Cytokines in Drosophila hematopoiesis and cellular immunity.

Hematopoiesis is a complex, multistep process in which progenitor cells undergo distinct cellular changes of proliferation and differentiation to give rise to mature blood cells in circulation. Many of the genetic and molecular events that drive these changes have been characterized in mammals, frogs, and zebra fish, and more recently in the insect model system Drosophila melanogaster. Blood cells in Drosophila are actively involved in fighting infections and the cellular immune responses are intimately tied to the process of hematopoiesis. In this article, we briefly review the fundamental similarities in Drosophila and mammalian hematopoiesis and highlight the potential roles of four cytokines/growth factors in Drosophila hematopoiesis and cellular immunity.

Mutational analysis of the CD6 ligand binding domain.

CD6 belongs to the scavenger receptor cysteine-rich protein superfamily (SRCRSF), which includes a large number of cell surface proteins. The extracellular region of CD6 is composed of three SRCR domains. The membrane proximal SRCR domain of CD6 (CD6D3) specifically binds activated leukocyte cell adhesion molecule (ALCAM), a cell surface protein which is a member of the immunoglobulin superfamily (IgSF). CD6-ligand interactions have been implicated in immune cell adhesion, T cell maturation and the regulation of T cell activation. We tested 13 CD6D3 mutant proteins for binding to ALCAM and a panel of conformationally sensitive anti-CD6D3 monoclonal antibodies (mAbs). CD6D3 residues were classified according to their importance for structural integrity and ligand binding. The results were analyzed in the light of SRCR domain sequence comparison. A number of residues critical for ligand binding or important for structural integrity cluster in the C-terminal region of CD6D3 which is not conserved in other SRCR proteins.

Identification of residues in CD6 which are critical for ligand binding.

CD6 is a member of the scavenger receptor cysteine rich protein superfamily (SRCRSF). This family includes many cell surface proteins whose three-dimensional structures and functions are presently not well understood. The extracellular region of CD6 includes 3 SRCR domains. The membrane proximal SRCR domain specifically binds the activated leukocyte cell adhesion molecule (ALCAM), a CD6 ligand belonging to the immunoglobulin superfamily. CD6-ALCAM interactions mediate immune cell adhesion and are implicated in T cell maturation and the regulation of T cell function. On the basis of SRCRSF sequence comparison, a mutagenesis analysis of the membrane proximal SRCR domain of CD6 (CD6D3) has been carried out. Fifteen mutants were characterized. Three CD6 residues were identified in a region of low sequence conservation which, when mutated, abolish ligand binding but not the binding to a panel of conformationally sensitive anti-CD6 mAbs. This study provides the first analysis of residues critical for ligand binding to a member of the SRCRSF.

CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics.

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell-APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 0.43 s-1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate-dynamic T cell-APC contacts and to facilitate scanning of APC for antigen.

Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59.

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent.

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution.

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Expression cloning of an equine T-lymphocyte glycoprotein CD2 cDNA. Structure-based analysis of conserved sequence elements.

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones.

Influenza hemagglutinin (HA) undergoes a conformational change that is required for viral entry. The rearrangement includes exposure of the fusion peptide, a hydrophobic segment buried in the trimer interface of the native protein. Since fusion peptide release triggers the membrane fusion event crucial for viral replication, inhibition of fusion peptide exposure should prevent infection. We reasoned that small molecules that bind to HA and stabilize its nonfusogenic conformation would block viral activity. A computer-assisted method was used to select putative HA ligands. One of the selected compounds, 4A,5,8,8A-tetrahydro-5,8-methano-1,4-naphthoquinone, prevented the conversion of X31 HA to a conformation recognized by alpha-fusion peptide antisera. Several derivatives of this compound, including both benzoquinones and hydroquinones, also showed inhibition. The most effective compounds tested have IC50S between 1 and 20 microM. Representative compounds also inhibited virus-induced syncytia formation, HA-mediated hemolysis, and viral infectivity in vitro. The inhibitors are attractive leads for the development of antiviral drugs and can serve as probes of the mechanism of the conformational change of HA.

Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin.

At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer.