Altered calsequestrin glycan processing is common to diverse models of canine heart failure.

Calsequestrin-2 (CSQ2) is a resident glycoprotein of junctional sarcoplasmic reticulum that functions in the regulation of SR Ca(2+) release. CSQ2 is biosynthesized in rough ER around cardiomyocyte nuclei and then traffics transversely across SR subcompartments. During biosynthesis, CSQ2 undergoes N-linked glycosylation and phosphorylation by protein kinase CK2. In mammalian heart, CSQ2 molecules subsequently undergo extensive mannose trimming by ER mannosidase(s), a posttranslational process that often regulates protein breakdown. We analyzed the intact purified CSQ2 from mongrel canine heart tissue by electrospray mass spectrometry. The average molecular mass of CSQ2 in normal mongrel dogs was 46,306 ± 41 Da, corresponding to glycan trimming of 3-5 mannoses, depending upon the phosphate content. We tested whether CSQ2 glycan structures would be altered in heart tissue from mongrel dogs induced into heart failure (HF) by two very different experimental treatments, rapid ventricular pacing or repeated coronary microembolizations. Similarly dramatic changes in mannose trimming were found in both types of induced HF, despite the different cardiomyopathies producing the failure. Unique to all samples analyzed from HF dog hearts, 20-40 % of all CSQ2 contained glycans that had minimal mannose trimming (Man9,8). Analyses of tissue samples showed decreases in CSQ2 protein levels per unit levels of mRNA for tachypaced heart tissue, also indicative of altered turnover. Quantitative immunofluorescence microscopy of frozen tissue sections suggested that no changes in CSQ2 levels occurred across the width of the cell. We conclude that altered processing of CSQ2 may be an adaptive response to the myocardium under stresses that are capable of inducing heart failure.

Are genuine changes in protein expression being overlooked? Reassessing Western blotting.

This study used purified calsequestrin 1 and AMP kinase (AMPK) proteins to demonstrate how Western blotting outcomes can be influenced when either the density of proteins detected lie within a nonproportional region of a standard curve or a standard curve is not taken into account for data analyses. It outlines the likelihood of true changes being overlooked through the simple mistake of using band density alone and/or through analyzing too much sample. To demonstrate this, extrapolation of a typical linear, although nonproportional, standard curve resulted in approximately fourfold error. The standard curve method was used to estimate the concentration of AMPK beta1 in rat extensor digitorum longus muscle as being of the order of 60 microM. The article suggests that adopting a more sensitive Western blotting protocol will improve the reliability of quantitative Western blotting outcomes.

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle.

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.

A 63 kDa skeletal muscle protein associated with eye muscle inflammation in Graves' disease is identified as the calcium binding protein calsequestrin.

It is generally accepted that thyroid-associated ophthalmopathy (TAO) is an autoimmune disease of the eye muscle (EM) and the surrounding orbital connective tissue in which circulating antibodies play an important role. Antibodies against EM membrane proteins of 63-67kDa mol. wt. seem to be the best markers of ophthalmopathy in patients with autoimmune thyroid disease. We purified a 63 kDa EM protein using SDS-polyacrylamide gel electrophoresis technology and TAO patients' sera as probes, digested the protein with cyanogen bromide and sequenced immunoreactive peptides. We also screened a human EM library with a rabbit antiserum against 63-65 kDa proteins and affinity purified antibodies from a TAO patient's serum that reacted with a 55 kDa EM membrane protein. From partial sequence information and from DNA sequencing of positive cDNA clones, the protein was identified as calsequestrin, a 63 kDa calcium binding protein localized in the sarcoplasmic reticulum of the muscle fiber. As determined by Northern blotting, calsequestrin was expressed in EM and other skeletal muscle but not thyroid or fibroblasts. Calsequestrin is different from the "64 kDa protein", which has been identified as succinate dehydrogenase flavoprotein subunit, which has a corrected mol. wt. of 67 kDa. Serum antibodies against calsequestrin were found in 40% of patients with clinically active TAO, but in only 4% of those with stable eye disease, and in 5% of normal subjects, by immunoblotting. Although it is possible that autoimmunity against calsequestrin plays a role in the progressive EM damage that characterizes ophthalmopathy it is more likely that the antibodies are secondary to a reaction against some other cell membrane protein, such as the novel thyroid and eye muscle shared protein G2s or the TSH receptor.

