Interaction of myosin and paramyosin

The interaction of myosin and paramyosin was investigated by enzymological and ultrastructural techniques. The actin-activated Mg⁺² ATPase of rabbit skeletal muscle myosin can be inhibited by clam adductor paramyosin. Both proteins must be rapidly coprecipitated to form filaments for this inhibition. Slowly formed cofilaments are fully activatable by F-actin. In both cases, the cofilaments possess unique structural characteristics when compared to homofilaments. The mode of inhibition appears to be competitive when different concentrations of paramyosin and F-actin are compared. The apparent affinity of the myosin heads for actin is reduced by the presence of paramyosin within rapidly reconstituted thick filaments. These results suggest that paramyosin may serve as part of a relaxing mechanism within invertebrate muscles. It is unlikely that paramyosin plays a role in the initiation and maintenance of catch within specialized molluscan muscles.     

Mechanisms of Contraction in Molluscan Muscle

The muscles of molluscs show a wide variety of structural types,a variation that is paralleled by the behavior of the muscles.All these muscles have certain features in common; they containlong filaments of two types, but differ in the length and thicknessof the thick filaments and in the proportion of the protein,paramyosin, in the muscle. Structural and physiological experimentssuggest that the basic contractile mechanism is the same inall molluscan muscles. The muscle-relaxing mechanism, however,may be specialized, particularly in certain smooth muscles whichhave the very slow relaxation characteristic of "catch" muscles.     

Structural features associated with movement and ‘catch’ of sea-urchin spines

Spine catch ligaments of a sea urchin Arbacia punctulata were extended under constant load. Ligaments from an undisturbed animal may show any extension rate from zero (catch state) to rapid extension to failure. Replacing the preparation bath with Ca²⁺- and Mg²⁺-free sea water reversibly abolishes the catch state. The fine structure of the outer muscle layer and inner ligament cone associated with the spine base is described. The unstriated paramyosin muscles bear thin flanges and form compact interlocking rows. Subsurface cisternae are associated with the plasma membrane. The muscles are innervated by glia-free axons ending in bulbous terminals containing lucent synaptic vesicles. The ligament comprises cylindrical bundles of collagen fibrils: one or more minute muscle fibers (paramyosin) lie parallel with and closely adjoining each bundle. The mean diameter of these muscles is 0.3 μg and they occupy 2–3 % of the ligament's cross-sectional area. Axons containing electronopaque secretory droplets accompany the muscles between the collagen bundles: the cell bodies of these neurones generally lie on the outer surface of the ligament. When an urchin points a spine, the ligament on the side of the contracting spine muscle shortens but does not buckle. A function of the intraligamental muscles is to effect this non-buckled shortening. The catch mechanism (which resides entirely within the ligament) may be due either to the intraligamental muscles and/or to a locked polymer mechanism in which matrix molecules between collagen fibrils are reversibly crosslinked by divalent cations.     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

The paramyosin function in glycerinated muscle fibers during blocking with antibodies of its contacts with structural proteins

Antibodies to paramyosin (APM) induce a partial decrease of the isometric tension in glycerinated fibres of the Anodonta cygnea catch muscle in the presence of ATP and Ca2+; the myofibrillar Mg2+ ATPase increases concomitantly. Assumedly paramyosin inhibits the cross-bridges unlocking, retaining them mechanically in a locked state. The fibres of barnacle giant muscle in an ATP deficient solution respond to APM by transient isometric tension development. A model for the participation of paramyosin in the contractile process is proposed. In both muscle types paramyosin hinders the functioning of certain elements of the contractile machinery.     

Paramyosin and the catch mechanism

1. Catch is a mechanism found in many molluscan smooth muscles in which tension is maintained at relatively low energy cost. 2. Paramyosin forms the core of thick filaments. In catch muscle paramyosin concentrations are high and the thick filaments are relatively long. 3. The mechanism of catch is not understood, but the consensus is that tension during catch is borne by slowly-cycling cross-bridge attachments to actin. 4. Stimulation by acetylcholine increases intracellular Ca2+ and initiates a contraction characterized by a relatively rapid cross-bridge cycling. Reduction of Ca2+ can lead to relaxation or catch. Relaxation occurs only when a second neurotransmitter, serotonin, is present. 5. The catch state is released by serotonin, via activation of adenylate cyclase, increased levels of cAMP and phosphorylation of one or more contractile proteins, possibly paramyosin. Other targets for phosphorylation are discussed. 6. The contractile cycle of catch muscles, therefore, is controlled by both Ca2+ and cAMP.     

5-HT induced accumulation of 3',5'-AMP and the phosphorylation of paramyosin in the ABRM of Mytilus edulis.

In Mytilus edulis 5-Ht induced relaxation of a muscle in catch is preceded by an increase in 3',5'-AMP content. In vitro two proteins of the contractile apparatus are phosphorylated by 3',5'-AMP dependent protein kinases. The 295000 dalton protein cannot be identified, the other one is paramyosin. Phosphorylated paramyosin inhibits actomyosin ATPase of smooth mollusc muscles at low and high Ca++ concentrations.     

Paramyosin in invertebrate muscles. I. Identification and localization

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion, we identified paramyosin in two smooth invertebrate "catch" muscles (Mytilus anterior byssus retractor and Mercenaria opaque adductor) and five invertebrate striated muscles (Limulus telson levator, Homarus claw muscle, Balanus scutal depressor, Lethocerus air tube retractor, and Aequipecten striated adductor). We show that (a) the paramyosins in all of these muscles have the same chain weights and (b) they are immunologically similar. We stained all of these muscles with specific antibody to Limulus paramyosin using the indirect fluorescent antibody technique. Paramyosin was localized to the A bands of the glycerinated striated muscles, and diffus fluorescence was seen throughout the glycerinated fibers of the smooth catch muscles. The presence of paramyosin in Homarus claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor is shown here for the first time. Of the muscles in this study, Limulus telson levator is the only one for which the antiparamyosin staining pattern has been previously reported.     

Some physical properties of paramyosin from earthworms

Paramyosin was prepared from earthworms (Lumbricus terrestris) by two different methods that have been used in the past. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the older method yields slightly degraded material (mostly β- and γ-paramyosin) while the newer method yields essentially intact, i.e., α-, paramyosin. Physical studies, particularly circular dichroism, light scattering, and sedimentation velocity show that the native molecule is a double α-helical coiled coil of molecular weight 200,000, length 1200 Å, and diameter 20 Å. These properties are the same as reported previously for molluscan paramyosin. Also like clam paramyosin, the worm protein molecule loses its helix content and dissociates into its two constituent polypeptide chains upon exposure to sufficient concentration of Gdn-HCl. Furthermore, the same partially denatured states can be reached from either native or completely denatured proteins, indicating that they are all equilibrium states. However, the Gdn · HCl-induced denaturation profile for the worm paramyosin is quite different from the clam. The helix content of worm paramyosin diminishes monophasically with increasing concentration of Gdn-HCl, showing that the molecule does not possess a region of special stability such as its clam analog boasts. This conclusion is supported by experiments on papain digestion of worm paramyosin, wherein no resistent core is seen.     

The Regulation of Catch in Molluscan Muscle

Molluscan catch muscles are smooth muscles. As with mammalian smooth muscles, there is no transverse ordering of filaments or dense bodies. In contrast to mammalian smooth muscles, two size ranges of filaments are present. The thick filaments are long as well as large in diameter and contain paramyosin. The thin filaments contain actin and appear to run into and join the dense bodies. Vesicles are present which may be part of a sarcoplasmic reticulum. Neural activation of contraction in Mytilus muscle is similar to that observed in mammalian smooth muscles, and in some respects to frog striated muscle. The relaxing nerves, which reduce catch, are unique to catch muscles. 5-Hydroxytryptamine, which appears to mediate relaxation, specifically blocks catch tension but increases the ability of the muscle to fire spikes. It is speculated that Mytilus muscle actomyosin is activated by a Ca⁺⁺-releasing mechanism, and that 5-hydroxytryptamine may reduce catch and increase excitability by influencing the rate of removal of intracellular free Ca⁺⁺.     

