Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin- activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.     

Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.     

Characterization of a second myosin from Acanthamoeba castellanii.

We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii. The sequence of ion exchange chromatography, actomyosin precipitation, actin extraction, and gel permeation chromatography yields per 100 g of cells about 11 mg of myosin-II which is 90 to 96% pure. ATPase activity is highest in the presence of Ca2+, but the enzyme is also active in EDTA provided high concentrations of K+ are present. The molecule consists of two 175,000 molecular weight heavy chains, one or two 17,500 molecular weight light chains, and two 16,500 molecular weight light chains. Myosin-II is rich in acidic residues and contains about 32 residues of cysteine/mol. The sedimentation coefficient is 5.9 S. Intrinsic viscosity is 126 cc/g. By equilibrium ultracentrifugation, the molecular weight averages depended upon the initial loading concentration in a way that suggested a 400,000 molecular weight species is in equilibrium with a 200,000 molecular weight species. By electron microscopy the molecule was seen to have two globular heads at one end of a tail 90 nm long. In KCl solutions of less than 0.25 M, the myosin-II tails self-associate to form the backbone of very small (6.6 x 205 nm) bipolar filaments with central bare zones 97 nm long. Myosin-II binds to actin filaments, forming periodic arrowhead-shaped complexes, but its Mg2+ ATPase activity is activated only 50% or less by actin. When radioactive myosin-II is incubated up to 90 min in unlabeled Acanthamoeba homogenates, it is not degraded into smaller fragments, such as the 190,000 molecular weight myosin-I. Our observations and the detailed enzymatic data presented by Maruta and Korn ((1977) J. Biol. Chem. 252, 6501-6509) argue that the smaller Acanthamoeba myosin-I (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem, 248, 4682-2690) does not arise by fragmentation of myosin-II in the homogenate or extract.     

Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail

Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation, including one that binds to another site near the tip of the myosin-II tail. This demonstrates that antibodies can inhibit polymerization of myosin-II, but only when they bind to key sites on the tail of the molecule.     

Microinjection into Acanthamoeba castellanii of monoclonal antibodies to myosin-II slows but does not stop cell locomotion

To study the in vivo role of myosin-II in Acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin-II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 microns/min to approximately 3 microns/min, injection of monoclonal antibodies specific for myosin-II decreased motility further to approximately 0.8 micron/min. This effect was seen whether or not the monoclonal antibody to myosin-II inhibited the actomyosin-II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin-II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin-II-injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer-injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin-II-injected cells until, on the average, approximately 50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer-injected cells (to approximately 1 micron/min). These cells tended to recover endosomes more rapidly (approximately 25 min) than cells injected with antimyosin-II monoclonal antibodies. The inability of antibodies to myosin-II to inhibit completely any of the movements studied suggests that although myosin-II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.     

Probing myosin head structure with monoclonal antibodies

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.     

Identification of functional regions on the tail of Acanthamoeba myosin- II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites

We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7- nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.     

Location of the head-tail junction of myosin

The tails of double-headed myosin molecules consist of an alpha-helical/coiled-coil structure composed of two identical polypeptides with a heptad repeat of hydrophobic amino acids that starts immediately after a conserved proline near position 847. Both muscle and nonmuscle myosins have this heptad repeat and it has been assumed that proline 847 is physically located at the head-tail junction. We present two lines of evidence that this assumption is incorrect. First, we localized the binding sites of several monoclonal antibodies on Acanthamoeba myosin-II both physically, by electron microscopy, and chemically, with a series of truncated myosin-II peptides produced in bacteria. These data indicate that the head-tail junction is located near residue 900. Second, we compared the lengths of two truncated recombinant myosin-II tails with native myosin-II. The distances from the NH2 termini to the tips of these short tails confirms the rise per residue (0.148 nm/residue) and establishes that the 86-nm tail of myosin-II must start near residue 900. We propose that the first 53 residues of heptad repeat of Acanthamoeba myosin-II and other myosins are located in the heads and the proteolytic separation of S-1 from rod occurs within the heads.     

