Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.     

Evidence for a calsequestrin-like calcium-binding protein in human spermatozoa

Human spermatozoa were investigated for the presence of protein(s) recognized by antibodies against calsequestrin, the high capacity, moderate affinity Ca2(+)-binding protein, originally described in striated muscle fibers. Western immunoblots of detergent-soluble sperm extracts probed with polyclonal antibodies raised against human skeletal muscle calsequestrin identified a strongly cross-reactive protein. This protein resembles muscle calsequestrin in many respects. In fact, its migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is pH dependent, its apparent molecular mass being 64 kDa in alkaline SDS-PAGE and 44 kDa in neutral SDS-PAGE; its isoelectric point is acidic (4.6); it is metachromatically stained blue by the carboxycyanine dye, Stains-All; it is a Ca2(+)-binding protein (45Ca blot overlay). Indirect immunofluorescence experiments showed that the immunoreactive protein has an intracellular localization confined to the tail mid-piece. From these findings we conclude that human sperm cells express a protein structurally and antigenically related to skeletal muscle calsequestrin; a basis for a novel interpretation of Ca2(+)-mediated events in spermatozoa is thus provided.     

Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.     

Myotonic dystrophy and myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic reticulum. The functional role of DMPK is not fully understood, however, it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc.     

Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions.

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.     

Myotonic dystrophy protein kinase (DMPK) and its role in the pathogenesis of myotonic dystrophy 1

Myotonic dystrophy 1 (DM1) is an autosomal, dominant inherited, neuromuscular disorder. The DM1 mutation consists in the expansion of an unstable CTG-repeat in the 3'-untranslated region of a gene encoding DMPK (myotonic dystrophy protein kinase). Clinical expression of DM1 is variable, presenting a progressive muscular dystrophy that affects distal muscles more than proximal and is associated with the inability to relax muscles appropriately (myotonia), cataracts, cardiac arrhythmia, testicular atrophy and insulin resistance. DMPK is a Ser/Thr protein kinase homologous to the p21-activated kinases MRCK and ROCK/rho-kinase/ROK. The most abundant isoform of DMPK is an 80 kDa protein mainly expressed in smooth, skeletal and cardiac muscles. Decreased DMPK protein levels may contribute to the pathology of DM1, as revealed by gene target studies. Here we review current understanding of the structural, functional and pathophysiological characteristics of DMPK.     

MKBP, a Novel Member of the Small Heat Shock Protein Family, Binds and Activates the Myotonic Dystrophy Protein Kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as αB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by αB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

Identification of two distinct proteins that are immunologically related to the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel.

The alpha 1 subunit of the dihydropyridine-sensitive calcium channel is a protein which is critical for excitation-contraction coupling and L-type calcium current in skeletal muscle. Using antibodies generated against peptides from three regions of the deduced amino acid sequence of the alpha 1 subunit, we have identified two distinct proteins in rabbit skeletal muscle. Both proteins appeared to be recognized by antibodies against the amino (N) terminus of the alpha 1 subunit sequence. One protein was also recognized by antibodies against an internal (I) region of the predicted sequence but not by antibodies against the carboxyl (C) terminus. In contrast, the other protein was recognized by antibodies against the carboxyl terminus but not by the antibodies against the internal region. We have designated these proteins pNI and pNC based on their patterns of antibody recognition. No protein was detected which was recognized by all three antibodies. pNI is the protein commonly identified as the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Of note is that pNI, which apparently lacks sequences from the predicted carboxyl tail, is the protein present in preparations which we have previously demonstrated contain dihydropyridine-sensitive calcium channel activity. pNC is herein identified as a skeletal muscle protein that is immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Its function is unknown. In addition to their distinct patterns of antibody recognition, pNI and pNC were also distinguishable by several other properties. pNC migrated as a protein of approximately 160 kDa in 5% sodium dodecyl sulfate-polyacrylamide gels versus approximately 165 kDa for pNI. pNI was enriched in transverse tubule membranes, whereas pNC was found to be enriched in triad and junctional sarcoplasmic reticulum membrane fractions and was not found in transverse tubule membranes. Under conditions in which pNI bound to wheat germ agglutinin-Sepharose, pNC did not bind. The results demonstrate that there are two proteins in skeletal muscle which are immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel but which are distinguishable by several biochemical and immunological characteristics.     

Characterization of DHP binding protein in crayfish striated muscle

The dihydropyridine calcium channel blocker, [3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.     