Subunit structure of oyster paramyosin

Paramyosin from the oyster Crassostrea commercialis was studied by equilibrium sedimentation. In non-denaturing solvents the minimum molecular weight is 208000. Dissociation into subunits requires complete disruption of the alpha-helix. This occurs at pH7 in guanidine hydrochloride solutions of concentration greater than 7m in the presence of a disulphide-bond-reducing agent. Solutions of the protein in concentrated guanidine hydrochloride are polydisperse and contain species of low molecular weight (approx. 25000) comprising approx. 5% to 10% of the protein. The molecular weight of the main component is estimated to be 97000 and the paramyosin molecule contains two of these subunits. From the present observations no decision can be made as to whether or not the small component (or components) represents part of the paramyosin molecule. Preferential binding of guanidine hydrochloride to the extent of 0.13g./g. of protein was shown in solutions of paramyosin in 7.85m-guanidine hydrochloride.     

Comparison of solubility properties of alpha-paramyosin, beta-paramyosin and acid-extracted paramyosin.

The solubility properties of paramyosin in the zones of pH and ionic strength in which aggregation occurs were initially studied using preparations isolated by a method originally described by Bailey (Bailey, K. (1956), Pubbl. Stn. Zool. Napoli 29, 26). Other preparations yielding apparently different protein components have been described by Hodge (Hodge, A.J. (1952), Proc. Natl. Acad. Sci., U.S.A. 38, 850) using acid conditions, and Stafford and Yphantis (Stafford, W.F., AND Yphantis, D. (1972), Biochem. Biophys. Res. Commun. 49, 848) have identified alpha-, beta-, and gamma-paramyosin using various times and temperatures of extraction with or without ethylenediaminetetraacetic acid. We have found that acid-extracted paramyosin is very similar if not identical to alpha-paramyosin, but that both acid and alpha forms differ considerably from beta- and gamma-paramyosin. Beta-Paramyosin precipitates abruptly from solution in narrow zone of pH below neutrality, and increases in ionic strength shift the zone of precipitation toward lower pH values. In contrast, both acid and alpha-paramyosin show gradual aggregation with changing pH at lowerionic strength (less than 0.3) but sharp transitions similar to beta-paramyosin at higher ionic strength (greater than 0.3). Transitions were also found at lower pH (ca. 4.0) which were not mirror images of transitions at higher pH (ca. 7.0). Viscosity measurements show that acid extracted paramyosin is close in behavior to a native extract obtained by extraction in mild, nondenaturing media containing mixed antibiotics. Each of these extracts differed considerably from beta-paramyosin. Mild, nonhydrolytic procedures employed by others to remove small, noncovalent bonded components or to separate protein complexes were not effective in converting alpha- to beta-paramyosin. Comparison of extraction procedures strongly supports the suggestion of Stafford and Yphantis that beta- and gamma-paramyosin are hydrolytic products of alpha-paramyosin and that the proteases responsible may be of bacterial origin.     

The Role of Microscopy in the Investigation of Paramyosin as a Vaccine Candidate against Schistosoma japonicum

There has been growing interest in paramyosin as a vaccine component to combat schistosomiasis. Immunological and molecular techniques have been used in the past to investigate the effectiveness of a paramyosin vaccine as an anti-schistosomal treatment. However, recent localization studies at ultrastructural and morphological levels have highlighted a number of questions concerning the role of paramyosin within schistosome parasites. Debates about how a non-surface protein such as paramyosin might provide protection against schistosome infections have recently been addressed by microscopy results. Immunolocalization studies have indicated multiple functions of paramyosin within the parasite and provided insights into how a vaccine may target the parasite, as discussed here by Geoffrey Gobert.     

Paramyosin in invertebrate muscles. II. Content in relation to structure and function

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.     

Paramyosin-enhanced clearance of Brugia malayi microfilaremia in mice

Progress in development of a vaccine against human filariasis has been hampered by lack of knowledge of the biochemical structure of specific Ag that induce protective immunity in experimental hosts. In the current study, antiserum to infective third-stage larvae of Brugia malayi was used to select potentially protective Ag shared by microfilariae (mf) and adult worms. A major Ag of 97 kDa (Bm 97) was identified by immunoblotting and isolated by electroelution. Immunization of mice with 2 micrograms electroeluted Bm 97 induced partial resistance to subsequent i.v. challenge with live B. malayi mf (40 to 60% reduction in parasitemia compared to controls, p less than 0.05). Immunoblot studies of B. malayi mf and adult worm lysates showed reactivity of a 97-kDa molecule with monospecific antiserum to Schistosoma mansoni paramyosin. In addition, mouse antibody to Bm 97 reacted with a 97-kDa molecule contained in wild-type Caenorhabditis elegans but not in two mutant strains deficient for paramyosin. Subcutaneous injection of mice with paramyosin (5 micrograms twice at a 2-wk interval) purified from C. elegans or B. malayi by salt precipitation induced resistance to microfilaremia (21 to 60% lower intensities than controls, p less than 0.01). These data indicate that the invertebrate muscle protein paramyosin enhances clearance of blood-borne stages of lymphatic filariae. Examination of the ability of paramyosin to induce resistance in third-stage larvae-challenged hosts is warranted.     

