Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin.

Nineteen monoclonal antibodies which bind to native dystrophin in the plasma membrane of frozen muscle sections were obtained using a recombinant fusion protein as immunogen. On Western blots of normal mouse muscle extracts, the antibodies bind specifically to a 400,000 Mr protein which is absent from dystrophic mouse (mdx) muscle. At least four distinct epitopes have been identified by cleavage mapping methods. Although the fusion protein contained 25% of the human dystrophin sequence (Cys816-Asp1747; Mr 108,000), most of the monoclonal antibodies (15 out of 19) recognize a single fragment of Mr 27,500.     

Construction of dystrophin fusion proteins to raise targeted antibodies to different epitopes.

For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of antibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749-2248 or the nucleotide sequence 5456-6953 of the mRNA, which corresponds to the major deletion-prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.     

Purification of fusion proteins expressed by pEX3 and a truncated pEX3 derivative

A derivative of the pEX3 expression vector was constructed that codes for the first 407 amino acids of the 1051 amino acids of the pEX3 fusion protein. The amount of truncated fusion protein (40 mg/g cells), obtained by expression in E. coli, was similar to that produced by the original pEX3 vector. The truncated fusion protein was purified more easily from E. coli contaminants than the original fusion protein by washing with 2 M urea and 0.5% Triton X-100.     

A truncated dystrophin lacking the C-terminal domains is localized at the muscle membrane.

A Duchenne muscular dystrophy patient who displayed near-normal dystrophin staining at the sarcolemma with N-terminal, but not with C-terminal, anti-dystrophin monoclonal antibodies was found to have a frameshift deletion of exons 42 and 43. This deletion introduces an early termination codon, and a 225-kD protein was detected by western blotting with N-terminal antibodies only. The results suggest that an N-terminal truncated dystrophin fragment encoded by exon 1-41 is able to associate with the muscle cell membrane. The current idea that the C-terminal domains of dystrophin are important or essential for its integration with the sarcolemma may have to be reexamined in the light of these observations.     

Agrobacterium rhizogenes lacZ-rolC gene expression in Escherichia coli: detection of the product in transgenic plants using RolC-specific antibodies

The rolC sequence of the Agrobacterium rhizogenes Ri plasmid was fused in-frame to the 3' end of the lacZ gene in plasmid pEX3. The fusion protein RolC-beta-galactosidase was accumulated as insoluble inclusion bodies in Escherichia coli. Antibodies were raised in rabbits against the fusion protein. After affinity purification, RolC-specific antibodies were found to react with a 22-kDa polypeptide prepared from roots of transgenic tobacco plants possessing a rolC gene. The result of differential centrifugation suggested that RolC is present in the soluble fraction of transformed cells.     

Epitope mapping of Escherichia coli cell division protein FtsZ with monoclonal antibodies.

A fusion between lacZ and ftsZ of Escherichia coli was constructed to obtain a beta-galactosidase-FtsZ fusion protein. This fusion protein was used to raise antibodies against cell division protein FtsZ. Six monoclonal antibodies were obtained, and they reacted with FtsZ from cytoplasm and membrane fractions. The epitopes in FtsZ were localized by studying the reactions of the monoclonal antibodies with fusion proteins truncated at the carboxy terminus and with fragments that were obtained by CNBr cleavage of purified FtsZ. Five different epitopes were defined. Epitopes I and III reacted with the same monoclonal antibody, without showing apparent amino acid homology. Epitope II was defined by monoclonal antibodies that cross-reacted with an unknown cytoplasmic 50-kDa protein not related to FtsZ. Epitopes IV and V were recognized by different monoclonal antibodies. All monoclonal antibodies reacted strongly under native conditions, so it is likely that the five epitopes are situated on the surface of native FtsZ. By using these data and computer analysis, a provisional model of FtsZ is proposed. The FtsZ protein is considered to be globular, with a hydrophobic pocket containing GTP-binding elements. Epitopes I and II are situated on each side of the hydrophobic pocket. Because the carboxy terminus contains epitope V, the carboxy terminus of FtsZ is likely oriented toward the protein's surface.     

