Effect of C4 Depletion on the Utilization of the Terminal Components of Guinea-pig Complement by Endotoxin

ENDOTOXIN and antigen-antibody complexes (Ag-Ab) are potent inflammatory agents which induce marked pathophysiological effects in a number of animal species. Both these agents lead to increased vascular permeability, local accumulations of polymorphonuclear leucocytes and alterations in the coagulability of blood¹. There is mounting evidence that these similarities derive from endotoxins and Ag-Ab being activators of the complement (C) system, an important mediator of inflammation¹'². When endotoxins or preformed Ag-Ab are reacted with normal guinea-pig (GP) serum so as to induce equivalent consumption of whole haemolytic C, there are, however, striking differences in the consumption profiles of the individual C components³. Although interaction of Ag-Ab with GP serum results in the consumption of CI, C4 and C2 as well as C3 to C9, the interaction of endotoxin with GP serum leads to a marked depletion of C3 to C9 with little or no detectable loss of C1,C4 and C2 (ref. 3).     

Surviving Mousepox Infection Requires the Complement System

Poxviruses subvert the host immune response by producing immunomodulatory proteins, including a complement regulatory protein. Ectromelia virus provides a mouse model for smallpox where the virus and the host''s immune response have co-evolved. Using this model, our study investigated the role of the complement system during a poxvirus infection. By multiple inoculation routes, ectromelia virus caused increased mortality by 7 to 10 days post-infection in C57BL/6 mice that lack C3, the central component of the complement cascade. In C3^−/− mice, ectromelia virus disseminated earlier to target organs and generated higher peak titers compared to the congenic controls. Also, increased hepatic inflammation and necrosis correlated with these higher tissue titers and likely contributed to the morbidity in the C3^−/− mice. In vitro, the complement system in naïve C57BL/6 mouse sera neutralized ectromelia virus, primarily through the recognition of the virion by natural antibody and activation of the classical and alternative pathways. Sera deficient in classical or alternative pathway components or antibody had reduced ability to neutralize viral particles, which likely contributed to increased viral dissemination and disease severity in vivo. The increased mortality of C4^−/− or Factor B^−/− mice also indicates that these two pathways of complement activation are required for survival. In summary, the complement system acts in the first few minutes, hours, and days to control this poxviral infection until the adaptive immune response can react, and loss of this system results in lethal infection.     

Immune regulation of complement components in vivo

Immune regulation of individual complement components has been studied in F₁ hybrids obtained from mating normal males with females homozygous for a genetically controlled deficiency of those components. Experiments have been performed with C5-deficient mice, C6-deficient rabbits, and C4-deficient guinea pigs. Prior to mating, complement-deficient females were rendered hyperimmune to the component they lacked and their F₁ offspring were treated postnatally with antibody to the pertinent complement component. We had previously shown that antibody treatment could suppress C5 production in mice but in experiments presented here, similar antibody treatment had no effect on in vivo biosynthesis of C6 in rabbits and C4 in guinea pigs. Variation in the susceptibility of these three components of complement to regulation by antibody might reflect differences in the inducing antibody, the ontogeny of the complement component, the sites of origin, or the genetic mechanisms responsible for the deficiency states. Lack of the ability to suppress with antibody in vivo does not denote an inability to suppress with antibody in analogous in vitro systems.     

Polymorphism of the sixth component of complement (C6) in the dog

Isoelectric focusing was used to identify five alleles at the locus determining the production of the sixth component of complement (C6) in the dog. Four of these alleles,C6 ¹, C6², C6⁴,andC6 ⁵,were studied in family pedigrees and shown to be inherited in a codominant autosomal fashion. All alleles except forC6 ⁴occurred commonly in the multiple breeds tested. This investigation was supported by Grant HL 17265 awarded by the National Institute of Heart, Lung, and Blood Diseases, DHEW, and by Grants CA 18105 and CA 31787 awarded by the National Cancer Institute.     

Record Chromosome Number in a Mammal?

