Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement

The structure of the human properdin molecule was investigated by hydrodynamic, spectroscopic, and transmission electron microscope studies. Sucrose density gradient ultracentrifugation of purified, functionally active properdin showed a single component sedimenting at 5.5 S. Electron microscopic examination by two different methods, however, revealed polydispersity of the protein which consisted of cyclic dimers, trimers, tetramers, pentamers, and higher cyclic oligomers. Approximately 80% of the oligomers were dimers, trimers, and tetramers. Monomers could not be detected. These polymers could be partially separated by gel filtration on Sephacryl S-300 and all fractions were active in terms of binding to C3b. The specific activity increased with oligomer size. When reexamined after incubation at 37 degrees C for 4 h or at 4 degrees C for 2 weeks, the chromatographic behavior of the oligomers and their electron microscopic appearance were unchanged, suggesting that in vitro no rapid equilibration occurred. The protomer is clearly visualized within polymers as a flexible, rod-like structure 26.0 nm in length and 2.5 nm in diameter, with pronounced thickening at each end. The monomer is bivalent with respect to binding to other properdin monomers and the binding sites are localized to the ends of the structure. A model could be devised which is consistent with the distinct geometry of the intersubunit contacts observed in micrographs. The circular dichroism spectrum of properdin suggests the presence of little alpha helix or beta structure and shows positive ellipticity at 231 nm. In contrast to previous investigators, we conclude that isolated human properdin is polydisperse and consists of a set of cyclic polymers constructed from a single highly asymmetric and flexible protomer.     

Changes in the immunochemical properties of highly purified properdin in human serum.

The fate of highly purified properdin (P) upon introduction into normal human serum or properdin-depleted serum (RP) was investigated. It was observed that, concomitant with the activation of the alternate pathway components, properdin underwent immunochemical alterations characterized by a shift in mobility from gamma2 to beta2 position and by an increase in the sedimentation rate from 5.1S to between 6.8 and 9.3S. The immunoelectrophoretic behavior of C3 was also altered with the appearance of a beta2 arc in addition to the beta1C arc. The immunochemical properties of altered P resemble those of "native" properdin in fresh serum. The principle in serum (designated factor F) mediating these changes is a euglobulin with an approximate sedimentation rate and molecular weight of 9.0S and 250,000 daltons, respectively. The alteration in the immunochemical properties of P may be due to aggregation of P molecules or a complex formation between P and a serum euglobulin (probably C3) mediated by factor F and it is associated with loss of ability of P in initiate the alternate pathway of complement activation upon interaction with serum.     

Association of activated properdin with complexes of properdin with C3

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.     

The activation mechanism of human complement system by immune precipitate formed with rabbit IgG antibody

Immune precipitate (Ippt) formed between egg albumin and rabbit IgG antibody activated both pathways of the human complement system. On incubation with diluted serum, Ippt combined with several factors in the serum to form a complex which acquired C3- and C5-cleaving activities. In serum chelated with ethyleneglycol tetraacetic acid (EGTA), C3- and C5-cleaving activities of properdin system enzymes were formed on Ippt. Kinetic studies on the formation and the decay of C3- and C5-cleaving enzymes on Ippt revealed that C3- and C5-cleaving activites were almost dependent on the properdin system enzymes. The experiments in which C3-cleaving activity formed on Ippt was inhibited by anti-properdin or anti-B but not by anti-C4 supported the above results. The participation of the classical pathway was considered to accelerate the assembly of the properdin system enzymes.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.     

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.     

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.     

Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis

C-reactive protein (CRP) is a serum protein that shows rapid increases of as much as 1000-fold in concentration in response to infection, traumatic injury, or inflammation. CRP reacts with the phosphocholine moiety of pneumococcal cell wall C-polysaccharide, and this reaction can lead to complement activation in vitro and protection against pneumococcal infection in vivo. We have previously studied the chemiluminescence response of human neutrophils to Streptococcus pneumoniae as a measure of in vitro opsonophagocytosis by CRP and complement. CRP in the presence of complement was an effective opsonin for S. pneumoniae serotype 27 (Pn27), but not for serotypes 3 or 6. Because Pn27 differs from most serotypes of S. pneumoniae in containing phosphocholine in its capsular polysaccharide, we have determined the sites of CRP and C3 fixation to Pn27 and S. pneumoniae serotype 4 (Pn4), and related these to the ability of CRP and complement to opsonize these serotypes in vitro. By using a chemiluminescence (CL) assay to measure opsonophagocytosis, CRP was shown to enhance the response of human neutrophils and monocytes to Pn27 in the presence of normal human serum. The CL response of neutrophils and monocytes to Pn4 was not affected by the addition of CRP to serum. The addition of anti-capsular antibody to Pn4 and Pn27 enhanced the CL responses of both neutrophils and monocytes to both bacteria. The localization of bound CRP and C3 on Pn4 and Pn27 was determined by immunoelectron microscopy. CRP bound to Pn4 only in the cell wall region and C3 was located in this area whether or not CRP was present. Anti-capsular antibody deposited C3 in the capsule of Pn4. In contrast, Pn27 bound CRP throughout the capsule and cell wall areas. C3 was deposited in the cell wall region of Pn27 by serum alone and in the cell wall region and capsule when CRP or anti-capsular antibody was present. Because C3 fixation to the capsule was consistently associated with enhanced responses by phagocytic cells, it appears that the site of CRP binding and subsequent complement activation may be critical in the opsonophagocytosis of S. pneumoniae. These findings extend the correlation between capsular C3 and opsonization to a nonimmune system. By using CRP and different pneumococcal serotypes we have shown that the same molecules that are effective in the stimulation of phagocytic cells when bound to the capsule are not effective when bound to the cell wall.     

Is IL-2 regulated by a serum inhibitor?

Normal murine serum inhibits the proliferation of cloned cytotoxic T lymphocytes driven by pure interleukin 2 (IL-2), indicating that a component of normal murine serum is directly inhibitory to IL-2-dependent proliferation. However, the effect is not specific to such cells, since an IL-2-independent variant cell, and a number of lymphoid tumor cell lines are similarly inhibited. Addition of purified IL-2 does not overcome the inhibition, although its degree is reduced. Fractionation of murine serum showed that there are at least two inhibitory activities, which migrate with globular proteins of molecular weights greater than or equal to 10(6) and 4 X 10(4), respectively, on gel chromatography. Neither of the activities was specific for IL-2-dependent cells. Furthermore, murine IL-2 is stable in murine serum in vitro, although it disappears rapidly from the circulation after intravenous injection. It is therefore unlikely that serum inhibitor of IL-2 is an important immunoregulator in vivo.     

Induction of chitinase and β-1,3-glucanase in Parthenocissus quinquefolia cells cultured in vitro

Chitin, chitosan and peptidoglycan induced chitinase (EC 3. 2. 1. 14) activity in Parthenocissus quinquefolia cells cultured in vitro, while cellulose did not. The real inducers seemed to be oligomers released from the large size polymers by hydrolytic enzymes secreted into the medium during the cell growth and division. This effect was mimicked by the addition to the medium of a partially purified Parthenocissus chitinase/lysozyme (EC 3. 2. 1. 17), which was also able to hydrolyse chitosan. Oligomers of chitin and of chitosan induced the activity to the same level and with the same time course, while peptidoglycan oligomers induced less activity. Oligomers also induced β-1,3-glucanase (EC 3. 2. 1. 6) activities. The changes with time of both activities and the relative effects of the three kinds of polymers suggested that the induction of both enzymes involves a common element early in the signal pathway.     

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.     

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Entamoeba histolytica: purification of cathepsin B

A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.     

Streptococcal pyrogenic exotoxin B cleaves properdin and inhibits complement-mediated opsonophagocytosis

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. The reduction of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we investigated the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using Western blotting and an affinity column immobilized with SPE B, we found that both SPE B and C192S, an SPE B mutant lacking protease activity, bound to serum properdin, and that SPE B, but not C192S, degraded serum properdin. Further study showed that SPE B-treated, but not C192S-treated, serum blocked the alternative complement pathway. Reconstitution of properdin into SPE B-treated serum unblocked the alternative pathway. GAS opsonized with SPE B-treated serum was more resistant to neutrophil killing than GAS opsonized with C192S-treated or normal serum. These results suggest that a novel SPE B mechanism, one which degrades serum properdin, enables GAS to resist opsonophagocytosis.     

