The complement system in teleosts

Complement, an important component of the innate immune system, is comprised of about 35 individual proteins. In mammals, activation of complement results in the generation of activated protein fragments that play a role in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Fish appear to possess activation pathways similar to those in mammals, and the fish complement proteins identified thus far show many homologies to their mammalian counterparts. Because information about complement proteins, regulatory proteins, and complement receptors in fish is far from complete, it is unclear whether all the complement functions that have been identified in mammals also occur in fish. However, it has been clearly demonstrated that fish complement can lyse foreign cells and opsonise foreign organisms for destruction by phagocytes. There are also indications that complement fragments participate in inflammatory reactions. Fish possess multiple isoforms of several complement proteins, such as C3 and factor B. It has been hypothesised that the function of this diversity in complement proteins serves to expand their innate immune recognition capacity and response. Understanding the functions of complement in fish and the roles the individual proteins, including the various isoforms, play in host defence, is important not only for understanding the evolution of this system but also for the development of new strategies in fish health management.     

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M. truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins. Following the setting up of the purification process, microsomal proteins were separated on 2-DE. Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting. A high percent (83%) of successful protein identification was obtained when using M. truncatula clustered EST database for queries. During the purification process, the enrichment in membrane-associated proteins was monitored on 2-D gels. The membrane location of microsomal proteins was further confirmed using PMF identification. This study reports a fractionation process for characterizing microsomal root proteins of M. truncatula, which could be an interesting tool for investigating the molecular mechanisms involved in root symbioses.     

A BRAIN-SPECIFIC PROTEIN FROM OCTOPUS VULGARIS, LAM

Abstract— Several major brain-specific proteins have been detected in cephalopods by electrophoretic analysis of the soluble proteins extracted from the optic lobes and other organs of octopus and by 2-dimensional fractionation of the soluble proteins from optic lobes and hepatopancreases of octopus and squid. One of the brain-specific proteins from octopus, identified as 0-1, has been purified by chromatography on DEAE-cellulose, Sephadex G-150, and DEAE-Sephadex. The protein appears to be pure on the basis of several physicochemical criteria. Amino acid analysis indicates a high content of glutamic and aspartic acids or their amides (or both) and the lack of tryptophan. A molecular weight of 17,000 has been calculated from sodium dodecyl sulphate-gel electrophoresis, gel filtration and ultracentrifugation analysis. The preparation of a specific rabbit antiserum against 0-1 has allowed its determination by agar immunodiffusion and complement fixation techniques. With the latter procedure it has been shown that the protein is absent outside the nervous system, is present in a concentration of several mg/g wet weight in octopus brain and is widely distributed within the octopus central and peripheral nervous system and in several molluscan species. It is also present in optic lobes of octopus at early stages of development.     

Tissue-specific regulation of inflammation.

Proteins of the complement system are important effectors and modulators of inflammation. The complement cascade is triggered by microbes, tissue debris, and specific antibodies. Serum complement proteins are derived primarily from liver, but extrahepatic complement synthesis is important in homeostasis and in local host defenses. Tissue-specific regulation of expression of complement genes is governed by mechanisms similar to those that regulate other "acute phase reactants." That is, tissue injury or infection elicit changes in expression of these acute phase proteins, which, although variable in kinetics, magnitude, and direction, are a consequence of an elaborate system of cell-to-cell communication. This communication is mediated via a complex network of cytokines, including the interferons, interleukins, several growth factors, and sex hormones. The cell biological and molecular biological details of these mechanisms are now under active investigation. An understanding in molecular terms of the balance between proinflammatory and counterregulatory forces on complement gene expression should provide new insight into the functions of complement and the design of novel therapies for disorders of inflammation.     

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.     

Crystal structure and mutational analysis of the DaaE adhesin of Escherichia coli

DaaE is a member of the Dr adhesin family of Escherichia coli, members of which are associated with diarrhea and urinary tract infections. A receptor for Dr adhesins is the cell surface protein, decay-accelerating factor (DAF). We have carried out a functional analysis of Dr adhesins, as well as mutagenesis and crystallographic studies of DaaE, to obtain detailed molecular information about interactions of Dr adhesins with their receptors. The crystal structure of DaaE has been solved at 1.48 A resolution. Trimers of the protein are found in the crystal, as has been the case for other Dr adhesins. Naturally occurring variants and directed mutations in DaaE have been generated and analyzed for their ability to bind DAF. Mapping of the mutation sites onto the DaaE molecular structure shows that several of them contribute to a contiguous surface that is likely the primary DAF-binding site. The DAF-binding properties of purified fimbriae and adhesin proteins from mutants and variants correlated with the ability of bacteria expressing these proteins to bind to human epithelial cells in culture. DaaE, DraE, AfaE-III, and AfaE-V interact with complement control protein (CCP) domains 2-4 of DAF, and analysis of the ionic strength dependence of their binding indicates that the intermolecular interactions are highly electrostatic in nature. The adhesins AfaE-I and NfaE-2 bind to CCP-3 and CCP-4 of DAF, and electrostatic interactions contribute significantly less to these interactions. These observations are consistent with structural predictions for these Dr variants and also suggest a role for the positively charged region linking CCP-2 and CCP-3 of DAF in electrostatic Dr adhesin-DAF interactions.     

Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

Iodination of reovirus, cytoplasmic polyhedrosis virus (CPV), and wound tumor virus (WTV), and their respective subviral forms, followed by analysis of the labeled polypeptides by using polyacrylamide gel electrophoresis, has been used to compare the protein contents of these three diplornaviruses. This approach, when combined with electron microscopy and buoyant density determinations, appears capable of localizing individual polypeptides in some of the viral and subviral forms. CPV (p = 1.435 g/cm(3)) seems to resemble reovirus cores (p = 1.440 g/cm(3)) in both ultrastructure and polypeptide composition. CPV is composed of five polypeptides with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000. The polyhedral matrix, which in nature encapsulates the virions, is, in turn, composed mainly of two polypeptide species with molecular weights of about 30,000 and 20,000, and several minor proteins. The proteins of WTV consist mainly of four species of polypeptide with molecular weights of about 156,000, 122,000, 63,000, and 44,000, and several minor components. These molecular weight determinations are consistent with the hypothesis that, as has been suggested for reovirus, the viral proteins of CPV and WTV seem to be coded for by monocistronic mes senger RNA molecules transcribed from distinct segments of the double-stranded RNA viral genomes.     

Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock.

In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement than its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.     

Microglia in cerebral ischemia: molecular actions and interactions

The precise role of microglia in stroke and cerebral ischemia has been the subject of debate for a number of years. Microglia are capable of synthesizing numerous soluble and membrane-bound biomolecules, some known to be neuroprotective, some neurotoxic, whereas others have less definitive bioactivities. The molecular mechanisms through which microglia activate these molecules have thus become an important area of ischemia research. Here we provide a survey review that summarizes the key actions of microglial factors in cerebral ischemia including complement proteins, chemokines, pro-inflammatory cytokines, neurotrophic factors, hormones, and proteinases, as well several important messenger molecules that play a part in how these factors respond to extracellular signals during ischemic injuries. We also provide some new perspectives on how microglial intracellular signaling may contribute to the seemingly contradictory roles of several microglial effector molecules.     

The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar)

Fish embryos and hatchlings are exposed to pathogens long before maturation of their lymphoid organs. Little is known about defence mechanisms during the earliest stages of life, but innate mechanisms may be essential for survival. The complement system in fish is well developed and represents a major part of innate immunity. Complement factor 3 (C3) is central subsequent to activation of all pathways of the complement system, leading to inflammatory reactions, such as chemotaxis, opsonisation and lysis of pathogens. Hepatocytes represent the major source of C3, but modern molecular biological methods have confirmed that C3 is synthesised at multiple sites. Our main objective was to study the ontogeny of C3 in Atlantic salmon by mapping the commencement of synthesis and localisation of proteins. Eggs, embryos, hatchlings and adult fish were analysed for the presence of C3 mRNA and proteins. From immunohistochemical studies, C3 proteins were detected at several extrahepatic sites, such as the skeletal muscle, developing notochord and chondrocytes of the gill arch. Immunoblotting revealed presence of C3 proteins in the unfertilised egg, but C3 mRNA was only detected after fertilisation by real-time RT-PCR. Taken together, the results implicated the maternal transfer of C3 proteins as well as novel non-immunological functions during development.     

Backbone dynamics of complement control protein (CCP) modules reveals mobility in binding surfaces

The regulators of complement activation (RCA) are critical to health and disease because their role is to ensure that a complement-mediated immune response to infection is proportionate and targeted. Each protein contains an uninterrupted array of from four to 30 examples of the very widely occurring complement control protein (CCP, or sushi) module. The CCP modules mediate specific protein-protein and protein-carbohydrate interactions that are key to the biological function of the RCA and, paradoxically, provide binding sites for numerous pathogens. Although structural and mutagenesis studies of CCP modules have addressed some aspects of molecular recognition, there have been no studies of the role of molecular dynamics in the interaction of CCP modules with their binding partners. NMR has now been used in the first full characterization of the backbone dynamics of CCP modules. The dynamics of two individual modules-the 16th of the 30 modules of complement receptor type 1 (CD35), and the N-terminal module of membrane cofactor protein (CD46)-as well as their solution structures, are compared. Although both examples share broadly similar three-dimensional structures, many structurally equivalent residues exhibit different amplitudes and timescales of local backbone motion. In each case, however, regions of the module-surface implicated by mutagenesis as sites of interactions with other proteins include several mobile residues. This observation suggests further experiments to explore binding mechanisms and identify new binding sites.     

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up-regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.     

A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase.

