Identification of Gly-Pro-Glu (GPE), the aminoterminal tripeptide of insulin-like growth factor 1 which is truncated in brain, as a novel neuroactive peptide

A truncated form of IGF-1 which lacks the aminoterminal tripeptide Gly-Pro-Glu (GPE) is found in human brain. It was proposed that GPE may result from neural specific processing and also have a function within the CNS. GPE was synthesized and shown to inhibit glutamate binding to the N-methyl-D-aspartate (NMDA) receptor. Whilst the carboxyterminal glutamate was necessary for NMDA receptor binding, the aminoterminal glycine potentiated receptor crossreaction. Furthermore, GPE had a potent stimulatory effect on the potassium induced release of acetylcholine from rat cortical slices. A less potent stimulation of dopamine release from striatum was also observed. The specific competitive NMDA receptor antagonist, (+/-)2-amino-7-phosphonoheptanoate (AP7), inhibited the action of GPE on dopamine but not on acetylcholine release. These studies have identified GPE as a novel neuroactive peptide with a potent action on acetylcholine release and support the general concept that the proteolytic products of the IGF-1 precursor play a role in the regulation of brain function.     

Cell cycle analysis with bromodeoxyuridine pulselabeling in asynchronous hybridoma cultures

Summary The cell cycle kinetics of a mouse hybridoma was examined by immunocytochemical staining of incorporated bromodeoxyuridine in an asynchronous culture. The cell cycle phase traverse times were extracted from a time series of bivariate distributions of incorporated bromodeoxyuridine and total DNA content; the G₁, S and G₂/M phase traverse times were 7, 9 and 4 h, respectively in the exponential growth phase.     

Dental amalgam and mercury

Summary Mercury concentration in intraoral air and urine of seven females with dental amalgam was measured before and after intake of one hard-boiled egg. A considerable decrease in mercury concentration in intraoral air was found. Twenty women with about equal dental amalgam status, with or without subjective symptoms related to dental amalgam, were also studied. Mercury concentrations in intraoral air and urine were measured. For all the 27 women the basal intraoral air concentration of mercury ranged over 0.6–10.4 g/m³ (median value 4.3 g/m³). This corresponds to a release of 0.02–0.38 ng/s (median value 0.16 ng/s). In urine, the mercury concentration varied from < 0.8–6.9 g/g creatinine (median value 1.9 g/g creatinine). Data from both parameters were significantly correlated to the total number of teeth areas with dental amalgam. Protein values in urine indicated no renal damage. Maximum concentrations of mercury vapour in intraoral air for the 27 women who had chewed chewing gum for 5 min varied between 2–60 g Hg/m³ (median value 19 g Hg/m³). This corresponds to 0.07–2.20 ng Hg/s and a median value of 0.70 ng Hg/s.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH‐Π interactions

Engineering of novel carbohydrate‐binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein‐carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi‐specific carbohydrate‐binding module (CBM) X‐2 L110F bound to a branched oligomer of xyloglucan (XXXG). X‐2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β‐glucans. The structural observations of the present study compared with previously reported structures of X‐2 L110F in complex with linear oligomers, show that the π‐surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X‐2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH‐π interactions can influence binding specificity and that flexibility is a key feature for the multi‐specificity displayed by X‐2 L110F, allowing for the accommodation of branched ligands. Proteins 2014; 82:3466–3475. © 2014 Wiley Periodicals, Inc.     

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

[11C]PIB-amyloid binding and levels of Aβ40 and Aβ42 in postmortem brain tissue from Alzheimer patients

β-Amyloid (Aβ) deposits are one of the major histopathological hallmarks of Alzheimer's disease (AD). The amyloid-imaging positron emission tomography (PET) tracer [¹¹C]PIB (N-methyl[¹¹C]2-(4′-methylaminophenyl)-6-hydroxy-benzothiazole) is used in the assessment of Aβ deposits in the human brain. [¹¹C]PIB-amyloid interaction and insoluble Aβ40 and Aβ42 peptide levels in the brain were quantified in postmortem tissue from nine AD patients and nine age-matched control subjects in the temporal, frontal and parietal cortices and the cerebellum. Autoradiographical studies showed significantly higher densities of specific [¹¹C]PIB-amyloid binding in gray matter in the temporal and parietal cortex (62 fmol/mg tissue) in AD patients as compared to control subjects, whereas the density was somewhat lower in the frontal cortex (56 fmol/mg tissue). No specific binding could be detected in the AD cerebellum or in the tissues from the control subjects (≤5 fmol/mg tissue). Insoluble Aβ40 and total Aβ levels (i.e. sum of Aβ40 and Aβ42) were significantly higher in patients than in controls in all measured cortical regions as determined using ELISA, which was confirmed using immunohistochemistry. The present findings show a more regional selective distribution of [¹¹C]PIB amyloid binding than previously reported. Moreover, it is suggested that some of the [¹¹C]PIB binding and insoluble Aβ seen in control subjects may be amyloid in the blood vessels.     