Differential sensitivity of stomatal and non-stomatal components to NaCl or Na2SO4 salinity in horsegram, Macrotyloma uniflorum (Lam.)

14CO2 assimilation rate (P), leaf diffusive conductance (gs), photosynthetic electron flow, and activities of enzymes of Calvin cycle were studied in a horsegram [Macrotyloma uniflorum (Lam.)] in response to salinity induced by NaCl or Na2SO4. A significant reduction in P and gs by both salt treatments was registered. Na2SO4 caused a greater reduction in gs than the NaCl salinity. Studies with isolated chloroplasts confirmed a greater sensitivity to NaCl than to Na2SO4. Salinity inhibited the photosynthetic electron transport. The activity of ribulose-1,5-bisphosphate carboxylase (E.C.4.1.1.39) was under salinity inhibited more than the activities of other three enzymes of the Calvin cycle, ribulose-5-phosphate kinase (E.C.2.7.1.19), ribose-5-phosphate isomerase (E.C.5.3.16), and NADP-glyceraldehyde-3-phosphate dehydrogenase (E.C.1.2.13). These inhibitions lead to a reduced capacity for ribulose-1,5-bisphosphate regeneration. Isolated chloroplasts extracted from salt stressed plants and supplemented with the substrates of Calvin cycle could elevate P, but the P was always lower than in the controls. Decreased P in horsegram exposed to high salinity can be attributed to both stomatal and non-stomatal components, however, the sensitivity to the salt source, NaCl or Na2SO4, was different.     

Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.     

干旱胁迫对不同葡萄砧木光合特性和荧光参数的影响

干旱胁迫导致葡萄砧木实生苗叶片光合能力下降.在正常供水和轻度干旱下,砧木的P_n 以3309C最高,其次是1103P,420A较低,各砧木的G_s和T_r差异不显著;中度干旱下,则以1103P的P_n最高,3309C最低;而严重干旱胁迫下,1103P的P_n比3309C高出124%,水分利用效率是3309C的1.95倍.干旱胁迫下,3种砧木的共同趋势是可变荧光 (F_o) 升高,最大荧光 (F_m)、实际光能转化效率 (ФPSⅡ）和可变荧光与最大荧光比 (F_v/F_m) 降低,但品种变幅不同.中度干旱使3309C的F _o升高17.1%,F_v/F_m降低了8.5%,而1103P的F_o升高6.8%,F_v/F_m降低了5.8%;严重干旱则使3309C的F_o升高36.2%,F_v/F_m降低了20.1%,而1103P的F_o升高9.9%,F_v/F_m降低了10.2%.干旱胁迫对不同葡萄砧木光合和荧光参数的影响与其抗旱性密切相关,其中F_v/F_m和P_n的相关系数最大（r=0.9883）.     

A 34-amino acid peptide of the third component of complement mediates properdin binding

In this study, a peptide of 34 amino acids from the Mr 40,000 C terminus alpha-chain fragment of C3 was found to mediate properdin (P) binding. Treatment of the Mr 40,000 fragment with CNBr generated one major Mr 17,000 fragment that was capable of binding P. Amino acid sequence data placed the Mr 17,000 fragment within residues 1385 to 1540 of the C3 sequence. After analyzing this sequence for highly conserved segments within the C3 from other species (which bind P) and segments of low similarity within human C4, mouse C5, and alpha 2-macroglobulin (which do not bind P), a 34-amino acid (1402 to 1435) peptide was synthesized. This synthetic peptide bound to P and inhibited its binding to C3b. In addition, it exhibited negative regulatory activity on the alternative pathway as it inhibited the lysis of rabbit erythrocytes by normal human serum. These results show that the P-binding site is located within the residues 1402 to 1435 of the C3 sequence.