CROSS has reported^{1, 2} chromosome complements in rodents ranging up to 2n=84 (in the North American geomyid Geomys breviceps) and 2n=86 (in the heteromyid Dipodomys merriami). Matthey has contested³ this claim; he maintained that in Geomys bursarius 2n =70 or 72 and he doubted whether a higher number had been confirmed. Numbers in the 2n=70 to 80 range have since been reported⁴ in several genera of Canidae and Ursidae. In general, rodent chromosomes fall into the lower group numbers but, among murines, Lophuromys aquillus is reported to have a chromosome complement of 70 (ref. 5) and Cricetomys gambianus one of 78 (ref, 6). In the gerbilline, Meriones shawii, the number can vary up to 78 (ref. 7).     

Intermedilysin-Receptor Interactions during Assembly of the Pore Complex: ASSEMBLY INTERMEDIATES INCREASE HOST CELL SUSCEPTIBILITY TO COMPLEMENT-MEDIATED LYSIS*

Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9.The cholesterol-dependent cytolysins (CDC)² are a family of structurally related pore-forming toxins that are important virulence factors for a variety of Gram-positive pathogens (1–4). The CDCs are secreted by the bacterium as soluble monomers and then bind to cholesterol-rich eukaryotic cell membranes (5). Once bound, the monomers laterally diffuse and interact with one another to form a large oligomeric prepore structure comprised of 35–40 CDC monomers. One of the hallmarks of this family of toxins is the absolute requirement of their pore-forming mechanism on membrane cholesterol (1). Membrane cholesterol serves to target the CDCs to the eukaryotic cell membrane and is necessary to convert the prepore oligomer to the inserted pore complex (6). Two classes of CDCs currently exist. The first class is typified by perfringolysin O (PFO) from Clostridium perfringens that appears to bind directly to cholesterol-rich membranes, an interaction mediated by three short loops in domain 4 (7). The second group includes intermedilysin (ILY) from Streptococcus intermedius and vaginolysin from Gardnerella vaginalis (8). These CDCs bind to the glycosylphosphatidylinositol-anchored protein human CD59 (hCD59). It has been shown for ILY that it first binds hCD59 and then inserts its domain 4 loops in a cholesterol-dependent fashion (7). Why the latter two CDCs have evolved to specifically bind hCD59 and whether they remain engaged with this receptor throughout the assembly of the pore complex remains unclear. S. intermedius is a pathogen frequently associated with abscesses of the oral cavity as well as with life-threatening abscesses of the head, neck, and liver (9, 10). ILY appears to be important in establishing these deep-seated abscesses as S. intermedius isolated from these sites produces levels of ILY 6–10 times greater than strains isolated from peripheral site infections or the oropharynx (9). ILY binds only human cells, whereas other CDCs, such as PFO, bind to most cholesterol-rich eukaryotic membranes. The species selectivity of ILY is because of its specificity for human hCD59 and appears to be encoded in domain 4 of the toxin (11, 12).CD59 is an 18–20-kDa surface-expressed glycoprotein tethered to the cell membrane via a glycosylphosphatidylinositol anchor. It is widely distributed on most human and nonhuman cell types. It is associated with a number of important cellular functions that include serving as an adaptor molecule for a candidate C1q receptor (C1qRO^–₂) (13, 14) and acting as a cell-signaling molecule (15). Its primary role, however, is regulating the terminal pathway of complement by inhibiting the formation of the membrane attack complex (MAC) on host cells by binding to C8α and C9, thus preventing the formation of the MAC pore (16–18). In various autoimmune diseases and inflammatory conditions, excessive complement activation can saturate the available CD59 resulting in MAC-mediated host cell injury (19). CD59 exhibits species selectivity such that it most effectively inhibits only the homologous MAC (20). ILY recognition of the same or similar structural differences in CD59 is the basis for its species selective activity (11).ILY binding to hCD59 triggers a series of conformational changes in ILY leading to its membrane oligomerization into the prepore complex (6). This is accompanied by the cholesterol-dependent insertion of three loops at the base of domain 4 and the insertion of the undecapeptide, events that are necessary for the conversion of the prepore to a pore complex (7). It is not known, however, whether ILY remains engaged with hCD59 throughout its assembly into the pore complex. Whether ILY remains engaged during and after the assembly of the pore complex may also impact the ability of the eukaryotic cell to protect itself from the host MAC because a previous study suggested the ILY-binding site on hCD59 overlaps that for complement proteins C8α and C9 (11). To address these questions, we investigated the interaction of ILY with hCD59 during the assembly of the ILY pore complex. We further determined whether nonlytic assembly intermediates of ILY increase MAC-mediated damage to host cells by short circuiting the protective function of hCD59. These studies show ILY remains engaged during the assembly of its prepore complex and disengages from its receptor upon pore formation. In addition, we show that engagement of hCD59 by ILY prior to pore formation significantly increases the host cell sensitivity to the host MAC-mediated lysis.     

