含有补体控制蛋白结构域的蛋白质的结构和功能

补体控制蛋白(CCP)结构域分布广泛,含有CCP结构域的蛋白质在补体调节、机体排异和抵御微生物侵袭,甚至肿瘤发生发展等方面具有重要的功能。现在发现的含CCP结构域的蛋白质大约有100多种。我们综述了CCP结构域的基本特征,简要介绍了有代表性的含CCP结构域的蛋白质的功能。     

Kinetics of Expression of Two Major Plasmodium berghei Antigens in the Mosquito Vector, Anopheles stephensi

ABSTRACT. Expression of a 21 kDa determinant (Pbs21), first detected on the surface of ookinetes, and of the circumsporozoite protein (CSP) was studied by immunofluorescence and Western blots during the developmental cycle of Plasmodium berghei in the mosquito A nopheles stephensi . The expression of Pbs21 was predominantly localised on the ookinete surface one day after the infectious blood meal, and thereafter reactivity declined to a minimum on days 2 and 3, the time of onset of oocyst development. A gradual increase in fluorescence was observed on the oocysts from day 6 that was retained until day 17 post-infection. In contrast, sporozoites released from oocysts or salivary glands showed little or no antibody labelling with anti-Pbs21. Circumsporozoite protein was not detectable in any rnidgut preparations until 5–6 days after feeding, when reactivity was observed against immature oocysts. Expression then continued and increased throughout oocyst and sporozoite development. Western blots confirmed that Pbs21 was expressed minimally during the oocyst development but was not detectable in sporozoites. Co-localisation of anti-Pbs21 and anti-CSP monoclonal antibodies to the 50 kDa and 60 kDa bands in Western blots of sporozoite suggests immunological cross-reactivity between the CSP and the anti-21 kDa antibodies.     

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP''s binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites.     

Phg1/TM9 Proteins Control Intracellular Killing of Bacteria by Determining Cellular Levels of the Kil1 Sulfotransferase in Dictyostelium

Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified) sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.     

