Plasminogen is a complement inhibitor

Plasminogen is a 92-kDa single chain glycoprotein that circulates in plasma as a zymogen and when converted to proteolytically active plasmin dissolves preformed fibrin clots and extracellular matrix components. Here, we characterize the role of plasmin(ogen) in the complement cascade. Plasminogen binds the central complement protein C3, the C3 cleavage products C3b and C3d, and C5. Plasminogen binds to C3, C3b, C3d, and C5 via lysine residues, and the interaction is ionic strength-dependent. Plasminogen and Factor H bind C3b; however, the two proteins bind to different sites and do not compete for binding. Plasminogen affects complement action in multiple ways. Plasminogen enhanced Factor I-mediated C3b degradation in the presence of the cofactor Factor H. Plasminogen when activated to plasmin inhibited complement as demonstrated by hemolytic assays using either rabbit or sheep erythrocytes. Similarly, plasmin either in the fluid phase or attached to surfaces inhibited complement that was activated via the alternative and classical pathways and cleaved C3b to fragments of 68, 40, 30, and 17 kDa. The C3b fragments generated by plasmin differ in size from those generated by the complement protease Factor I, suggesting that plasmin-mediated C3b cleavage fragments lack effector function. Plasmin also cleaved C5 to products of 65, 50, 30, and 25 kDa. Thus, plasmin(ogen) regulates both complement and coagulation, the two central cascade systems of a vertebrate organism. This complement-inhibitory activity of plasmin provides a new explanation why pathogenic microbes utilize plasmin(ogen) for immune evasion and tissue penetration.     

Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.     

Anti-analgesic and anti-amnesic effect of complement C3a

In the present study, we found that complement C3a exerted central effects after intracerebroventricular administration in mice. At doses of 3 and 10 pmol/mouse, the peptide showed an antagonistic effect on analgesia induced by morphine and U-50488H, known to be mu- and kappa-opioid receptor agonists, respectively. Moreover, complement C3a improved scopolamine- and ischemia-induced amnesia at a dose of 10 pmol/mouse. Anti-analgesia was not observed by C3a des-Arg at 10 pmol/mouse. The present findings suggest that complement C3a may act as a peptide with anti-opioid activity in the central nervous system.     

Functional characterization of a ficolin-mediated complement pathway in amphioxus

The ficolin-mediated complement pathway plays an important role in vertebrate immunity, but it is not clear whether this pathway exists in invertebrates. Here we identified homologs of ficolin pathway components from the cephalochordate amphioxus and investigated whether they had been co-opted into a functional ficolin pathway. Four of these homologs, ficolin FCN1, serine protease MASP1 and MASP3, and complement component C3, were highly expressed in mucosal tissues and gonads, and were significantly up-regulated following bacterial infection. Recombinant FCN1 could induce hemagglutination, discriminate among sugar components, and specifically recognize and aggregate several bacteria (especially gram-positive strains) without showing bactericidal activity. This suggested that FCN1 is a dedicated pattern-recognition receptor. Recombinant serine protease MASP1/3 formed complexes with recombinant FCN1 and facilitated the activation of native C3 protein in amphioxus humoral fluid, in which C3 acted as an immune effector. We conclude that amphioxus have developed a functional ficolin-complement pathway. Because ficolin pathway components have not been reported in non-chordate species, our findings supported the idea that this pathway may represent a chordate-specific innovation in the evolution of the complement system.     

Isolation of a hagfish gene that encodes a complement component.

It has been widely accepted that cyclostomes are the most primitive vertebrates extant with the ability to produce antibodies. We isolated cDNA clones that encode a putative 'antibody' from one of the cyclostomes, Eptatretus burgeri. The amino acid sequence predicted from the nucleotide sequences of the cDNA clones indicated that this gene does actually encode the proteins isolated as hagfish 'antibodies' by various investigators. However, these proteins are not similar to mammalian immunoglobulins but have some characteristics common to complements C3, C4 and C5 in higher vertebrates. We discuss the relationships of the isolated gene for hagfish complement with the mammalian genes for complements C3, C4 and C5. We also discuss the possibility of the presence of antibodies in cyclostomes.     

Covalent capture: a natural complement to self-assembly

The utility of peptide self-assembly can be extended by covalent capture of these supramolecular materials. Disulfide bond formation, native chemical ligation, olefin metathesis, radical capture and oxidative lysine cross-linking have been used recently to help stabilize and characterize a variety of self-assembled peptides. These include natural peptides, proteins and protein mimics such as alpha-helical coiled coils, amyloid-like beta-sheet fibres, portions of p53, glutathione S-transferase and elastin as well as unnatural peptide constructs such as cyclic peptide nanotubes and cylindrical micelles of peptide amphiphiles.     