Mapping of Functional Domains in Herpesvirus Saimiri Complement Control Protein Homolog: Complement Control Protein Domain 2 Is the Smallest Structural Unit Displaying Cofactor and Decay-Accelerating Activities

Herpesvirus saimiri encodes a functional homolog of human regulator-of-complement-activation proteins named CCPH that inactivates complement by accelerating the decay of C3 convertases and by serving as a cofactor in factor I-mediated inactivation of their subunits C3b and C4b. Here, we map the functional domains of CCPH. We demonstrate that short consensus repeat 2 (SCR2) is the minimum domain essential for classical/lectin pathway C3 convertase decay-accelerating activity as well as for factor I cofactor activity for C3b and C4b. Thus, CCPH is the first example wherein a single SCR domain has been shown to display complement regulatory functions.The complement system is an ancient and yet highly evolved effector mechanism of immune defense that forms an imperative branch of innate immunity (23, 46). In addition, recent findings have clearly revealed its role as a vital viaduct between the innate and acquired immune systems (6, 18). Thus, it is not surprising that the system helps in purging a wide array of invaders, including viruses. Consequently, for their successful survival, many viruses have developed mechanisms to subvert the host complement system (7, 24, 26, 29, 39, 45). Herpesviruses and poxviruses, in particular, subvert host complement by encoding structural and/or functional homologs of human complement regulators belonging to the regulator-of-complement-activation (RCA) family, by capturing host membrane complement regulators and by using cellular receptors for entering cells (1, 8, 15, 23).The RCA proteins are formed by multiple tandem repeats of bead-like complement control protein (CCP) domains or short consensus repeats (SCRs) separated by short linkers. It has been suggested that the sequence variations enforced upon these SCR domain folds and the interdomain dynamics dictate the functionality of the complement regulators (17, 19, 44, 49). Because sequence similarity in herpesviral complement regulators varies between 43% and 89% and in poxviral complement regulators exceeds 91%, it is likely that the structural diversity in herpesviral complement regulators may have resulted in functional differences in these proteins and/or have resulted in variation in structural requirements for complement regulation. In the herpesviridae family, detailed functional characterization has been performed for complement regulators of Kaposi''s sarcoma-associated herpesvirus (Kaposica/KCP) (28, 42), herpesvirus saimiri (HVS) (CCPH) (10, 38), and rhesus rhadinovirus (RCP) (31). All these proteins showed conservation of complement regulatory activities, indicating thereby that structural diversity has not resulted in loss of complement regulatory functions in these proteins. However, it is not clear whether sequence variations within the herpesviral complement regulators have resulted in differences in the domain requirements for complement regulatory activities, since mapping of functional domains has been performed only for Kaposica (30, 43). In the present study, we therefore have mapped the complement regulatory domains of HVS CCPH to get further insight into diversity in domain requirements for functional activities.HVS is a classical prototype of the gamma 2-herpesviruses or rhadinoviruses. It causes rapidly progressing fulminant lymphoma, lymphosarcoma, and leukemia of T-cell origin in marmosets, owl monkeys, and other species of New World primates but not in its natural host, the squirrel monkey (9, 16). Unlike other herpesviruses, it encodes two complement regulators: an RCA homolog (ORF 4; CCPH) that regulates the early steps of complement activation (2, 10) and a CD59 homolog (ORF 15) that inhibits the late steps of complement activation (4, 36). The RCA homolog is formed of four SCR modules (Fig. ​(Fig.1).1). As a result of alternative splicing, the protein is expressed as a full-length membrane-bound form (mCCPH) containing the transmembrane region as well as a spliced secretory form (sCCPH) lacking the transmembrane region (2). Earlier, we showed that sCCPH inhibits complement by targeting C3 convertases: (i) it supports serine protease factor I-mediated inactivation of C3b and C4b, the subunits of C3 convertases (cofactor activity), and (ii) it accelerates the irreversible decay of the classical pathway (CP)/lectin pathway and to a limited extent the alternative pathway (AP) C3 convertases (decay-accelerating activity [DAA]) (38).Open in a separate windowFIG. 