Physical and biological properties of an epsilon-heavy chain mRNA and a kappa-light chain mRNA coding for rat immunoglobulin E

The mRNA coding for epsilon-heavy chain and kappa-light chain have been highly enriched from a rat IgE-producing myeloma, IR-162. Based on denaturing gel analyses, the 20 S epsilon-heavy chain mRNA has an estimated molecular weight of 850,000, equivalent to about 2500 nucleotides. The 14S rat kappa-light chain mRNA has an estimated molecular weight of 410,000, equivalent to about 1200 nucleotides. Only about two-thirds of the length of these mature cytoplasmic rat mRNA code for protein. The 20 S mRNA stimulates the in vitro synthesis of a single major serologically related protein which is large enough to be epsilon-heavy chain. It is unglycosylated and has an apparent molecular weight of about 62,000. The in vivo unglycosylated epsilon-heavy chain, obtained in the presence of tunicamycin, has an apparent molecular weight of about 59,000, compared with about 76,000 for the glycosylated heavy chain of the secreted rat IgE. Therefore, the in vitro synthesized epsilon-heavy chain protein is about Mr = 3000 larger than the in vivo unglycosylated epsilon-heavy chain, equivalent to about 25 extra amino acids. This is consistent with the synthesis of an epsilon-heavy chain putative precursor. Likewise, the 14 S mRNA stimulates the in vitro synthesis of a single putative precursor protein, which is serologically related to kappa chain, is unglycosylated, and is about an extra 20 amino acids. This is the first report on the physical and biological properties of an epsilon-heavy chain mRNA, as well as any rat immunoglobulin mRNA.     

Glycosylation causes an apparent block in translation of immunoglobulin heavy chain

Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells.     

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

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

Protein side chain conformation predictions with an MMGBSA energy function

The prediction of protein side chain conformations from backbone coordinates is an important task in structural biology, with applications in structure prediction and protein design. It is a difficult problem due to its combinatorial nature. We study the performance of an “MMGBSA” energy function, implemented in our protein design program Proteus, which combines molecular mechanics terms, a Generalized Born and Surface Area (GBSA) solvent model, with approximations that make the model pairwise additive. Proteus is not a competitor to specialized side chain prediction programs due to its cost, but it allows protein design applications, where side chain prediction is an important step and MMGBSA an effective energy model. We predict the side chain conformations for 18 proteins. The side chains are first predicted individually, with the rest of the protein in its crystallographic conformation. Next, all side chains are predicted together. The contributions of individual energy terms are evaluated and various parameterizations are compared. We find that the GB and SA terms, with an appropriate choice of the dielectric constant and surface energy coefficients, are beneficial for single side chain predictions. For the prediction of all side chains, however, errors due to the pairwise additive approximation overcome the improvement brought by these terms. We also show the crucial contribution of side chain minimization to alleviate the rigid rotamer approximation. Even without GB and SA terms, we obtain accuracies comparable to SCWRL4, a specialized side chain prediction program. In particular, we obtain a better RMSD than SCWRL4 for core residues (at a higher cost), despite our simpler rotamer library. Proteins 2016; 84:803–819. © 2016 Wiley Periodicals, Inc.     

Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy

We have used immunoelectron microscopy to determine the location of an intermediate chain in the isolated outer arm dynein from Chlamydomonas flagella. When the purified alpha beta dimer of the outer arm was incubated with antibodies recognizing two distinct epitopes on its 69-kDa intermediate chain and then negatively stained and examined by electron microscopy, both antibodies appeared to have bound to the base of the Y-shaped stem that connects the two heads of the particle. These results indicate that this intermediate chain is located at the base of the stem. Inasmuch as this polypeptide is tightly associated with the 78-kDa intermediate chain and several light chains in an intermediate chain-light chain complex, it is likely that this entire assemblage is located at the base of the particle. Thus, these polypeptides are in a potentially important position with regard to the ATP-insensitive (structural end) binding of dynein to microtubules and to dynein-dynein interactions within the axoneme.     

Modification of the side chain of micromolide, an anti-tuberculosis natural product

This paper describes a series of modifications of the side chain of micromolide, an anti-tuberculosis natural product. Most of the synthesized compounds showed significantly decreased activities, which suggests that the long aliphatic side chain of micromolide and its double bond are essential to its activity.     