Protons induce calsequestrin conformational changes.

Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes extensive calcium-induced conformational changes at neutral pH that cause distinct intrinsic fluorescence changes. The results reported in this work indicate that pH has a marked effect on these calcium-induced intrinsic fluorescence changes, as well as on calorimetric changes produced by the addition of Ca(2+) to calsequestrin. The addition of Ca(2+) at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrinsic fluorescence was not affected by the addition of Ca(2+), and the same intrinsic fluorescence as that measured in millimolar calcium at neutral pH was obtained. The magnitude and the cooperativity of the calcium-induced intrinsic fluorescence changes decreased as either [H+] or [K+] increased. The evolution of heat production, determined by microcalorimetry, observed upon increasing the molar ratio of Ca(2+) to calsequestrin in 0.15 M KCl, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content changes produced by Ca(2+). We propose that protons bind to calsequestrin and induce protein conformational changes that are responsible for the observed proton-induced intrinsic fluorescence and calorimetric changes.

Skeletal muscle of patients with Duchenne's muscular dystrophy: evidence of a mitochondrial proteolytic factor responsible for calmitine deficiency.

We studied the effect of mitochondrial extracts of skeletal muscle obtained from patients with Duchenne's muscular dystrophy (DMD) on calmitine of the mitochondrial matrix isolated from skeletal muscle of control mice. Our results in vitro clearly show that calmitine of the mitochondrial matrix of control muscle was degraded in the presence of mitochondrial extracts of muscle from DMD patients. The diseased muscle apparently contains an abnormal calmitine-specific proteolytic factor responsible for the calmitine deficiency previously observed in this tissue. As calmitine binds calcium and probably plays a role in regulating the balance of bound and free calcium within mitochondria, a calmitine deficiency could result in an overload of mitochondrial free calcium. Certain enzymes involved in ATP synthesis would be inhibited, resulting in the muscular degeneration characteristic of this myopathy. Our results suggest the cause of mitochondrial calcium overload and the events leading to muscular degeneration in this disease model. Abnormal protease activity could be the factor triggering all of these processes in the DMD patient. These findings suggest that it may now feasible to search for an efficient pharmacologic treatment for DMD.

Evidence that spinach leaves express calreticulin but not calsequestrin.

The presence of either calreticulin (CR) or calsequestrin (CS-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca(2+)-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn(2+) (i.e., under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca(2+). Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca(2+)-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.

Molecular cloning of human calmitine, a mitochondrial calcium binding protein, reveals identity with calsequestrine.

The cDNA of a mitochondrial calcium binding protein, "calmitine", has been cloned from a human skeletal muscle cDNA library. One cDNA of 1.8 kb has been isolated and sequenced. It encodes for a protein of 390 amino acid residues of 41,746 KDa and contains a leading peptide of 28 amino acids. The sequencing showed the possibility for 21 phosphorylation sites, 4 myristylation sites, and one N glycosylation site. Sequence comparison with other proteins revealed the identity of calmitine with calsequestrine, the sarcoplasmic reticulum low affinity, but high Ca2+ binding capacity, protein isolated in 1971. Subcellular fractionation showed a marked increase in these Ca2+ binding proteins in mitochondria as compared with the sarcoplasmic reticulum; furthermore the mitochondrial matrix is highly enriched with that protein. Therefore, our data either suggest a bicompartimentation of calmitine or indicate that the localization of calsequestrine should be reconsidered in the light of our data. Calmitine represents the Ca2+ reservoir of mitochondria, the function of which could be similar to what has been reported for calsequestrine in the sarcoplasmic reticulum.

Characterization study of the ryanodine receptor and of calsequestrin isoforms of mammalian skeletal muscles in relation to fibre types.