1.Schematic illustration of sCCPH and SDS-PAGE analysis of purified recombinant sCCPH and its deletion mutants. (Top) Schematic representation of the structure of the soluble form of CCPH (sCCPH), which is composed of four SCRs. The domains are numbered, and the minimum domains shown to be important for C3b and C4b cofactor activities (CFA) and CP DAA are identified. (Bottom) Expressed and purified sCCPH and its deletion mutants were analyzed by 12% (left) and 13% (right) SDS-PAGE under reducing conditions and stained with Coomassie blue. Molecular weights as determined by SDS-PAGE: for sCCPH, 32,000; for SCR1-3, 26,000; for SCR2-4, 27,500; for SCR1-2, 17,000; for SCR2-3, 17,500; for SCR3-4, 16,500; for SCR1, 9,500; for SCR2, 7,000; for SCR3, 8,000; and for SCR4, 8,000. Molecular mass is expressed as kilodaltons in the figure.(This work was done in partial fulfillment of the Ph.D. thesis requirements of A.K.S., University of Pune, Pune, India.)In order to map the functional domains of sCCPH, we have generated a series of soluble triple, double, and single SCR deletion mutants. In brief, the deletion mutants of sCCPH comprising SCR1-3, -2-4, -1-2, -2-3, and -3-4 as well as SCR1, -2, -3, and -4 were constructed from the full-length HVS sCCPH clone (38) by PCR amplification and cloning into the bacterial expression vector pET29. The authenticity of each of the clones was confirmed by DNA sequencing, and then they were transformed into the Escherichia coli BL21 strain for expression. The mutants carried the histidine tag at the C terminus and hence were purified to homogeneity by using histidine affinity chromatography. Refolding of the purified proteins was performed by using the rapid dilution method as previously described (38, 47, 48), and the refolded proteins were loaded onto a Superose 12 gel filtration column (Pharmacia) to obtain monodisperse populations of the expressed mutants (38, 48). The preservation of various functions in mutants (see below) suggests that the mutants have maintained their proper conformation. The expressed proteins were >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. ​(Fig.11).To identify the domains required for cofactor activities of sCCPH against C3b and C4b, we utilized a fluid phase assay wherein C3b or C4b was incubated with each of the deletion mutants and factor I, and inactivation of C3b/C4b (cleavage of the α′-chain) was determined by running the samples on SDS-PAGE gels. It is clear from the data presented in Fig. ​Fig.22 that sCCPH and the mutants SCR1-3, -2-4, and -1-2 supported the cleavage of the α′-chain of C3b. A very weak cleavage was also supported by SCR2-3 and -3-4. The cleavage of the α′-chain of C4b, however, was supported by sCCPH and the mutants SCR1-3, -2-4, -1-2, and -2-3 but not by SCR3-4 (Fig. ​(Fig.2).2). Together, these data point out that SCR1 and -2 considerably contribute to the C3b and C4b cofactor activities of sCCPH but that SCR3 and SCR4 in the case of C3b cofactor activity and SCR3 in the case of C4b cofactor activity contribute to its optimal activity. These results, however, did not elucidate whether a single domain(s) could impart the cofactor activities. We therefore expressed the single-domain mutants (SCR1, SCR2, SCR3, and SCR4) and analyzed their cofactor activities. The results presented in Fig. ​Fig.33 indicate that SCR2, by itself, possesses the ability to support factor I-mediated inactivation of C3b and C4b; SCR3 also displayed very weak cofactor activity against C3b when used at higher concentrations (88 μM; data not shown). These results suggest that structural elements involved in the interaction of sCCPH with factor I are primarily located within SCR2 and -3. Admittedly, the single-domain mutants possess very weak cofactor activities and other domains too contribute to the optimal activity; the cofactor activities of SCR2 for C3b and C4b were 781- and 212-fold lower than that for sCCPH (Fig. ​(Fig.3).3). It should be mentioned here that earlier observations on mapping of the human RCA proteins (factor H, C4b-binding protein, membrane cofactor protein, and complement receptor 1) (3, 11-13, 21), Kaposica (30), and vaccinia virus CCP (VCP) (27) indicated that a minimum of two (in Kaposica) or three (in all other RCA proteins) successive SCR domains are necessary for factor I cofactor activities. Thus, sCCPH is the first complement regulator in which a single SCR domain has been shown to display the factor I cofactor function.Open in a separate windowFIG. 