Immunolocalization of myotonic dystrophy protein kinase in corbular and junctional sarcoplasmic reticulum of human cardiac muscle

The subcellular localization of myotonic dystrophy protein kinase has been examined in human cardiac muscles with confocal laser-scanning microscopy and electron microscopy. A polyclonal antibody was produced against the synthesized peptide from a human kinase cDNA clone. We checked the antibody specificity for cardiac myotonic dystrophy protein kinase using an immunoblotting technique. Immunoblotting of extract from human cardiac muscles showed mainly 70 kDa and 55 kDa molecular weight bands. Confocal images of the protein kinase immunostaining showed striated banding patterns similar to those of skeletal muscles. In addition, the kinase was strongly detected around the intercalated disc. Immunoelectron microscopy showed that the kinase was mainly expressed in both corbular and junctional sarcoplasmic reticulum, but not in network sarcoplasmic reticulum. These results suggest that myotonic dystrophy protein kinase may be involved in the modulation of Ca2+ homeostasis in cardiac myofibres. © 1998 Chapman & Hall     

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells.     

Subcellular localization of the retinoblastoma protein

The subcellular localization of the retinoblastoma (RB) protein has been studied in primate cell lines by immunofluorescence staining using different monoclonal and polyclonal antibodies. The protein appeared as granules of heterogeneous size over the interphase nuclei. Computer assisted digital overlap analysis indicated that it was predominantly localized in euchromatic areas with low DNA density. The largest RB positive grains lined up on the heterochromatin/euchromatin boundary. During mitosis, the RB protein dissociated from the condensing chromosomes. It was dispersed throughout the cytoplasm during metaphase and anaphase, and it reassociated with the decondensing chromatin during telophase. A new monoclonal antibody, designated aRB1C1, was raised against a bacterial TrpE/human retinoblastoma protein. It specifically recognized the nonphosphorylated and differentially phosphorylated forms of the RB protein in immunoprecipitation experiments. A collection of RB expressing cell lines gave a positive staining reaction with the antibody, whereas the RB negative Weri-RB-27 retinoblastoma and OHS osteosarcoma cells failed to react. Wild-type RB complementary DNA was introduced into Weri-RB-27 by retrovirus mediated gene transfer. Similar experiments were performed with the DU145 prostatic carcinoma cell line that expresses a mutant RB protein. Reconstituted cells of both lines expressed the normal size RB protein and gave a positive immunofluorescence reaction with the aRB1C1 and other anti-RB antibodies. The new monoclonal antibody, however, showed cell type dependent differences of the staining pattern compared to other anti-RB antibodies, suggesting differentiation dependent accessibility to its epitope.     

Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils.

Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) induces tyrosine phosphorylation of several polypeptides, including a prominent band of approximately 41 kDa. A polypeptide of identical electrophoretic mobility was recognized by a monoclonal antibody raised against a sequence corresponding to amino acids 325-345 of ERK-1, one of a family of mitogen-activated protein (MAP) kinases. To establish the possible identity of these polypeptides, extracts from control and fMLP-treated cells were immunoprecipitated with immobilized antiphosphotyrosine antibodies. Reactivity with anti-ERK-1 antibodies was observed only in the precipitate of chemoattractant-stimulated cells. These data imply that a MAP kinase constitutes at least part of the tyrosine-phosphorylated 41-kDa polypeptide. By using an in vitro renaturation assay, treatment of intact cells with fMLP was found to stimulate several protein kinases, including one of approximately 41 kDa. Renaturation of samples immunoprecipitated with antiphosphotyrosine antibodies revealed the presence of an active protein kinase in chemoattractant-stimulated, but not in control cells. The immunoprecipitated kinase comigrated with the 41-kDa tyrosine phosphorylated polypeptide and the anti-ERK-1 reactive band. We conclude that a MAP kinase closely related or identical to ERK-1 is tyrosine phosphorylated and activated when human neutrophils are stimulated by chemotactic peptides. The rapid phosphorylation of this kinase, which is apparent within seconds, is compatible with a role in the activation of the respiratory burst and/or other neutrophil responses.     

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.     

The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses.

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3.Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.     