Complement contributes to inflammatory tissue destruction in a mouse model of Ross River virus-induced disease

Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus, are mosquito-borne viruses that cause significant human disease worldwide, including explosive epidemics that can result in thousands to millions of infected individuals. Similar to infection of humans, infection of C57BL/6 mice with RRV results in severe monocytic inflammation of bone, joint, and skeletal muscle tissues. We demonstrate here that the complement system, an important component of the innate immune response, enhances the severity of RRV-induced disease in mice. Complement activation products were detected in the inflamed tissues and in the serum of RRV-infected wild-type mice. Furthermore, mice deficient in C3 (C3^−/−), the central component of the complement system, developed much less severe disease signs than did wild-type mice. Complement-mediated chemotaxis is essential for many inflammatory arthritides; however, RRV-infected wild-type and C3^−/− mice had similar numbers and composition of inflammatory infiltrates within hind limb skeletal muscle tissue. Despite similar inflammatory infiltrates, RRV-infected C3^−/− mice exhibited far less severe destruction of skeletal muscle tissue. In addition to these studies, complement activation was also detected in synovial fluid from RRV-infected patients. Taken together, these findings indicate that complement activation occurs in the tissues of humans and mice infected with RRV and suggest that complement plays an essential role in the effector phase, but not the inductive phase, of RRV-induced arthritis and myositis.     

Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca²⁺-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca²⁺-dependent manner (with optimal 61 μm free Ca²⁺ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca²⁺, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (K_d = 1.7 × 10^–5 m) than for the C2F interaction (K_d = 2.6 × 10^–9 m). In contrast, it was the otoferlin C2D domain that bound the Ca_v1.3 II-III loop, in a Ca²⁺-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca²⁺ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Ca_v1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref. 1). In mechanosensory hair cells, calcium microdomains (2) and possibly nanodomains (3) are formed when voltage-gated calcium channels open upon depolarization. Calcium at these sites is thought to activate protein interactions, leading to vesicle fusion. Some of the key players in this process are the target-SNARE² proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin (4). Vesicle-SNARE synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal synapse, modulating calcium channels and vesicle release by a Ca²⁺-dependent interaction with other SNARE proteins in the presence of lipid molecules (4–6). However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not detected (7). Instead, fast neurotransmitter release in auditory and vestibular hair cells, facilitated largely by an L-type voltagegated calcium channel, Ca_v1.3 (8, 9), is thought to be modulated by a newly discovered protein, otoferlin, acting as the Ca²⁺ sensor and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic deafness (10). Gene sequences of different deaf families show that the OTOF gene can undergo mutation at multiple locations (11–13). Recently, it has been demonstrated that otoferlin is necessary for synaptic exocytosis from hair cells (14). Further, an engineered mutation in the C2B domain of otoferlin has been shown to cause deafness in mice (15). However, the precise function of otoferlin as a synaptic protein is not well understood.Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in humans have been documented to cause DFNB9 deafness (11, 12). Previous studies suggested that a region of otoferlin containing all three C2 domains, D, E, and F, binds directly to the t-SNARE molecules syntaxin 1A and SNAP-25 in response to an increase in Ca²⁺ concentration (14). However, it is not understood how a single amino acid substitution in one domain of otoferlin, such as C2F (11) or C2D (12), might independently lead to deafness. Here, we examine the role of otoferlin as a Ca²⁺ sensor as well as a facilitator of vesicle fusion, as indicated by protein-protein interactions and their [Ca²⁺] dependence.     