The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors

The small leucine-rich repeat proteins, fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-sulfated tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N and C termini, respectively, and PRELP is known to bind to heparin via this domain. Another heparin-binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C terminus. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full-length osteoadherin. The tyrosine sulfate domains also bound heparin-binding proteins such as basic fibroblast growth factor-2, thrombospondin I, MMP13, the NC4 domain of collagen IX, and interleukin-10. Fibronectin with large heparin-binding domains did not bind, neither did CILP containing a heparin-binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the sulfated tyrosine residues shown by different binding properties of 10-kDa fragments subfractionated by ion-exchange chromatography. These interactions may sequester growth factors, cytokines, and matrix metalloproteinases in the extracellular matrix as well as contribute to its organization.The integrity of the extracellular matrix depends on a multitude of interactions between molecular constituents leading to the formation of major macromolecular assemblies important for tissue functions. A major component in most types of extracellular matrix is the network of fibrillar structures primarily composed of collagen I in fibrous tissues and bone, whereas cartilage contains the similar collagen II.These collagen fibrils contain a number of associated molecules, often bound to their surface. One such molecule is the distinct collagen IX, containing three triple helical domains each surrounded by non-triple helical domains. Another set of molecules binding to triple helical collagen is the members of the small leucine-rich repeat protein family, such as fibromodulin (1), lumican (2), decorin (3), biglycan (4), PRELP (5), chondroadherin (6), and possibly osteoadherin. The typical LRR³ protein contains 10–11 repeats of some 25 amino acids with leucine residues at conserved locations. This domain represents a common denominator for the family and contains structures providing for interaction with, e.g. triple helical collagen (7–9). The LRR proteins contain an extension at either the N- or C-terminal end or, in a few cases, at both ends. These extensions may contribute to a second function exemplified by PRELP, where the N-terminal with a stretch of clustered arginine residues provides binding to heparin/heparan sulfate containing optimally five or more disaccharides with three sulfate groups each (10). In decorin and biglycan, the N-terminal extension have substituents of glycosaminoglycan chains of dermatan/chondroitin sulfate that can contribute to collagen binding (11) as well as provide putative self interactions with a similar chain on another molecule. In particular, it has been shown that decorin and biglycan will bind via their protein core to the N-terminal globular domain of collagen VI (4) and direct the formation of the collagen VI-beaded filament network, provided that the glycosaminoglycan chains are intact.There are a number of proteins known to interact with heparin. Whereas heparin is not present in the extracellular matrix, these proteins may bind to stretches within the heparan sulfate chains enriched in disaccharides having high sulfate content. The heparan sulfate is found particularly as a component of cell surface proteoglycans such as glypicans (12) and syndecans (13) and of the extracellular matrix proteoglycans perlecan (14) and agrin (15). Important ligands for these chains are growth factors exemplified by members of the FGF family. Other molecules that bind to heparan sulfate include fibronectin, having two such domains with molecular weights of around 20,000 (16). Also several members of the metalloproteinase family contain heparin-binding motifs as do many cytokines.The most common heparin-binding sequence contains clusters of basic amino acids, often with additional proline residues. PRELP and chondroadherin as well as the other proteins mentioned represent examples having such sequences. A different type of motif, first found in thrombospondin I, contains consecutive repeats of a WXZ sequence, where the tryptophan may be mannosylated (17, 18). This is referred to as the thrombospondin type I motif heparin-binding structure. Thrombospondin I in addition contains a heparin-binding basic cluster of amino acids (19). CILP on the other hand only contains the thrombospondin type 1 motif. These domains can bind to heparin with high affinity, an interaction that can be disrupted by high salt.A very different type of extension is found in the N-terminal part of fibromodulin and osteoadherin. These proteins contain a number of tyrosine residues, which may and often do, carry a sulfate group. Thus, fibromodulin contains up to nine such residues and osteoadherin as many as eight, where six are located in the N-terminal and two in the C-terminal extension (20). Any given preparation contains molecules within the same species with a range of levels of O-sulfate-substituted tyrosine. The functional significances of these domains have been unknown. We now show that these domains can mimic heparin in several interactions.     

Genome-Wide Mouse Mutagenesis Reveals CD45-Mediated T Cell Function as Critical in Protective Immunity to HSV-1

Herpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc^L3X), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc^L3X mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc^L3X mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc ^L3X mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4⁺ and CD8⁺ T cells and could be attributed to function of CD4⁺ T helper 1 (Th1) cells in CD8⁺ T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development.     

Ubr1 and Ubr2 Function in a Quality Control Pathway for Degradation of Unfolded Cytosolic Proteins

Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.     

Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.     

Killing of Gram-Negative Bacteria with Normal Human Serum and Normal Bovine Serum: Use of Lysozyme and Complement Proteins in the Death of Salmonella Strains O48

Serum is an environment in which bacterial cells should not exist. The serum complement system provides innate defense against microbial infections. It consists of at least 35 proteins, mostly in pre-activated enzymatic forms. The activation of complement is achieved through three major pathways: the classical, alternative, and lectin. Lysozyme, widely present in body fluids, catalyzes the hydrolysis of β 1,4 linkage between N-acetyloglucosamine and N-acetylmuramic acid in the bacterial cell wall and cooperates with the complement system in the bactericidal action of serum. In this study, ten strains of serotype O48 Salmonella, mainly associated with warm-blooded vertebrates and clinically important causing diarrhea in infants and children, were tested. The results demonstrated that the most efficient killing of Salmonella O48 occurred when all the components of normal bovine serum (NBS) and normal human serum (NHS) cooperated. To prove the role of lysozyme in the bactericidal activity of bovine and human serum, the method of serum adsorption onto bentonite (montmorillonite, MMT) was used. In order to investigate structural transitions accompanying the adsorption of serum components, we applied X-ray diffraction methods. The results of this investigation suggested that apart from lysozyme, other proteins (as, e.g., C3 protein or IgG immunoglobulin) were adsorbed on MMT particles. It was also shown that Ca²⁺ cations can be adsorbed on bentonite. This may explain the different sensitivities of the serovars belonging to the same O48 Salmonella serotype to NBS and NHS devoid of lysozyme.     