Localization of an actin binding domain in smooth muscle myosin light chain kinase

Phosphorylation of the regulatory light chain of myosin II by myosinlight chain kinase is important for regulating many contractile processes.Smooth muscle myosin light chain kinase has been shown to be associated withboth actin and myosin filaments in vitro and in vivo. In this report wedefine an actin binding region by using molecular deletions to generaterecombinant mutant proteins that were analyzed by co-sedimentation withF-actin. An actin binding region restricted to residues 2-42 in the animoterminus of the rabbit smooth muscle myosin light chain kinase wasidentified.     

Formation of chain structures with an anisotropic pairwise interaction between grains

Results are presented from the numerical study of the processes accompanying the formation of chain structures in systems with an anisotropic pairwise interaction similar to the interaction caused by ion focusing. The simulations were performed for extended and bounded chain structures in a wide range of parameters corresponding to conditions of experiments with laboratory dusty plasma. The development of various instabilities in such systems is analyzed in detail for the first time.     

The beta chain of chicken fibrinogen contains an atypical thrombin cleavage site

A cDNA corresponding to almost the entire coding region of the mRNA for the beta chain of chicken fibrinogen was sequenced. At the protein level, significant homology to the beta subunits of other vertebrate fibrinogens was found, with the highest degree of amino acid identity localized in the C-terminal region. In general, features conserved in the fibrinogens from other species also characterize the chicken sequence, including the cysteine motifs bordering an alpha-helical permissive region of fixed length and a single glycosylation site in the C-terminal region. However, the site of thrombin-catalyzed cleavage, which in other species consists of an Arg-Gly peptide bond, is instead an Arg-Ala bond in the chicken beta chain. The Ala was confirmed directly from a sequencing analysis of the purified beta chain of chicken fibrin. This finding may explain the observed slow clotting time of chicken fibrinogen relative to that of other species.     

Genetic control of the serum concentration of an H-2-antigen-like polypeptide chain

Mouse serum contains a protein complex consisting of at least three polypeptide chains. One of the chains with an approximate molecular weight of 40 000 is similar to the heavy chain of normal H-2 antigens. The serum concentration of this 40 000 dalton chain is under genetic control. Formal genetic analyses in B10.M and B10.S mice, their F₁ progeny and in backcrosses show that there are two codominantly expressed alleles at a single locus regulating the serum concentration. Measurements of the 40 000 dalton chain in recombinant mice suggest that the controlling locus is situated to the right of theS region.     

Purification and translation of an immunoglobulin lambda chain messenger RNA from mouse myeloma.

Here we describe the 500-fold purification of an mRNA encoding an immunoglobulin lambda light chain derived from the mouse myeloma tumor, RPC-20. Purification involves the isolation of membrane-bound polysomes, oligo(dT)-cellulose chromatography, and sucrose gradient centrifugation under conditions favoring denaturation of polynucleotide complexes. The mRNA purified in this way directs the cell-free synthesis of a polypeptide which is five or six amino acids longer than the mature form of RPC-20 light chain. In addition to directing the synthesis of a precursor-like polypeptide, the mRNA migrates on electrophoresis as a band containing approximately 1150 nucleotides, about 500 more than required to encode the mature form of the light chain.     

Expression and chain assembly of human laminin-332 in an insect cell-free translation system

Laminins are a family of large heterotrimeric glycoproteins comprising alpha, beta, and gamma chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the beta3-gamma2 heterodimer and the alpha3-beta3-gamma2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The alpha3-beta3 and the alpha3-gamma2 heterodimer were not detected, suggesting that the alpha3 chain can assemble with only beta3-gamma2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the beta-gamma heterodimer to the alpha-beta-gamma heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.     

Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.     

IgE immunotoxins. Effect of an IgE-ricin A chain conjugate on rat skin histamine content

Immunotoxins--toxins covalently conjugated to specific antibodies--have been studied as possible agents in the treatment of cancer. The avid binding of IgE antibodies to FcR on mast cells and basophils suggested the possible use of an IgE-immunotoxin in the treatment of malignant mastocytosis or as a method to generate mast cell-depleted animals for study. To this end, the effect of a covalent conjugate of rat myeloma IgE and ricin A chain on rat cutaneous mast cells was examined in vivo. IgE-ricin A chain was capable of binding to and sensitizing cutaneous mast cells in vivo as indicated by a bluing response to intracutaneous anti-ricin A chain. IgE-ricin A chain, given either as a single dose or, even more effectively, as two split doses, significantly reduced cutaneous histamine content for 6 to 8 days. Neither a mixture of IgE and ricin A chain that were not conjugated nor the induction of cutaneous mast cell degranulation with anti-IgE affected cutaneous histamine levels. Therefore, IgE-ricin A chain produces a prolonged depletion of cutaneous histamine levels.     