2.Analysis of factor I cofactor activity of sCCPH and its deletion mutants for human complement proteins C3b and C4b. Cofactor activity was assessed by incubating 3.0 μg of human C3b (upper panels) or C4b (lower panels) with sCCPH/SCR1-3/SCR2-4 (4.0 μM) or SCR1-2/2-3/3-4 (24 μM) in the presence or absence of factor I (100 ng) for the indicated time periods at 37°C in 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl. The reactions were stopped by addition of sample buffer containing dithiothreitol, and the amount of C3b or C4b cleaved was visualized by subjecting the samples to SDS-PAGE analysis on 10% or 11.5% gel, respectively, and staining with Coomassie blue. During C3b cleavage, the α′-chain is cleaved into N-terminal 68-kDa and C-terminal 46-kDa fragments. The 46-kDa fragment is then cleaved into a 43-kDa fragment. These cleavages indicate inactivation of C3b. In the case of C4b, the α′-chain is cleaved into N-terminal 27-kDa, C-terminal 16-kDa (not visible in the gel), and central C4d fragments. These cleavages indicate the inactivation of C4b.Open in a separate windowFIG. 3.Analysis of factor I cofactor activity (CFA) of single SCR mutants of sCCPH for human complement proteins C3b and C4b. (Upper panels) Cofactor activity was assessed by incubating 3.0 μg of human C3b or C4b with the single SCR mutants (44 μM) in the presence or absence of factor I (100 ng) for 4 h at 37°C in PBS (10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl). The reactions were stopped by addition of sample buffer containing dithiothreitol, and the amount of C3b or C4b cleaved was visualized by subjecting the samples to 13% SDS-PAGE and stained with Coomassie blue. Cleavage of the α′-chain of C3b and C4b and generation of cleavage products indicate the inactivation of these proteins. (Middle panels) Human C3b (3.0 μg) or C4b (3.0 μg) and factor I (100 ng) were incubated in PBS with increasing concentrations of sCCPH or the SCR2 mutant at 37°C for 1 h, and the cleavage products were analyzed as described above. (Lower panels) The intensity of the α′-chains of C3b and C4b in the middle panels was determined densitometrically and is represented graphically. The closed and open circles represent sCCPH and the SCR2 mutant, respectively.As discussed above, in addition to the inactivation of subunits of C3 convertases (C3b and C4b), sCCPH also regulates C3 convertases by accelerating their decay. It possesses considerable DAA for the CP/lectin pathway C3 convertase (C4b,2a) and a poor decay activity for the AP C3 convertase (C3b,Bb). Thus, we next examined the DAAs of the various sCCPH mutants to map the domains required for this function. To measure the CP C3 convertase decay activity, the C4b,2a enzyme was formed on sheep erythrocytes and allowed to decay in the presence of various mutants. The remaining enzyme activity was then measured by incubating the reaction mixture with EDTA sera (a source of C3 to C9) and measuring hemolysis. Apart from sCCPH, mutants SCR1-3, -1-2, and -2-3 showed substantial DAA for the CP C3 convertase (Fig. ​(Fig.4).4). These data suggested that SCR1-3 is primarily responsible for this activity. On a molar basis, SCR1-3 was 1.6-fold less efficient than sCCPH. Because both SCR1-2 and SCR2-3 possessed the decay activity, it was likely that similar to the cofactor activities, a single SCR domain of sCCPH might also possess the DAA for the CP C3 convertase. Hence, we also assessed the DAAs of the single-domain mutants. Interestingly again, SCR2 was the only single domain that distinctly displayed CP DAA (Fig. ​(Fig.4);4); however, on a molar basis, it was 26-fold less active than sCCPH. Previous data on the involvement of SCR domains in decay acceleration of CP C3 convertase in human RCA proteins (decay-accelerating factor, complement receptor 1, and C4b-binding protein) (3, 5, 20) and viral RCA homologs (Kaposica and VCP) (27, 30) have shown that a minimum of two or three consecutive domains are necessary for the activity. Thus, sCCPH is the only prototype to date in which a single SCR is adequate to impart the CP DAA.Open in a separate windowFIG. 