We have investigated high-affinity ryanodine-binding sites in membrane preparations from representative fast-twitch and slow-twitch muscles of the rabbit and rat, as well as from human mixed muscle. Our results, obtained in high-ionic strength binding buffer, demonstrate extensive similarities in binding affinity for [3H]ryanodine (Kd: about 10 nM) and a two-fold to four-fold difference in membrane density of the ryanodine receptor between fast-twitch and slow-twitch muscle of the rat and rabbit, respectively. The [3H]ryanodine-pCa relationship for the Ca(2+)-activation curve of ryanodine binding was found to be similar for all mammalian muscles, as tested at 20 nM ryanodine. With 10 mM caffeine or 50 microM doxorubicin the pCa for half-maximal activation of [3H]ryanodine binding invariably shifted from an average pCa value of 6.5 to pCa 7.1-7.3. IC50 values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca(2+)-release channel blocker, did not differ significantly (range 0.3-1.0 microM). The Ca(2+)-dependence curve (range 1 nM-10 mM free Ca2+) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cisternae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cisternae from the same species. Cardiac ryanodine receptor is also clearly distinguishable for electrophoretic mobility, Cleveland's peptide maps, and, most strikingly, for total lack of cross-reactivity with polyclonal antibody to fast skeletal RyR. By the same properties, the ryanodine receptor of fast- and slow-twitch muscle appear to be the same or a similar protein. On investigating the composition of calsequestrin in rat and human skeletal muscles, both in membrane-bound form and after purification by phenyl-Sepharose chromatography, we have been able to show that, independent of the animal species, the cardiac isoform, as characterized by the identical amino-terminal amino-acid sequence, pattern of immunoreactivity, and lack of Ca(2+)-dependent shift in mobility on SDS-PAGE, is exclusively expressed in slow-twitch fibres, together with the main fast-skeletal calsequestrin isoform. While our experimental findings strongly argue for the presence of only one population of skeletal-specific Ca(2+)-release channels in junctional terminal cisternae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of Ca(2+)-release, depending on its isoform composition.

Crystallization of canine cardiac calsequestrin.

Calsequestrin is the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum membranes. Two X-ray quality crystal forms of canine cardiac calsequestrin were obtained by the hanging drop method using KCl as a precipitant. One form is monoclinic (space group P2(1), a = 73.4 A, b = 104.4 A, c = 60.2 A, beta = 120.4 degrees) with two molecules in the asymmetric unit and a solvent content of approximately 40%. The second form is trigonal (P3(1)21 or P3(2)21, a = b = 99.3 A, c = 89.8 A) with a single molecule in the asymmetric unit and 55% solvent content. Cross rotation function calculations show that despite the different space groups the packing of the molecules in both crystals is likely to be similar suggesting the existence of a stable dimer. The monoclinic crystals diffract beyond 3 A using a laboratory rotating anode source, while under the same conditions the trigonal crystals diffract only to approximately 4.5 A. This is the first report of successful preparation of X-ray quality crystals of a high capacity Ca2+ binding protein.

Distribution of Ca(2+)-modulating proteins in sarcoplasmic reticulum membranes after denervation.

The early response to the loss of motor innervation to the muscle is connected with an altered Ca(2+)-homeostasis. Our study, based on Western blotting, indicates that denervation influenced expression of some sarcoplasmic Ca(2+)-modulating proteins. Evidence has been brought for an increase of the level of calsequestrin and of the putative ryanodine receptor paralleled with a slight decrease of the total amount of Ca(2+)-pump protein. The expression of unchanged Ca(2+)-pump isoform and unaltered quantities of other non-junctional Ca(2+)-binding proteins support the hypothesis that changed cellular Ca2+ homeostasis include also an alteration of Ca(2+)-modulating systems, mainly from the junctional region of sarcoplasmic membranes.

Purification and characterization of calsequestrin from chicken cerebellum.

Chicken cerebellum microsomal fractions contain a protein tentatively identified as calsequestrin (CS) (Volpe et al., Neuron 5, 713-721, 1990). Here we report, for the first time, the purification of cerebellum CS from whole tissue homogenate by DEAE-Cellulose chromatography and Ca(2+)-dependent elution from phenyl-Sepharose. The purified cerebellum CS displays the shift and increase in intrinsic fluorescence characteristic of skeletal muscle CS, and is shown to be a high-capacity, low-affinity Ca2+ binding protein (Kd = 1 mM).

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.

Purification and characterization of a calsequestrin-like calcium-binding protein from carp (Cyprinus carpio) sarcoplasmic reticulum.

1. A calsequestrin-like calcium-binding protein was purified from carp sarcoplasmic reticulum by column chromatographies using DEAE-cellulose and Butyl-Toyopearl 650S. 2. The mol. wt was estimated to be 50 kDa, which was larger than that of rabbit calsequestrin (42 kDa). 3. Carp calsequestrin-like protein bound Ca2+ with a higher affinity (apparent Kd = 400 microM) and lower capacity (25 mol/mol) compared with rabbit calsequestrin (1 mM and 40-50 mol/mol, respectively). 4. Anti-carp calsequestrin-like protein rabbit antiserum reacted with rabbit calsequestrin in immunoblotting analysis. 5. Carp calsequestrin-like protein was rich in acidic amino acids, as was rabbit calsequestrin.