Structure of the Tyrosine-sulfated C5a Receptor N Terminus in Complex with Chemotaxis Inhibitory Protein of Staphylococcus aureus

Complement component C5a is a potent pro-inflammatory agent inducing chemotaxis of leukocytes toward sites of infection and injury. C5a mediates its effects via its G protein-coupled C5a receptor (C5aR). Although under normal conditions highly beneficial, excessive levels of C5a can be deleterious to the host and have been related to numerous inflammatory diseases. A natural inhibitor of the C5aR is chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). CHIPS is a 121-residue protein excreted by S. aureus. It binds the N terminus of the C5aR (residues 1-35) with nanomolar affinity and thereby potently inhibits C5a-mediated responses in human leukocytes. Therefore, CHIPS provides a starting point for the development of new anti-inflammatory agents. Two O-sulfated tyrosine residues located at positions 11 and 14 within the C5aR N terminus play a critical role in recognition of C5a, but their role in CHIPS binding has not been established so far. By isothermal titration calorimetry, using synthetic Tyr-11- and Tyr-14-sulfated and non-sulfated C5aR N-terminal peptides, we demonstrate that the sulfate groups are essential for tight binding between the C5aR and CHIPS. In addition, the NMR structure of the complex of CHIPS and a sulfated C5aR N-terminal peptide reveals the precise binding motif as well as the distinct roles of sulfated tyrosine residues sY11 and sY14. These results provide a molecular framework for the design of novel CHIPS-based C5aR inhibitors.The human complement system is a key component of the innate host defense directed against invading pathogens. Complement component C5a is a 74-residue glycoprotein generated via complement activation by cleavage of the α-chain of its precursor C5. C5a is a strong chemoattractant involved in the recruitment of neutrophils and monocytes, activation of phagocytes, release of granule-based enzymes, and in the generation of oxidants (1, 2). C5a exerts its effect by activating the C5a receptor (C5aR).³ Although this is a highly efficient process, excessive or erroneous activation of the C5aR can have deleterious effects on host tissues. C5a has been implicated in the pathogenesis of many inflammatory and immunological diseases, including rheumatoid arthritis, inflammatory bowel disease, immune complex disease, and reperfusion injury (3, 4). Consequently, there is an active ongoing search for compounds that suppress C5a-mediated inflammatory responses.Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121-residue protein excreted by S. aureus, which efficiently inhibits the activation of neutrophils and monocytes by formylated peptides and C5a (5, 6). CHIPS specifically binds to the formylated peptide receptor (FPR) and the C5aR with nanomolar affinity (K_d = 35.4 ± 7.7 nm and 1.1 ± 0.2 nm, respectively) (7), thereby suppressing the inflammatory response of the host. A CHIPS fragment lacking residues 1-30 (designated CHIPS_31-121) has the same activity in blocking the C5aR compared with wild-type CHIPS (8). CHIPS_31-121 is a compact protein comprising an α-helix packed onto a four-stranded anti-parallel β-sheet (8). C5a has an entirely different fold (PDB ID code 1KJS) and is comprised of an anti-parallel bundle of four α-helices stabilized by three disulfide bonds (9, 10). Preliminary experiments indicated that CHIPS binds exclusively to the extracellular N-terminal portion of the C5aR (7). In contrast, the binding of C5a by its receptor involves two separate binding sites: C5a residues located in the region between 12-46 (11, 12) bind to a primary binding site partly coinciding with the binding site of CHIPS, while the C terminus of C5a (residues 69-74) binds to the activation domain of the C5aR located in the receptor core (13). Because of their dissimilarity in sequence and structure, the binding sites of CHIPS and C5a are not identical (11). The present working model is that CHIPS interferes with the primary binding site of C5a located at the N terminus of the C5aR, thereby preventing the C-terminal tail of C5a from contacting the activation domain of the C5aR and blocking downstream signaling. Currently, the development of C5aR inhibitors has been focused primarily on mimicking C5a in order to directly interrupt C5a-mediated C5aR signaling (3, 4, 14). Understanding the interactions between CHIPS and the C5aR may provide valuable insights toward the development of new C5aR antagonists.Postma et al. (15) proposed that residues involved in CHIPS binding are located between residues 10-18 of the C5aR. Specifically, the acidic residues Asp-10, Asp-15, and Asp-18 and residue Gly-12 appear to be critical for binding. High affinity binding was observed between ¹²⁵I-labeled CHIPS and the N-terminal portion of the C5aR (residues 1-38) expressed on the cell surface of HEK293 cells (K_d = 29.7 ± 4.4 nm). In contrast, very moderate affinity between CHIPS and a synthetic C5aR N-terminal peptide (residues 1-37; K_d = 40 ± 19 μm), measured by isothermal titration calorimetry (ITC), was recently reported by Wright et al. (16). The discrepancy in the magnitude of these dissociation constants may be explained by the presence of two sulfate groups on tyrosine 11 and 14 of the C5aR N terminus expressed on the cell surface of HEK293 cells, which are absent in the synthetic C5aR peptide utilized by Wright et al. (16). Farzan et al. (17) stressed the critical role of these sulfate groups in activation of the C5aR by C5a. Previous mutational studies employing FITC-labeled CHIPS, however, suggested that the sulfate groups had only a limited effect on the binding affinity (15).To resolve these discrepancies, we set out to chemically synthesize several sulfated and unsulfated peptides representing the N terminus of the human C5aR. We have measured the binding affinities of these peptides to CHIPS_31-121 by ITC and used the C5aR peptide with the highest affinity to determine the structure of the complex between CHIPS_31-121 and the C5aR N terminus by NMR spectroscopy.     