A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing lambda phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (Mr 78,000) is still unidentified.     

Recent advances on the complement system of teleost fish

The complement system plays an essential role in alerting the host of the presence of potential pathogens, as well as in their clearing. In addition, activation of the complement system contributes significantly in the orchestration and development of an acquired immune response. Although the complement system has been studied extensively in mammals, considerably less is known about complement in lower vertebrates, in particular teleost fish. Here we review our current understanding of the role of fish complement in phagocytosis, respiratory burst, chemotaxis and cell lysis. We also thoroughly review the specific complement components characterized thus far in various teleost fish species. In addition, we provide a comprehensive compilation on complement host-pathogen interactions, in which we analyze the role of fish complement in host defense against bacteria, viruses, fungi and parasites. From a more physiological perspective, we evaluate the knowledge accumulated on the influence of stress, nutrition and environmental factors on levels of complement activity and components, and how the use of this knowledge can benefit the aquaculture industry. Finally, we propose future directions that are likely to advance our understanding of the molecular evolution, structure and function of complement proteins in teleosts. Such studies will be pivotal in providing new insights into complement-related mechanisms of recognition and defense that are essential to maintaining fish homeostasis.     

Avian Tumor Virus Proteins and RNA in Uninfected Chicken Embryo Cells

The content of proteins P19 and P15 (mol wt 19,000 and 15,000, respectively) of avian leukovirus in various types of uninfected chicken embryos has been determined by radioimmunoassay. All chicken embryos examined, including embryos which have thus far been classified as group specific (gs) antigen negative by complement fixation tests, contained these viral proteins as well as P27 as previously reported. The embryos known as “gs antigen-positive” type contained about five times as much of these viral proteins as did the “gs antigen-negative” type. The ratio of the three viral proteins was similar for all types of embryos, suggesting that the genes for these proteins are coordinately controlled. In contrast to the relatively high levels of viral internal proteins in gs antigen-negative cells, the amounts of virus-specific RNA detectable by molecular hybridization were extremely low. The levels of helper activity, which presumably reflect the level of viral envelope glycoprotein, were also generally low or undetectable in these cells. Thus, the expression of the gene for envelope glycoprotein does not appear to be controlled coordinately with the genes for viral internal proteins.     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Parasite interaction with host complement: beyond attack regulation

Many orthologous proteins of known mammalian receptors have been discovered in parasites. Besides disguising the parasite as self in terms of the host immune system, evidence is accumulating that these receptors link to signalling pathways in parasites that appear to be involved in their growth or development. Recently, several proteins of the host complement system, which forms part of the innate defence against invading microorganisms, have been shown to possess alternative functions. These complement proteins interact with signalling pathways involved in early development and differentiation, as well as organ and tissue regeneration. By altering cellular interactions and responses, complement is being shown to have novel roles besides the originally described inflammatory role. The possibility exists that, as for other host factors interacting with parasites and affecting their growth or development, host complement proteins could also have such an influence.     

Virus-induced proteins in pseudorabies-infected cells. II. Proteins of the virion and nucleocapsid.

Analysis of purified naked and enveloped nucleocapsids of pseudorabies virus with high-resolution techniques has allowed a reassessment of their protein composition. Enveloped particles are shown to contain at least 20 proteins whose molecular weights are in the range 20,000 to 230,000. Naked nucleocapsids contain one major and seven minor proteins in the molecular weight range 20,000 to 155,000. Phosphorylation of at least one virion protein is shown to take place in vivo. These results demonstrate that pseudorabies virus is similar in its protein complement to other herpesviruses which have recently been examined.     

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs. I. Isolation and properties of the low-molecular-weight apoproteins.

In a continuing study of protein-lipid interactions in egg yolk, the total apoprotein mixture (i.e. the 'apovitellenins') from the high-lipid, low-density lipoprotein (density 0.97 g/ml) of the yolk from hen's eggs has been isolated in a soluble form. By gel-filtration chromatography in 6M urea the mixture has been separated into several fractions from which three new low-molecular-weight proteins (I, Ia, and II), making up about 30% of the total, have been isolated. The most plentiful of these (I) consists of stable aggregates with several identical subunits each of molecular weight about 10 000. This protein is analogous to the principal protein from the corresponding lipoprotein of emu's egg yolk, i.e. emu's apovitellenin I. Hen's apovitellenin I has a slightly different amino acid composition from that of the emu; notably it contains a sulphydryl group. The hen's protein also forms more stable aggregates that are dissociated by detergent and by guanidine hydrochloride but are stable in urea. The molecular weight of Ia is similar to that of I and the amino acid composition is the same, with the exception that Ia has a higher proportion of amide groups. It aggregates less readily than I under the same conditions. The third new protein (II, 'hens's apovitellenin II') has a molecular weight of about 20 000. It has no tyrosine or methionine residues, but contains glucosamine and has several disulphide groups. It has been isolated in very small amount only.