In situ evidence of an alternative oxidase and an uncoupling protein in the respiratory chain of Aspergillus fumigatus

Aspergillus fumigatus is an unusual pathogen in immunocompetent individuals; its incidence has increased in the last decades in patients immunocompromised, like those with chronic granulomatosis disease and AIDS. The aim of this study was to identify differences between the respiratory chain of host and the fungus planning to use the later as a pharmacological target. We evaluated respiration, membrane potential and oxidative phosphorylation of mitochondria of the spheroplasts of A. fumigatus in situ, after permeabilization with digitonin. Firstly, a functional respiratory chain (complex I-V) was demonstrated: adenosine 5'-diphosphate (ADP) induced an oligomycin-sensitive transition from resting to phophorylating respiration in the presence of the oxidizable substrates malate, glutamate, alpha-ketoglutarate, pyruvate, dihydroorotate, succinate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and exogenous NADH. In addition, the ability of the fungus to oxidize exogenous NADH, as well as the insensitivity of its respiration to rotenone, in association with the sensitivity to flavone, indicate the presence of an alternative NADH-ubiquinone oxidoreductase; the partial sensitivity of respiration to antimycin A and cyanide, in association with the sensitivity to benzohydroxamic acid, indicates the presence of an alternative oxidase. The fatty acid-uncoupled respiration was partly reversed by bovine serum albumin (BSA) and guanosine 5'-triphosphate (GTP) and was insensitive to either carboxyatractyloside or ADP. These results, together with evidences obtained using antibodies raised against uncoupling protein (UCP) from potato, indicate in addition, the presence of an uncoupling protein in the respiratory chain of A. fumigatus.     

Assembly and transport properties of invariant chain trimers and HLA-DR-invariant chain complexes.

The MHC class II-associated invariant chain behaves as a resident endoplasmic reticulum protein in the absence of class II molecules. In humans, two predominant forms exist; one, p35, differs from the other, p33, by an N-terminal cytoplasmic extension of 16 amino acids that contains a strong endoplasmic reticulum-retention signal. Here we show that one mechanism for retention of p33 is its association with p35 in mixed invariant chain trimers. However, even for p33 homotrimers transport from the endoplasmic reticulum is inefficient. In an MHC class II-positive B cell line, the formation of invariant chain trimers is rapid and is the first intermediate in the assembly of a nine-chain alpha beta-invariant chain complex. With time, three higher molecular weight complexes are progressively formed. These correspond to an invariant chain trimer with one alpha beta dimer, two alpha beta dimers, and three alpha beta dimers, respectively. No free alpha beta dimers are detectable early in biosynthesis. However, beginning at 2 h of chase, alpha beta dimers begin to appear concomitant with the disappearance of the completely assembled alpha beta-invariant chain complex. This conversion is virtually complete by 4 h, and presumably reflects the proteolytic degradation of the invariant chain component of the alpha beta-invariant chain complex and the generation of endosomal alpha beta dimers capable of binding antigenic peptides.     

Altering substrate chain length specificity of an acylhomoserine lactone synthase in bacterial communication

Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the "right" acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes M127T and F69L as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language.     

Properties of myosin light chain kinase prepared from rabbit skeletal muscle by an improved method

Myosin light chain kinase was prepared from rabbit skeletal muscle. DEAE-Sephadex, calmodulin-Sepharose 4B affinity gel and Ultrogel AcA 34 were used for the purification. It took 3 days for the preparation, and 6.2 mg of myosin light chain kinase was isolated from 600 g of frozen muscle. The molecular weight of the myosin light chain kinase estimated by sedimentation equilibrium analysis was 103,000 +/- 4,100. The isoelectric point was 5.0. Chemical modification of cysteine residues did not affect the catalytic activity, but modification of tyrosine residues diminished the activity. In order to activate myosin light chain kinase, it was necessary to bind calmodulin in an equimolar ratio and the dissociation constant was estimated to be 3.6 nM. The optimum pH for the catalytic activity was 7.5, and the activity was inhibited by NaCl and KCl. In the presence of 2.74 mg/ml myosin light chain and 75 mM KCl, the catalytic activity was found to be 88 s-1. The Vm and Km at 0.14 M KCl were 100 s-1 and 53 microM, respectively, for the isolated light chain as substrate and 70-80 s-1 and 19 microM for myosin as substrate.