ELEVATED PLASMA PHENYLALANINE CONCENTRATION AND LYSINE INCORPORATION INTO RIBOSOMAL PROTEIN OF DEVELOPING BRAIN

Hyperphenylalaninemia was induced in 7-day-old rabbits over a 6-hr period by intraperitoneal injection of phenylalanine. l -[U-¹⁴C]Lysine was injected intraperitoneally into these rabbits and into a control group. The rate of incorporation of l -[U-¹⁴C]lysme into brain ribosomal protein was decreased during a 5-hr period in the presence of elevated plasma phenylalanine concentrations. Lysine transport from the peritoneum to the plasma was unaffected by the high plasma phenylalanine concentrations.     

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B^18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B^18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg⁴⁴ and Tyr⁵¹ as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.     

Nature of the serum heat-labile accessory factor involved in the metabolic inhibition of Mycoplasma pneumoniae

Using the metabolic-inhibition (MI) test as an assay, sera from rabbits immunized with Mycoplasma pneumoniae were separated on the basis of a requirement for a heat-labile (56 C) accessory factor (HLAF). As in previous studies, this requirement was found in sera collected early in the immune response, but disappeared as the immunization procedure progressed. Results obtained following exposure of HLAF-requiring sera to zymosan, hydrazine, and ethylene-diaminetetraacetic acid (EDTA) suggested that complement components C3, C8, and possible other components which follow C3 in the complement cascade, were required to demonstrate MI activity. Results obtained when HLAF-non-requiring sera were treated in the same manner, suggested the presence of two antibody populations. One population required component C3, and possibly C5, C6, and C9, while the other population required none of the complement components.     

Calmodulin Binds to Extracellular Sites on the Plasma Membrane of Plant Cells and Elicits a Rise in Intracellular Calcium Concentration

Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca²⁺ fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca²⁺ levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom.Calmodulin (CaM)² is a conserved multifunctional calcium sensor that mediates intracellular Ca²⁺ signaling and regulates diverse cellular processes by interacting with calmodulin-binding proteins (1–3). Interestingly, in both animals and plants, CaM may also act as an extracellular agent to regulate physiological events (4). Consistent with this notion, extracellular CaM has been detected within the cell walls of a broad range of plant species (4, 5).Functional studies have established that exogenously applied CaM can stimulate the proliferation of suspension-cultured plant cells (6) as well as affect intracellular activities of heterotrimeric G proteins and phospholipases in protoplasts (7, 8). Based on these findings, it has been proposed that, in plants, extracellular CaM may function as a signaling agent involved in the regulation of cell growth and development (4). However, as a 17-kDa hydrophilic protein, exogenously applied CaM could well be retrieved from the apoplasmic space and then exert its effects on components within the cytoplasm. Evidence against this hypothesis was provided by studies with Arabidopsis thaliana suspension-cultured cells in which it was shown that 24 h of incubation in exogenous CaM did not result in protein uptake or degradation (4).To exert an effect from the apoplasm, it would seem logical to assume that a protein(s) within the plant plasma membrane would have a CaM-binding site exposed to the apoplasm. Although a number of studies have addressed the molecular mechanism(s) by which extracellular CaM might act as a signal (6, 9) and attempts have been made to identify extracellular CaM-binding proteins (4, 6), currently there is no direct evidence in support of the hypothesis that specific CaM-binding sites exist at the surface of plant cells.To address this question, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection (10–13) at the surface of plant cells. These nanoparticles have several advantages over conventional fluorophores for light microscopic imaging, including their higher brightness and photostability (14, 15). In addition, because of their electron dense nature, QDs can be used for single labeling studies at the transmission electron microscope level (16, 17). Using this QD-CaM system, we demonstrate that QD-CaM binds selectively to sites on the outer surface of the plant plasma membrane. We also show by three independent methods that applied CaM can modulate Ca²⁺ fluxes across the plasma membrane, leading to alterations in cytoplasmic Ca²⁺ status. These findings support the hypothesis that, in plants, apoplasmic CaM can act as a signaling agent.     

The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins

Summary The hemolytic activity of the terminal complement proteins (C5b-9) towards erythrocytes containing high potassium concentration has been reported to be dramatically increased when extracellular Na⁺ is substituted isotonically by K⁺ (Dalmasso, A.P., et al., 1975,J. Immunol. 115:63–68). This phenomenon was now further investigated using resealed human erythrocyte ghosts (ghosts), which can be maintained at a nonlytic osmotic steady state subsequent to C5b-9 binding: (1) The functional state of C5b-9-treated ghosts was studied from their ability to retain trapped [¹⁴C]-sucrose or [³H]-inulin when suspended either in the presence of Na⁺ or K⁺. A dramatic increase in the permeability of the ghost membrane to both nonelectrolytes-in the absence of significant hemoglobin release-was observed for C5b-9 assembly in the presence of external K⁺. (2) The physical binding of the individual¹²⁵I-labeled terminal complement proteins to ghost membranes was directly measured as a function of intra- and extracellular K⁺ and Na⁺. The uptake of¹²⁵I-C7,¹²⁵I-C8, and¹²⁵I-C9 into membrane C5b-9 was unaltered by substitution of Na⁺ by K⁺. (3) The binding of the terminal complement proteins to ghosts subjected to a transient membrane potential generated by the K⁺-ionophore valinomycin (in the presence of K⁺ concentration gradients) was measured. No significant change in membrane binding of any of the C5b-9 proteins was detected under the influence of both depolarizing and hyperpolarizing membrane potentials. It can be concluded that the differential effect of Na⁺ versus K⁺ upon the erythrocyte membrane isnot due to an effect upon the binding of the complement proteins to the membraneper se, but upon the functional properties of the assembled C5b-9 pore site.     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.     

