hemocytes. Surface proteins of hemocytes control the abrupt transition of hemocytes from resting, nonadherent cells to activated, adherent cells during these cell-mediated responses. Two of these surface proteins, an integrin and a tetraspanin, interact during this adhesive transition. As demonstrated with a hemocyte adhesion assay and a surface plasmon resonance assay, the large extracellular loop of tetraspanin D76 binds to a hemocyte-specific integrin of Manduca sexta. The interaction between the large extracellular loop domain and hemocyte-specific integrin is interrupted not only by a monoclonal antibody (MS13) that binds to a domain of beta-integrin known to be a ligand-binding site for cell adhesion but also by double-stranded beta-integrin RNA. Transfected S2 cells expressing tetraspanin mediate adhesion of hemocytes. A monoclonal antibody to tetraspanin D76 perturbs the cell-mediated immune response of encapsulation. These studies involving antibody blocking, RNA interference, and binding assays imply a trans interaction of integrin and tetraspanin on hemocyte surfaces.

Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta.

Neuroglian, a member of the L1 family of cell adhesion molecules (L1-CAMs), is expressed on surfaces of granular cells and a subset of large plasmatocytes of Manduca sexta that act as foci for hemocyte aggregation during the innate immune response. Neuroglian expressed on surfaces of transfected Sf9 cells induced their homophilic aggregation, with the aggregation being abolished in the presence of recombinant immunoglobulin (Ig) domains of neuroglian. Neuroglian and its Ig domains also can interact with hemocyte-specific integrin (HS integrin) as demonstrated with an enzyme-linked immunoassay and a surface plasmon resonance (SPR) assay. Neuroglian double-stranded (ds) RNA not only depresses expression of neuroglian in hemocytes but also depresses the cell-mediated encapsulation response of these hemocytes to foreign surfaces. After injection of a monoclonal antibody (MAb 3B11) into M. sexta larvae that recognizes the Ig domains of neuroglian, the cell-mediated encapsulation response of hemocytes was likewise inhibited. The Ig domains of neuroglian are involved in both homophilic and heterophilic interactions, and subsets of these six different Ig domains may affect different functions of neuroglian.

Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte attachment to foreign surfaces.

Observations of hemocyte aggregation on abiotic surfaces suggested that certain plasmatocytes from larvae of Manduca sexta act as foci for hemocyte aggregation. To establish how these particular plasmatocytes form initial attachments to foreign surfaces, they were cultured separately from other selected populations of hemocytes. While all circulating plasmatocytes immunolabel with anti-beta-integrin monoclonal antibody (MAb), only these larger plasmatocytes immunolabel with a MAb to the adhesion protein neuroglian. Neuroglian-negative plasmatocytes and granular cells that have been magnetically segregated from the majority of granular cells adhere to each other but fail to adhere to foreign substrata; by contrast, neuroglian-positive plasmatocytes that segregate with most granular cells adhere firmly to a substratum. Hemocytes form stable aggregates around the large, neuroglian-positive plasmatocytes. However, if neuroglian-positive plasmatocytes are separated from most granular cells, attachment of these plasmatocytes to foreign surfaces is suppressed.

Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta.

Upon encountering an object recognized as foreign, insect hemocytes aggregate in multiple layers on the surfaces of the object in a process known as encapsulation. For encapsulation to occur, hemocytes must switch from their usual nonadherent state to an adherent state, presumably by regulating the activity of adhesion proteins. Although detailed knowledge exists regarding the adhesion receptors for cells of the mammalian immune system, comparable information on adhesion molecules of insect hemocytes and their function in immune responses is extremely limited. We report here the identification of an integrin present exclusively on the surface of hemocytes in the tobacco hornworm, Manduca sexta. Monoclonal antibodies MS13 and MS34, which bind to plasmatocytes and block encapsulation, were used for immunoaffinity chromatography to isolate their corresponding hemocyte antigen, which was revealed to be the same integrin beta subunit. A cDNA for this M. sexta integrin beta1 was cloned and characterized. Integrin-beta1 mRNA was detected by Northern analysis in hemocytes and not in other tissues tested. MS13 and MS34 were demonstrated to bind to a recombinant fragment of integrin beta1 consisting of the I-like domain, consistent with their blocking of a ligand-binding site and subsequent disruption of plasmatocyte adhesion. Injection of double stranded integrin-beta1 RNA into larvae resulted in decreased integrin beta1 expression in plasmatocytes and significantly suppressed encapsulation. These results indicate that activation of ligand-binding by the hemocyte-specific integrin plays a key role in stimulating plasmatocyte adhesion leading to encapsulation.