伯克霍尔德氏菌(Burkholderia sp.)2-萘酸单加氧酶基因(nmo)的克隆及表达

通过酶切连接将Burkholderia sp．JTl500的一段DNA片段(4．8kb)亚克隆到表达载体pUC18上,得到重组子pEKl23。测序后的pEKl23重组子4．8kb插入片段的序列已经登陆欧洲EMBL基因库,序列接受号为AJ566333。对这一DNA片段的序列分析显示,此DNA片段含有3个阅读框,且在这3个阅读框5’端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3,其阅读框长度为1158bp,编码386个氨基酸,与已报道的Ralstonia eutropha HF39羟化酶(单加氧酶,bec)氨基酸序列有64％的同源性。pEKl23对2-萘酸代谢途径中4个关键底物的转化实验结果显示,其基因产物仅对2-萘酸发生加氧转化反应,而且2-萘酸浓度有明显的降低,证实此基因是2-萘酸单加氧酶基因(nmo)。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中。SDS-PAGE结果表明,pXK3、pEKl23两重组子中2-萘酸单加氧酶表达量并没明显区别,但加氧酶酶活却存在显著的差别。推测在启动子后,单加氧酶阅读框前的两个阅读框的基因产物,对单加氧酶活有促进作用。     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Selection of antibody fragments specific for an alpha-helix region of acylphosphatase

The native state of common-type acylphosphatase (AcP) elicits two alpha-helices spanning residues 22-32 and 55-67 in the protein sequence. A peptide corresponding to the second alpha-helix (helix-2) of the protein was used to select phage antibodies consisting of a single chain fragment variable. The selection was performed in the presence of trifluoroethanol, a cosolvent known to induce the formation of helical structure in peptides and proteins. Phage scFv antibodies capable of binding the peptide specifically in a trifluoroethanol-induced alpha-helical conformation were isolated by affinity selection (biopanning). Some of these scFvs were also able to bind the native protein but not the peptide in a non-helical unstructured state. This indicates that the structural determinant recognized by the selected antibodies is the alpha-helical conformation of this specific region, rather than simply its amino acid sequence. This study shows that phage display libraries can be used to raise antibodies one can use as reagents able to target regions of a protein with a specific native-like secondary structure.     

Characterization of a cleavage product in the human choriogonadotropin beta-subunit

The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the alpha or beta-subunit. Under reducing conditions, immunoblotting with antibodies directed to the beta-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111-145 region. Using microsequencing techniques, we found that the fragment had a NH2 terminal portion corresponding to the sequence of the beta-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48-145 portion of the beta-subunit and is probably a cleavage product of the native protein with intrachain nicking.     

Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding.

We have expressed in mammalian cells a fragment (residues 1-302) of the alpha chain of platelet glycoprotein (GP) Ib containing the von Willebrand factor- (vWF) binding site. The secreted soluble protein had an apparent molecular mass of 45 kDa and reacted with conformation-dependent monoclonal antibodies that bind only to native GP Ib, thus demonstrating its proper folding. After insolubilization on nitrocellulose membrane, the recombinant GP Ib alpha fragment bound soluble vWF in the presence of ristocetin or botrocetin with a dissociation constant similar to that exhibited by GP Ib.IX complex on platelets. Moreover, the interaction was blocked by anti-GP Ib monoclonal antibodies known to inhibit vWF binding to platelets. The sequence of GP Ib alpha between residues 269-287 has a strong net negative charge due to the presence of 10 glutamic or aspartic acid residues; 5 of these are contained in the sequence of a synthetic peptide (residues 251-279) previously shown to inhibit vWF-platelet interaction. In order to evaluate the possible functional role of these acidic residues, we employed site-directed mutagenesis to express two mutant GP Ib alpha fragments containing asparagine or glutamine instead of aspartic or glutamic acid, respectively. Mutant 1, with substitutions between residues 251-279, failed to bind vWF whether in the presence of ristocetin or botrocetin; in contrast, vWF binding to Mutant 2, with substitutions between residues 280-302, was nearly normal in the presence of ristocetin, but markedly decreased in the presence of botrocetin. Thus, mammalian cells transfected with a truncated cDNA sequence encoding the amino-terminal domain of GP Ib alpha synthesize a fully functional vWF-binding site; acidic residues in the sequence 252-287 are essential for normal function.     