4.Analysis of CP and AP C3 convertase DAAs of sCCPH and its mutants. (Upper panel) The CP C3 convertase C4b,2a was formed on antibody-coated sheep erythrocytes (EA) by sequentially incubating them with human C1, C4, and C2 (Calbiochem). The C3 convertase on the cells was then allowed to decay by incubating EA-C4b,2a with various concentrations of sCCPH or its mutants for 5 min at 22°C, and the activity of the remaining enzyme was assessed by measuring the cell lysis following incubation for 30 min at 37°C with Guinea pig sera containing 40 mM EDTA (27, 32). (Lower panel) The AP C3 convertase C3b,Bb was formed on sheep erythrocytes (ES) by incubating them with human C3 (Calbiochem) and factors B and D in the presence of NiCl₂. The C3 convertase on the cells was then allowed to decay by incubating ES-C3b,Bb with various concentrations of sCCPH or its mutants for 10 min at 37°C, and the activity of the remaining enzyme was assessed by measuring the cell lysis following incubation with EDTA-sera for 30 min at 37°C (35, 37). The data obtained were normalized by considering the lysis that occurred in the absence of an inhibitor as 100% lysis.Although sCCPH is known to possess limited AP C3 convertase DAA, we sought to determine whether this limited activity is localized in a specific region or the full-length protein. To measure the AP DAA, the C3 convertase C3b,Bb was formed on the sheep erythrocytes and incubated with sCCPH or with each of its deletion mutants. The decay of the AP C3 convertase was assessed by adding EDTA sera and measuring hemolysis. Although the full-length protein displayed a limited AP C3 convertase, none of the deletion mutants exhibited any activity (Fig. ​(Fig.44).Inactivation of C3 convertases by the RCA proteins, owing to their cofactor and decay activities, requires interaction of these proteins with C3b and C4b. The ligand binding activity of the RCA proteins, however, does not always correlate with their cofactor and decay activities (12, 34), as apart from ligand binding, cofactor activity involves interaction of the RCA protein with factor I (40), and decay activity involves interaction of the RCA protein with C2a or Bb (22, 25). In order to determine whether cofactor and decay activity data of sCCPH and the various mutants correlate with the ligand binding data, we measured binding of these proteins to C3b and C4b by using a surface plasmon resonance-based assay (38). As observed earlier (38), sCCPH displayed higher affinity for C4b than for C3b (Fig. ​(Fig.55 and Table ​Table1).1). When we measured binding of various deletion mutants to C3b and C4b, only SCR2-4 showed binding to C3b, and SCR1-3 showed binding to C4b (Fig. ​(Fig.5).5). However, there were reductions of about 16- and 14-fold in the affinities of these deletion mutants for C3b and C4b, respectively, compared to that for sCCPH (Table ​(Table1),1), suggesting that all the four domains contribute to binding to C3b and C4b. Because most of the deletion mutants that displayed complement regulatory activities possessed negligible binding to C3b and C4b, it is clear that binding of the mutants does not correlate with their cofactor and decay activities. It is likely that during cofactor activity, interaction of the mutants with C3b and C4b is stabilized by the interaction of factor I with C3b/C4b and the mutants. Similarly, during DAAs, the mutants may possess better affinity for the convertases than their subunits C3b and C4b. Consistent with this argument, decay-accelerating factor has previously been shown to bind to CP C3 convertase with 1,000-fold higher affinity than to C4b (33).Open in a separate windowFIG. 5.Binding of sCCPH and its mutants to C3b and C4b. Binding was determined by a surface plasmon resonance-based assay (38). Sensograms were generated by immobilizing biotinylated C3b (1,200 response units [RUs]) and C4b (940 RUs) on streptavidin chips (Sensor Chip SA; Biacore AB; additional RUs of C3b [∼6,000 RUs] were deposited by forming AP C3 convertase on the chip and flowing native C3 [14]) and injecting sCCPH or its mutants in PBS-T (10 mM sodium phosphate and 145 mM NaCl, pH 7.4, containing 0.05% Tween 20) over the chip. Flow cells immobilized with bovine serum albumin-biotin (Sigma) served as control flow cells. (Left panels) Binding of sCCPH and its various mutants to C3b (top) and C4b (bottom). The sensograms were generated by injecting 500 nM and 2 μM of sCCPH and its various mutants over C3b and C4b chips, respectively. (Middle panels) Sensogram overlay for the interaction between sCCPH and C3b (top) or sCCPH and C4b (bottom). (Right panels) Sensogram overlay for the interaction between SCR2-4 and C3b (top) and SCR1-3 and C4b (bottom). The concentrations of proteins injected are indicated at the right of the sensograms. The solid lines in the top middle and top right panels represent the global fitting of the data to a 1:1 Langmuir binding model with a drifting baseline (A + B ↔ AB; Biaevaluation 4.1). The small arrows in the bottom middle and right panels indicate the time points used for evaluating the steady-state affinity data.

TABLE 1.

Kinetic and affinity data for the interactions of sCCPH and the deletion mutants with human complement proteins C3b and C4b^a