The proteoglycan decorin is synthesized and secreted by differentiated myotubes

Proteoglycans (PGs) are important components of the skeletal muscle extracellular matrix (ECM). Skeletal muscles are composed of muscle fibers and mononucleated cells. The latter are known to synthesize and secrete several PGs. Rat skeletal muscle ECM contains a chondrotin/dermatan sulfate PG which was immunoprecipitated by antibodies against rat decorin. The synthesis and secretion of PGs by a mouse cell line was analyzed during in vitro differentiation. PGs were characterized by biochemical and immunological techniques including immunocytolocalization experiments. At least three different PGs are synthesized and secreted by differentiated myotubes: a 220 to 460 kDa heparan sulfate, a 250 to 310 kDa chondroitin/dermatan sulfate, and a 75 to 130 kDa chondroitin/dermatan sulfate. This latter PG was specifically immunoprecipitated with antibodies against rat fibroblast decorin. Indirect immunocytolocalization analysis revealed that decorin was localized inside the cells, with a strong reaction around the nuclei. During differentiation the relative proportions of some PGs changed. Thus, a decrease in the relative proportion of the heparan sulfate PG was observed, whereas a significant increase in the relative proportion of decorin was detected. No change in the large chondroitin/dermatan PG was seen during the differentiation process. The possible cell sources of decorin found in rat skeletal muscle ECM are discussed.     

Patterns of dystrophin expression in developing, adult and regenerating tail skeletal muscle of Amphibian urodeles.

The patterns of expression of dystrophin were investigated by indirect immunofluorescence and by immunoblotting in developing, adult and regenerating tail skeletal muscle of newts Pleurodeles waltl and Notophthalmus viridescens. In this study, a monoclonal antibody H-5A3 directed against the C-terminal region (residues 3357-3660) and a polyclonal antibody raised to the central domain (residues 1173-1738) of the chicken skeletal muscle dystrophin were used. Western blot analysis showed that these antibodies recognized a 400 kDa band of dystrophin (and may be of dystrophin-related protein) in the adult muscle tissues and in newt tail regenerates. During skeletal muscle differentiation or epimorphic regeneration (blastema), anti-dystrophin immunoreactivity gradually accumulated over the periphery of the myofibers. Dystrophin and laminin were first and concomitantly observed at the ends of the newly formed myotubes where they were anchored on connective tissue septa or bone processes by dystrophin-rich myotendinous structures. It is noteworthy that neuromuscular junctions, which most probably also contain dystrophin, are established in urodeles near the ends of the myofibers as shown by histochemical localization of AChE activity or fluorescent bungarotoxin detection of AChRs. In the stump transition zone close to the tail amputation level where tissue regeneration of injured muscle fibers took place, dystrophin staining located on the cytoplasmic surface of myofibers progressively disappeared during the dedifferentiation process which seemed to occur during muscle regeneration as suggested by electron microscopy. Furthermore, double labeling experiments using anti-dystrophin and anti-laminin antibodies showed a good correlation between the remodeling processes of the muscle fiber basal lamina and the loss of dystrophin along the sarcolemma of damaged and presumably dedifferentiating muscle cells.     

Localization of a 215-kDa tyrosine-phosphorylated protein that cross-reacts with tensin antibodies.

Tyrosine phosphorylation of cytoskeletal proteins at adhesive junctions has been speculated to play a role in the regulation of cell signaling at these sites. Previously, monoclonal antibodies were generated against phosphotyrosine-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts, resulting in two antibodies which recognized antigens of 76 and 215 kDa that localized to focal contacts. We have now localized the 215-kDa antigen to a number of adhesive junctions in vivo, including the zonula adherens, intercalated discs, and myotendinous and neuromuscular junctions. In sections of skeletal muscle and in isolated myofibrils, the 215-kDa protein was localized to the I-band. By immunoprecipitation and immunoblot analysis, we determined that the 215-kDa antigen cross-reacts with a polyclonal anti-tensin antibody.     

Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24D2 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24D2 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24D2 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24D2 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells.     

Characterization of the Target Antigens of Phytomonas-specific Monoclonal Antibodies

ABSTRACT. Seven Phytomonas -specific monoclonal antibodies produced against Phytomonas serpens and Phytomonas françai were further characterised in order to identify and localise their target antigens. Four monoclonal antibodies recognized carbohydrate surface epitopes, in three of the cases associated with surface glycoproteins with apparent molecular weight of 80 kDa. One monoclonal antibody apparently bound to a surface/internal protein epitope, whereas the two others recognized intra-cellular proteins. The cell surface epitopes recognized by monoclonal antibodies were detected specifically in the genus Phytomonas. These epitopes, which are detected in culture, plant and insect forms, may be useful as targets for Phytomonas identification.