The CLIP-Domain Serine Protease Homolog SPCLIP1 Regulates Complement Recruitment to Microbial Surfaces in the Malaria Mosquito Anopheles gambiae

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1_cut that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals.     

Indirect Interactions Limit the Efficiency of Odonata as Natural Control Agents for Mosquito Larvae

Odonate nymphs are voracious predators and may be useful natural control agents. However, odonates are normally found in complex guilds with a high degree of conspecific and inter specific predation that may reduce their overall predation efficiency. The present study investigates whether indirect interactions, mediated via chemical predation signals, inhibit the consumption of mosquito larvae by two common odonates: Crocothemis erythraea and Ischnura evansi. Our results show that the predation rate of C. erythraea is reduced by chemical cues from Anax imperator, but the response of I. evansi to C. erythraea was statistically non-significant. This study shows that intra-guild interactions may limit the effectiveness of odonates as predators of mosquitoes.     

The Control of Vitellogenesis in the Mosquito, Aedes aegypti

Evidence from several laboratories indicates that oogenesisin mosquitoes is divided into at least two discrete stages.First, emergence triggers development of the oocytes up to theresting stage where they remain until the blood meal. This stageis mediated by the juvenile hormone from the corpora allata.The second stage is triggered by the blood meal and involvesthe release of a hormone from the brain as well as the vitellogeninstimulating hormone (VSH) from the ovary. The mature oocytesthen inhibit development of the penultimate oocytes until thefirst batch of eggs is laid. The evidence for an ovarian hormone,VSH, controlling vitellogenin synthesis by the mosquito fatbody is reviewed. This hormone activates and maintains vitellogeninsynthesis by the fat body in vivo and in vitro. The role ofVSH in mosquito reproduction and the possibility of similarhormones in other insects is discussed.     

Two defined alleles of the LRR receptor kinase GmNARK in supernodulating soybean govern differing autoregulation of mycorrhization

Plants regulate the extent of nodulation and root colonization by arbuscular mycorrhizal fungi (AMF), a phenomenon named autoregulation of symbiosis. We tested AMF colonization in split roots of various soybean genotypes [ Glycine max (L.) Merr. cv. Bragg, Enrei, Harosoy and Williams], where precolonization of one side of the split-root system by the AMF Glomus mosseae resulted in reduced mycorrhization of the other. AMF precolonization failed to control secondary mycorrhization in the supernodulating Bragg nonsense mutant nts1007 (Q106*), indicating that the GmNARK gene (predicted to encode a leucine-rich repeats (LRR) receptor kinase related to CLAVATA1 in Arabidopsis ) is involved in autoregulation of the AMF symbiosis. Here, we tested whether the allelic En6500 nonsense supernodulating mutant ( GmNARK K606*, derived from cv. Enrei) and supernodulating mutants of cv. Williams ( Nod1-3 and Nod2-4 ) with yet-undefined genetic lesions exhibit a similar symbiotic phenotype in mycorrhizal split-root systems. Surprisingly, these supernodulating mutants retained their ability to autoregulate AMF. To examine possible differences between two allelic mutants, we determined levels of IAA, abscisic acid, coumestrol, daidzein and genistein in mycorrhizal and uninoculated control roots. Compared with wild-type plants, both mutants showed reduced IAA accumulation in mycorrhizal roots. Roots of cv. Enrei and En6500 exhibited high levels of isoflavonoids not seen in Bragg or nts1007 . Taken together, these findings showed that supernodulation mutants, despite a common nodulation phenotype, differ in their ability to autoregulate AMF root colonization. This suggests either that the GmNARK gene product of some mutants is still partially functional (Q106* vs. K606*) or that varietal differences reflected in altered physiological responses suppress the loss of function.