Phosphorylation of threonine residues on cloned fragments of the Dictyostelium myosin heavy chain

A tail fragment of Dictyostelium discoideum myosin has been cloned and expressed as a fusion protein with the N-terminal region of MS-2 polymerase. The cloned fragment was phosphorylated with myosin heavy chain kinase II from aggregation-competent D. discoideum cells that specifically phosphorylate threonine residues on the myosin tail. Phosphopeptide maps showed the same site specificity of phosphorylation with the fusion protein as a substrate as with native myosin. An improved assay for the kinase was developed in which the fusion protein is precipitated with a monoclonal antibody that inhibits polymerization of the myosin tails without preventing their phosphorylation. Sites of phosphorylation were tentatively localized to a sequence in the C-terminal region of the heavy chain where four threonine residues are found.     

Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans.

A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum.     

The identification of epitopic sites in human alpha 1-proteinase inhibitor.

Human alpha 1-proteinase inhibitor (alpha 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized alpha 1-PI and mouse polyclonal antibodies against native alpha 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between alpha 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native alpha 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized alpha 1-PI as the antigen. The ability of the monoclonal antibodies to bind native alpha 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding alpha 1-PI and only fragment I. Antibodies in groups II and III bound alpha 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between alpha 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in alpha 1-PI. Specific monoclonal antibodies to three of these sites were obtained.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH

Membrane fusion of the flavivirus tick-borne encephalitis virus is triggered by the mildly acidic pH of the endosome and is mediated by envelope protein E, a class II viral fusion protein. The low-pH trigger induces an oligomeric rearrangement in which the subunits of the native E homodimers dissociate and the monomeric subunits then reassociate into homotrimers. Here we provide evidence that membrane binding is mediated by the intermediate monomeric form of E, generated by low-pH-induced dissociation of the dimer. Liposome coflotation experiments revealed that association with target membranes occurred only when liposomes were present at the time of acidification, whereas pretreating virions at low pH in the absence of membranes resulted in the loss of their ability to stably attach to liposomes. With the cleavable cross-linker ethylene glycolbis(succinimidylsuccinate), it was shown that a truncated soluble form of the E protein (sE) could bind to membranes only when the dimers were free to dissociate at low pH, and binding could be blocked by a monoclonal antibody that recognizes the fusion peptide, which is at the distal tip of the E monomer but is buried in the native dimer. Surprisingly, analysis of the membrane-associated sE proteins revealed that they had formed trimers. This was unexpected because this protein lacks a sequence element in the C-terminal stem-anchor region, which was shown to be essential for trimerization in the absence of a target membrane. It can therefore be concluded that the formation of a trimeric form of sE is facilitated by membrane binding. Its stability is apparently maintained by contacts between the ectodomains only and is not dependent on sequence elements in the stem-anchor region as previously assumed.     

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.     

Structural studies on human glutathione S-transferase pi. Family of native-specific monoclonal antibodies used to block catalysis.

The glutathione S-transferases are a family of related detoxification enzymes that have been shown to conjugate numerous electrophiles to the common cellular thiol glutathione. We have generated a panel of monoclonal antibodies against the human pi class isozyme of this enzyme, and, in this report, we characterize the binding of these antibodies to the glutathione S-transferase antigen. Of the 10 monoclonal antibodies that we have isolated, 7 are able to recognize the native form of the enzyme while the remaining 3 are only able to bind to glutathione S-transferase pi in assays that partially denature the antigen, such as an enzyme-linked immunosorbent assay or a Western blot. We synthesized seven partial protein fragments and asked whether the monoclonal antibodies could bind to these fragments in an immunoprecipitation reaction. The antibodies that can bind the native form of the enzyme all bind to the carboxyl-terminal domain of the protein. Two antibodies are able to inhibit the glutathione S-transferase-catalyzed reaction noncompetitively against glutathione. Incubation of a 10-fold molar excess of either antibody over enzyme can inhibit the reaction by 50%. We have also used the same protein fragments of glutathione S-transferase pi to show that amino acids 1-77 retain the capacity to bind glutathione in a glutathione-agarose binding assay.