Monoclonal antibodies binding to the tail of Dictyostelium discoideum myosin: their effects on antiparallel and parallel assembly and actin- activated ATPase activity

Eight monoclonal antibodies that bind to specific sites on the tail of Dictyostelium discoideum myosin were tested for their effects on polymerization and ATPase activity. Two antibodies that bind close to the myosin heads inhibited actin activation of the ATPase either partially or completely, without having an effect on polymerization. Two other antibodies bind to sites within the distal portion of the tail that has been shown, by cleavage mapping, to be important for polymerization. One of these antibodies binds close to the sites of heavy chain phosphorylation which is known to regulate both myosin polymerization and actin-activated ATPase activity. Both antibodies showed strong inhibition of polymerization accompanied by complete inhibition of the actin-activated ATPase activity. A unique effect was obtained with an antibody that binds to the end of the myosin tail. This antibody prevented the formation of bipolar filaments. It caused myosin to assemble into unipolar filaments with heads at one end and the antibody molecules at the other. Only at concentrations higher than required for its effect on polymerization did this antibody show substantial inhibition of the actin-activated ATPase. These results indicate that, using a monoclonal antibody as a blocking agent, parallel assembly of myosin can be dissected out from antiparallel association, and that essentially normal actin-activated ATPase activity could be obtained after significant reductions in filament size.     

Dynamic localization of myosin-I to endocytic structures in Acanthamoeba

We used fluorescence microscopy of live Acanthamoeba to follow the time course of the concentration of myosin-I next to the plasma membrane at sites of macropinocytosis and phagocytosis. We marked myosin-I with a fluorescently labeled monoclonal antibody (Cy3-M1.7) introduced into the cytoplasm by syringe loading. M1.7 binds myosin-IA and -IC without affecting their activities, but does not bind myosin-IB. Cy3-M1.7 concentrates at two different macropinocytic structures: large circular membrane ruffles that fuse to create macropinosomes, and smaller endocytic structures that occur at the end of stalk-like pseudopodia. These dynamic structures enclose macropinosomes every 30-60 s. Cy3-M1.7 accumulates rapidly as these endocytic structures form and dissipate rapidly after they internalize. Double labeling fixed cells with Cy3-M1.7 and polyclonal antibodies specific for myosin-IA, -IB, or -IC revealed that all three myosin-I isoforms associate with macropinocytic structures, but individual structures vary in their myosin-I isoform composition. Myosin-I and actin also concentrate transiently at sites where amoebae ingest yeast or the pseudopodia of neighboring cells (heterophagy) by the process of phagocytosis. Within 3 min of yeast attachment to the amoeba, myosin-I concentrates around the phagocytic cup, yeast are internalized, and myosin-I de-localizes. Despite known differences in the regulation of macropinocytosis and phagocytosis, the morphology, protein composition, and dynamics of phagocytosis and macropinocytosis are similar, indicating that they share common structural properties and contractile mechanisms.     

Generation of specificity-variant antibodies by alteration of carbohydrate in light chain of human monoclonal antibodies.

A hybridoma line, C5TN, produced human monoclonal antibody of which light chain had N-linked carbohydrate chain within the variable region. Some molecular-weight variants of light chain of the antibody were produced by C5TN variants resistant to cytotoxic effect of concanavalin A. The variant antibodies significantly altered the original cross-reactivity with antigens or lost the ability of antigen binding. The variants variously trimmed their carbohydrate chains by glycosidases, showed the changed reactivity or acquired the ability to bind for antigens. The carbohydrate-deficient antibodies from tunicamycin-treated C5TN and the variant clones behaved in a similar manner on antigen-binding reactivity. Furthermore, comparison of antibodies of which light chains have carbohydrate chains sensitive and resistant to some glycosidases showed that carbohydrate chain in variable region of light chain can influence their reactivity with antigen.     

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.     

Antigen binding of an ovomucoid-specific antibody is affected by a carbohydrate chain located on the light chain variable region

We cloned the variable regions of heavy and light chain genes of an anti-ovomucoid monoclonal antibody (MAb-OM21) produced by the mouse hybridoma cell line OM21. DNA sequence analysis showed that the light chain of the MAb-OM21 has only one potential N-glycosylation consensus sequence in the complementarity determining region 2 of the light chain. To find whether carbohydrate chains are located on the light chain, we assayed for the size of the light chain, after treatment with N-glycosidase, by western blotting, and also detection of the carbohydrate chains on the light chain was done using the lectin blot assay. A N-linked carbohydrate chain has been shown to bind to the light chain. To clarify the role of this carbohydrate chain in the light chain, we produced carbohydrate variant antibodies by N-deglycosylation using glycosidase or by expressing the antibody from different host cells. The N-deglycosylated variant antibody has greater antigen binding, and the antibody produced from the different host cells showed a reduced antigen binding activity and acquired the ability to react to ovalbumin. These results suggest that antigen binding of the ovomucoid specific antibody MAb-OM21 can be affected by the carbohydrate chain on the light chain variable region.     