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys⁴⁴⁷-Lys⁴⁷²) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp⁴⁴⁷-Tyr⁴⁵⁴ and Phe⁴⁶⁰-Lys⁴⁷¹ at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly⁴⁵⁵-Gly⁴⁵⁹. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na⁺/H⁺ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu⁴⁶⁵ contributing to the pore.The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1)⁴ is a ubiquitous integral membrane protein that regulates intracellular pH. It mediates removal of a single intracellular proton in exchange for an extracellular sodium ion (1). NHE1 has many functions aside from protection of cells from intracellular acidification (2). It promotes cell growth and differentiation (3), regulates sodium fluxes and cell volume after challenge by osmotic shrinkage (4), and has been demonstrated to be involved in modulating cell motility (5). In addition its activity is important in invasiveness of neoplastic breast cancer cells (6). NHE1 also plays critical roles in heart disease. It has a contributing role in heart hypertrophy and in the damage that occurs during ischemia and reperfusion. Inhibition of NHE1 with Na⁺/H⁺ exchanger inhibitors protects the myocardium during various disease states (7-10).NHE1 is composed of two general regions, an N-terminal membrane domain of ∼500 amino acids and a C-terminal regulatory domain of ∼315 amino acids (1, 8). The membrane domain is responsible for ion movement and an analysis of topology by cysteine scanning accessibility suggested it has 3 membrane-associated segments and 12 integral transmembrane segments (11) (Fig. 1A). The mechanism of transport of the membrane domain is of great interest both from a scientific viewpoint and in the design of improved NHE1 inhibitors that may be necessary for clinical use (1). In this regard, we have recently characterized the functionally important residues and the structure of both TM IV and TM VII. Prolines 167 and 168 of TM IV were critical to NHE1 function (12) and cysteine-scanning mutagenesis was used to show that Phe¹⁶¹ is a pore lining residue critical to transport. Analysis of the structure of TM IV showed that TM IV is composed of one region of β-turns, an extended middle region including Pro¹⁶⁷-Pro¹⁶⁸, and a helical region (13). TM VII was much more typical of a transmembrane helix although it was interrupted with a break in the helix at the functionally critical residues Gly²⁶¹-Glu²⁶² (14).Open in a separate windowFIGURE 1.Models of the Na⁺/H⁺ exchanger. A, simplified topological model of the transmembrane domain of the NHE1 isoform of the Na⁺/H⁺ exchanger as described earlier (11). EL, extracellular loop; IL, intracellular loop. B, model of amino acids present in TM XI.Another important TM segment of the Na⁺/H⁺ exchanger is TM XI (Fig. 1B). Several different lines of evidence have suggested that it is critical to NHE1 function. A recent study generated chimeras of NHE1 from various species and found that a region including TM XI was important in determining NHE1 inhibitor sensitivity (15). More specifically, mutagenesis of several amino acids of TM XI has shown that it is likely involved in either ion transport or proper targeting to the plasma membrane. Two mutants in TM XI, Y454C and R458C, are retained in the endoplasmic reticulum (16). In addition, mutation of Gly⁴⁵⁵ and Gly⁴⁵⁶ in TM XI shift the pH_i dependence of the exchanger to the alkaline side, whereas mutation of Arg⁴⁴⁰ in intracellular loop 5 at the N-terminal end of TM XI shifts the pH_i dependence to make it more acidic (17, 18). Also, the structure of the bacterial Na⁺/H⁺ exchanger NhaA has been elucidated. Both TM IV and TM XI play a critical role forming an assembly that cross, with each being a helix, an extended polypeptide and a short helix (19). We found that TM IV of NHE1 has a similar structure and function to that of TM IV of NhaA (2, 13), leaving open the possibility that TM XI of NHE1 is also similar in structure and function to TM XI of NhaA.For these reasons, we undertook a systematic examination of the structural and functional aspects of TM XI of the NHE1 isoform of the Na⁺/H⁺ exchanger. The sequence of human TM XI of NHE1 is ⁴⁴⁹QFIIAYGGLRGAIAFSLGYLLD⁴⁷⁰. In this study we use cysteine scanning mutagenesis and site-specific mutagenesis to identify and characterize critical pore lining residues of the protein. We also use nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of a synthetic peptide representing TM XI in dodecylphosphocholine (DPC) micelles. Evidence has suggested that TM segments of membrane proteins possess all the structural information required to form their higher order structures in their amino acid sequence (20). This has been demonstrated in earlier studies on membrane protein segments such as the cystic fibrosis transmembrane conductance regulator (21), a fungal G-protein-coupled receptor (22), bacteriorhodopsin (23, 24), and rhodopsin (25), where it was shown that isolated TM segments from membrane proteins had structures in good agreement with the segments of the entire protein. Also, the use of DPC micelles has been shown to be an excellent membrane mimetic environment for these studies (26, 27). Our study identifies Leu⁴⁶⁵ as contributing to the pore of the protein and shows that the structure of TM XI consists of two helices corresponding to Asp⁴⁴⁷-Tyr⁴⁵⁴ and Phe⁴⁶⁰-Lys⁴⁷¹ at the N and C termini, respectively, connected by a flexible region at residues 455-459. The structure of TM XI was similar to the x-ray structure of TM XI of NhaA.     

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal¹. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis^1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.