Combinatorial antibody libraries from cancer patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit breast cancer metastasis.

Combinatorial antibody libraries have the potential to display the entire immunological record of an individual, allowing one to detect and recover any antibody ever made, irrespective of whether it is currently being produced. We have termed this the "fossil record" of an individual's antibody response. To determine whether cancer patients have ever made antibodies with disease-fighting potential, we screened combinatorial antibody libraries from cancer patients for immunoglobulins that can identify metastatic tumor cells. This strategy yielded human antibodies specific for the activated conformation of the adhesion receptor integrin alphavbeta3 that is associated with a metastatic phenotype. In a remarkable example of convergent evolution, two of these antibodies were shown to contain the Arg-Gly-Asp integrin recognition motif of the natural ligand within the third complementarity-determining region of the heavy chain. These antibodies interfered with lung colonization by human breast cancer cells in a mouse model and inhibited existing metastatic disease. Our data imply that, at least at some time, these antibodies were part of a patient's surveillance system against metastatic cells, targeting the activated conformer of integrin alphavbeta3 and disrupting its functions. The ligand-mimetic nature of these antibodies, combined with specificity for a single receptor, is unique in the integrin-ligand repertoire. The convergent evolution of critical sequences in antibodies and other ligands that bind to the same target means that the immune response has sufficient power to find a best chemical solution for the optimization of binding energy, even though antibodies evolve in real time, as compared with billions of years for the natural ligand.

[Cloning, expression and characterization of human vascular endothelial growth factor receptor 1 tyrosine kinase].

Vascular endothelial growth factor receptor 1 (Flt1) plays an important role in angiogenesis. It was hypothesized that, upon binding to VEGF, Flt1 tyrosine kinase underwent dimerization and initiated the signal transduction in VEGF/VEGF receptor system. In this report, a soluble active Flt1 tyrosine kinase domain expressed in E. coli was obtained, and its properties were partly characterized. The cDNA of Flt1 tyrosine kinase domain was obtained from the total RNA extracted from human liver cancer tissues by using RT-PCR, and was cloned to vector pGEX-KG. A soluble active GST-fusion protein of Flt1 tyrosine kinase domain (GST-F) was obtained from E. coli BL21 (DE3) pLysS. Although it was reported that GST-F contains no phosphorylation site, it did autophosphorylate in vitro. Mg2+ and Mn2+ were essential for the activity. It was also found that GST-F phosphorylated a synthesized substrate PolyE4Y, but not MBP and Src-related-peptide. The optimal Mg2+ and Mn2+ concentration for polyE4Y phosphorylation was 15 mmol/L and 0.5 mmol/L, respectively. This work is helpful for developing the new anti-cancer drugs.

A new model for random screening inhibitors of vascular endothelial growth factor receptor 1 kinase.

AIM: To establish a 96-well plate based kinase assay using a recombinant vascular endothelial growth factor (VEGF) receptor 1 kinase domain protein. METHODS: A human VEGF receptor 1 kinase domain protein was expressed in E coli, and its activity was monitored by its ability of phosphorylating the polyE4Y substrate coated on the walls of 96-well plates with antibody recognition and a colorimetric readout. A random screening of a sample organic compound library was carried out, and the hits were characterized with a transformed cell line stably expressing VEGF receptor 1 protein. RESULTS: An efficient E coli expression system for human VEGF receptor 1 kinase domain protein was constructed, and the purified recombinant protein was used to establish a practical screening assay for kinase inhibitors in vitro. Two thousand eight hundred organic compounds were screened, and two disubstituted furans (A1 and A5) with new structure showed inhibition of VEGF receptor 1 kinase. Compound A1 inhibited only phosphorylation of substrate, while compound A5 inhibited both autophosphorylation and substrate phosphorylation. Both inhibitors affected phosphorylation in the transformed cells. CONCLUSION: The recombinant receptor kinase based assay is simple and effective in identifying kinase inhibitors.