Neuromuscular Systems in Molluscs

Molluscs have become increasingly popular in the study of centralneural mechanisms. More recently, there have been attempts torelate activity in central neurons with behavior in animalsof this phylum. The latter studies necessitate an understandingof the effectors of such behaviors. This requires not only informationabout the neuromuscular junction, but also an awareness of thecapabilities of the muscles themselves. Therefore, we have discussedsome structural and related functional characteristics of molluscanmuscle. We suggest that invertebrate mucles might be comparedon three scales: the amount of myofilament organization, theamount of vesicular specialization and organization, and theamount of paramyosin. We have considered some characteristicsof the widely-studied sustained contraction, known as "catch."Finally, we have discussed the neuromuscular junction—thetypes of junctions, the multiplicity of innervation, and someaspects of pharmacology. The results of such a study indicatedmany areas in which further research is essential before wecan understand behavior in terms of activity in the centralnervous system.     

Single charge change on the helical surface of the paramyosin rod dramatically disrupts thick filament assembly in Caenorhabditis elegans

Charge interactions between alpha-helical coiled-coil proteins have been postulated to determine the alignment of many filamentous proteins, such as myosin heavy-chain rod, paramyosin and alpha-keratin. Here we determined the sequence changes in nine mutations in the unc-15 paramyosin gene of Caenorhabditis elegans, including one nonsense, four missense, one deletion and three suppressor mutations. These mutation sites were located on a molecular model, constructed by optimizing charge interactions between paramyosin rods. Remarkably, single charge reversals (e.g., glutamic acid to lysine) were found that either disrupted or restored filament assembly in vivo. The positions of the mutations within the paramyosin molecule support the models of paramyosin assembly and further suggest that the C-terminal region containing a cluster of five mutations, and a site interacting with it, play a key role in assembly. One amino acid substitution in this C-terminal region, in which there is a "weak spot", led to a loss of reactivity with one monoclonal anti-paramyosin antibody. The results demonstrate how a single amino acid substitution can alter the assembly properties of alpha-helical molecules.     

B cell responses to paramyosin. Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis.

To examine the fine specificity of the human immune response to filarial paramyosin, the antigenicity of an expressed rcDNA (2.55 kb) of Dirofilaria immitis paramyosin was detailed by ELISA. Using sera from patients infected with Onchocerca volvulus, we analyzed both the entire paramyosin molecule and six subcloned fragments for their IgG, IgG subclasses, and IgE responses. Patients from both Guatemala (64% positive) and Ghana (100% positive) reacted to paramyosin with specific IgG levels above normal controls. Although there was no anti-paramyosin subclass restriction common to all patients, the IgG3 response in the Ghananians was significantly greater than that of Guatemalans (p less than 0.001). IgE anti-paramyosin responses showed positive correlations with IgG2 (p less than 0.001), IgG4 (p less than 0.002), and IgG1 (p less than 0.04) responses. Epitope mapping using the IgG response to the six subclones demonstrated preferential recognition of the amino terminal end of the molecule (nucleotides 1 to 360). IgG2 reactivity was clearly localized to the most amino-terminal 120 amino acids, and the IgG4 antibodies recognized amino acids immediately adjacent to this fragment. These studies examining the fine specificity of anti-filarial immune reactions should provide a method for understanding how parasites either evade or induce host immune responses.     

Phosphorylation of paramyosin

1. Myofibrils isolated from Mercenaria mercenaria were phosphorylated by endogenous kinase. Over a range of ionic strengths only paramyosin was phosphorylated. 2. Thiophosphorylation of paramyosin caused an inhibition of steady-state actin-activated ATPase activity of the myofibrils. 3. It is proposed that the endogenous kinase is the catalytic subunit of the cAMP-dependent protein kinase. 4. The sequence around the phosphorylation site was determined. 5. The phosphorylation site probably is close to the C-terminus of the paramyosin molecule.