Independent expression of a regulatory idiotype on heavy and light chains. A further immunochemical analysis with anti-heavy and anti-light chain antibodies

The heavy (H) and light (L) chains of murine monoclonal autoantibody 62 reacting with thyroglobulin independently express idiotypic (Id) determinants that are very similar if not identical with the Id62 expressed on the intact protein. In this report, we describe the production and characterization of rabbit antibodies to isolated H62 and L62 chains to further prove that individual chains express Id62 in an immunogenic form. The results demonstrate that both chains are capable of eliciting antibodies that, after appropriate adsorption, behave like conventional anti-Id62 antibodies prepared against the intact antibody molecule. By direct radioimmunoassay binding, competition of Id binding and Western blot anti-H62 and anti-L62 antibodies identify as Id-positive the same group of IgG1, bind in a reciprocal fashion to H- and L-chains of parental monoclonal antibody 62, and detect Id62-positive polyclonal serum autoantibodies to thyroglobulin. We conclude that monoclonal antibody 62 expresses independently a similar Id on both polypeptide chains and the intact antibody molecule, or its isolated chains, induce qualitatively similar anti-Id responses. These results are discussed in light of the possible structure/function implication such autoantibodies may have within the Id network.     

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy- chain phosphorylation, and (d) ADP release is fast. Thus, during steady- state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.     

Identification of functional regions on the tail of Acanthamoeba myosin- II using recombinant fusion proteins. II. Assembly properties of tails with NH2- and COOH-terminal deletions

We used purified fusion proteins containing parts of the Acanthamoeba myosin-II tail to localize those regions of the tail responsible for each of the three steps in the successive dimerization mechanism (Sinard, J. H., W. F. Stafford, and T. D. Pollard. 1989. J. Cell Biol. 107:1537-1547) for Acanthamoeba myosin-II minifiliment assembly. Fusion proteins containing the terminal approximately 90% of the myosin-II tail assemble normally, but deletions within the last 100 amino acids of the tail sequence alter or prevent assembly. The first step in minifilament assembly, formation of antiparallel dimers, requires the COOH-terminal approximately 30 amino acids that are thought to form a nonhelical domain at the end of the coiled-coil. The second step, formation of antiparallel tetramers, requires the last approximately 40 residues in the coiled-coil. The final step, the association of two antiparallel tetramers to form the completed octameric minifilament, requires residues approximately 40-70 from the end of the coiled-coil. A region of the tail near the junction with the heads is important for tight packing of the tails in the minifilaments. Divalent cations induce the lateral aggregation of minifilaments formed from native myosin-II or fusion proteins containing a nonmyosin "head," but under the same conditions fusion proteins composed essentially only of myosin tail sequences with very little nonmyosin sequences form paracrystals. The region of the tail necessary for this paracrystal formation lies NH2-terminal to amino acid residue 1,468 in the native myosin-II sequence.     

Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.     

Production of monoclonal antibodies against the FimA protein of Porphyromonas gingivalis in Nicotiana benthamiana

Porphyromonas gingivalis, a gram-negative anaerobic oral bacterium, causes periodontal disease by binding to saliva-coated oral surfaces. The FimA protein from P. gingivalis is a crucial pathogenic component of the bacterium and a target for vaccine development against periodontal disease. Complementary DNAs encoding the heavy and light chains of two monoclonal antibodies that bind specifically to the FimA protein were cloned into a plant expression vector under the control of the duplicated Cauliflower Mosaic Virus 35S promoter, and agroinfiltration was used to allow the vectors to infiltrate tobacco plants. The expressions of the heavy and light chains in the leaf tissue were detected using antibodies specific to each antibody chain. Western blot analysis showed the specific binding of the plant-derived monoclonal antibodies to the native FimA protein purified from P. gingivalis. Our finding that plant-derived monoclonal antibodies bound specifically to the native FimA protein indicates that plantderived monoclonal antibodies can protect against P. gingivalis invasion.     

Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.