Detection of complement activity by using a polysaccharide-protected membrane

The complement system of mammalian blood is a nonspecific part of the immune system involved in a number of disease conditions. We report the observation of pore creation caused by its activation in blood applied to the front gel layer of a bilayer membrane formed from dioleoylphosphatidyl choline and protected by a polysaccharide gel. The pores were detected by measuring the DC conductivity between nonblocking Ag/AgCl electrodes. The thickness of the protective gel was approximately 100 μm, and the complement response was seen within 3 min after application of activator. The lifetime of such membranes is limited only by hydrolysis of the phospholipid constituting the membrane. This easily prepared system is suitable for examining the kinetics of complement component interactions with inhibitors.     

Interaction of human complement with Sbi, a staphylococcal immunoglobulin-binding protein: indications of a novel mechanism of complement evasion by Staphylococcus aureus

Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and beta(2)-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption.     

Phenological records as a complement to aerobiological data

Phenological studies in combination with aerobiological studies enable one to observe the relationship between the release of pollen and its presence in the atmosphere. To obtain a suitable comparison between the daily variation of airborne pollen concentrations and flowering, it is necessary for the level of accuracy of both sets of data to be as similar as possible. To analyse the correlation between locally observed flowering data and pollen counts in pollen traps in order to set pollen information forecasts, pollen was sampled using a Burkard volumetric pollen trap working continuously from May 1993. For the phenological study we selected the main pollen sources of the six pollen types most abundant in our area: Cupressaceae, Platanus, Quercus, Plantago, Olea, and Poaceae with a total of 35 species. We selected seven sites to register flowering or pollination, two with semi-natural vegetation, the rest being urban sites. The sites were visited weekly from March to June in 2007, and from January to June in 2008 and 2009. Pollen shedding was checked at each visit, and recorded as the percentage of flowers or microsporangia in that state. There was an association between flowering phenology and airborne pollen records for some of the pollen types (Platanus, Quercus, Olea and Plantago). Nevertheless, for the other types (Cupressaceae and Poaceae) the flowering and airborne pollen peaks did not coincide, with up to 1 week difference in phase. Some arguments are put forward in explanation of this phenomenon. Phenological studies have shown that airborne pollen results from both local and distant sources, although the pollen peaks usually appear when local sources are shedding the greatest amounts of pollen. Resuspension phenomena are probably more important than long-distance transport in explaining the presence of airborne pollen outside the flowering period. This information could be used to improve pollen forecasts.     

Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans

Vaccinia virus complement control protein (VCP) inhibits both pathways of complement activation through binding the third and fourth components. A homolog of mammalian regulators of complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. This, the structure of any intact regulator of complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of complement.     

Circular dichroism of C5a anaphylatoxin of porcine complement

The far-ultraviolet circular dichroism spectrum of C5a anaphylatoxin of porcine complement implies that it has a substantial content of helical structure. The circular dichroism spectra of C5a in the 200–250 nm region at pH 7.2 and 3.7 are nearly identical and resemble those of C3a anaphylatoxin. Treatment of C5a with 2-mercaptoethanol progressively diminishes the ellipticity at 222 nm and its anaphylatoxic activity to limiting values. Removal of the reducing agent by dialysis completely restored both the ellipticity at 222 nm and the activity. This finding indicates that the integrity of the secondary conformation of the C5a molecule is largely dependent on disulfide bonds and is essential for its full activity.     

Characterization of a genetically identifiable component of complement in mice

The hemolytic properties of the sera of complement positive and complement negative strains of mice were studied. The results suggested that the complement negative sera lacked an activity of complement analagous to C'1 which when present is called hc¹. Certain physical and chemical properties of hc¹ were examined. It was destroyed when serum was heated to 56°C, but was resistant to heating when slightly purified. Hc¹ was still precipitable by specific antiserum after treatment with 8 molar urea, but was no longer hemolytically active. Neither the hemolytic nor precipitating activity of hc¹ was altered by treatment with either 0.1 molar ethylenediamine-tetraacetic acid or 0.1 molar 2-mercaptoethanol. Initial attempts made to purify hc¹ are described.     

Monocyte complement stimulator: a T-lymphocyte product which stimulates synthesis of the second complement component (C2).

Monocyte complement stimulator (MCS), a lymphokine previously shown to increase the rate of synthesis of the second complement component (C2) by human monocytes, is produced by sensitive T lymphocytes when exposed to antigen. MCS production requires cooperation of T lymphocytes with monocytes during the first 24 hr of exposure to antigen; both cell types must be capable of synthesizing protein during this time. MCS was found to differ from MIF in two ways: First, antigen-stimulated B lymphocytes and monocytes produce MIF but not MCS and second, MCS is destroyed by heating to 56 °C for 30 min while MIF retains its activity