Phosphorylation of cardiac and skeletal muscle calsequestrin isoforms by casein kinase II. Demonstration of a cluster of unique rapidly phosphorylated sites in cardiac calsequestrin.

Calsequestrin is an acidic Ca2(+)-binding protein of sarcoplasmic reticulum existing as different gene products in cardiac muscle and skeletal muscle. A unique feature of cardiac calsequestrin is a 31-amino acid-long COOH-terminal tail (Scott, B. T., Simmerman, H. K. B., Collins, J. H., Nadal-Ginard, B., and Jones, L. R. (1988) J. Biol. Chem. 263, 8958-8964), which is highly acidic and contains several consensus phosphorylation sites for casein kinase II. In the work described here, we tested whether this cardiac-specific sequence is a substrate for casein kinase II. Both cardiac and skeletal muscle calsequestrins were phosphorylated by casein kinase II, but cardiac calsequestrin was phosphorylated to a higher stoichiometry and at least 50 times more rapidly. The site of rapid phosphorylation of cardiac calsequestrin was localized to the distinct COOH terminus, where a cluster of three closely spaced serine residues are found (S378DEESN-DDSDDDDE-COOH). The slower phosphorylation of skeletal muscle calsequestrin occurred at its truncated COOH terminus, at threonine residue 363 (I351NTEDDDDDE-COOH). The similar sequence in cardiac calsequestrin (I351NTEDDDNEE) was not phosphorylated. Cardiac calsequestrin, as isolated, already contained 1.2 mol of Pi/mol of protein, whereas skeletal muscle calsequestrin contained only trace levels of Pi. The endogenous Pi of cardiac calsequestrin was also localized to the distinct COOH terminus. Our results indicate that the cardiac isoform of calsequestrin is the preferred substrate for casein kinase II both in vivo and in vitro.

Myonexin: an 80-kDa glycoprotein that binds fibronectin and is located at embryonic myotendinous junctions.

The distribution and function of an 80-kDa glycoprotein located at the surface of skeletal muscle cells and enriched in gelatin-binding fractions of skeletal muscle extracts are examined in the present study. The glycoprotein was purified by concanavalin A affinity chromatography followed by gel filtration and anion exchange chromatography. The purified protein did not display gelatin-binding although the protein bound to fibronectin in several assays. First, the glycoprotein bound to fibronectin-Sepharose and did not elute in high salt buffers although subsequent basic elutions displaced the 80-kDa protein from the column. Second, gel filtration of the 80-kDa glycoprotein in the presence of fibronectin showed separate peaks corresponding to the mass of the 80-kDa glycoprotein and fibronectin as well as a third, higher mass peak shown in immunoblots to contain both fibronectin and the 80-kDa glycoprotein. Third, immunoprecipitation with affinity-purified anti-80-kDa glycoprotein in the presence of the glycoprotein and radioiodinated fibronectin precipitated labeled fibronectin. The quantity of labeled fibronectin precipitated was reduced by the addition of nonradiolabeled fibronectin. Immunofluorescent microscopy using affinity-purified, anti-80-kDa showed this protein located at the myotendinous junctions of frog tadpoles and embryonic chicks. In chicks, it was discernible by immunofluorescence only during the morphogenetic stages that myotendinous junctions were being assembled. Amino acid analysis shows that the 80-kDa glycoprotein has a high concentration of acidic residues. There is only one cysteine per molecule in the 80-kDa glycoprotein and comparisons of reducing and nonreducing gels show that no disulfides are present, indicating that this is not an integrin protein. Amino terminal sequencing reveals that the protein contains marked similarity to the amino terminal of calsequestrin although the protein is distinct from calsequestrin in lacking Ca2(+)-dependent phenyl sepharose affinity and in its molecular weight and distribution. The observations indicate that the 80-kDa glycoprotein is a fibronectin receptor present at chick myotendinous junctions during junction morphogenesis. This apparently novel protein is named "myonexin" to reflect its location and likely function in attaching fibronectin to the surface of muscle cells.