The requirement for proteinase activity for human lymphocyte-mediated natural cytotoxicity (NK): evidence that the proteinase is serine dependent and has aromatic amino acid specificity of cleavage

We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.     

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.     

Validation of affinity reagents using antigen microarrays

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.     

Redox-based regulation of signal transduction: principles, pitfalls, and promises

Oxidants are produced as a by-product of aerobic metabolism, and organisms ranging from prokaryotes to mammals have evolved with an elaborate and redundant complement of antioxidant defenses to confer protection against oxidative insults. Compelling data now exist demonstrating that oxidants are used in physiological settings as signaling molecules with important regulatory functions controlling cell division, migration, contraction, and mediator production. These physiological functions are carried out in an exquisitely regulated and compartmentalized manner by mild oxidants, through subtle oxidative events that involve targeted amino acids in proteins. The precise understanding of the physiological relevance of redox signal transduction has been hampered by the lack of specificity of reagents and the need for chemical derivatization to visualize reversible oxidations. In addition, it is difficult to measure these subtle oxidation events in vivo. This article reviews some of the recent findings that illuminate the significance of redox signaling and exciting future perspectives. We also attempt to highlight some of the current pitfalls and the approaches needed to advance this important area of biochemical and biomedical research.     

Sequence-specific endonuclease Bam HI. Effect of hydrophobic reagents on sequence recognition and catalysis

The specificity of cleavage of Bam HI is altered in the presence of hydrophobic reagents, such as glycerol and M2SO. The enzyme with altered specificity, designated Bam HI.1, generated digestion patterns of various DNAs, which were distinct from those generated by Bam HI. Cleavage sites recognized in phiX174 RF DNA in the presence of these hydrophobic reagents are not related to the Bam HI palindrome. Bam HI.1 appears to be an endogenous form of Bam HI that can be expressed by altering the hydrophobicity of the reaction.     

A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies

Proteomic research includes the characterization of protein mixtures in order to understand complex biological systems and determine relationships between proteins, their function, and protein-protein interactions. Often the goal of such research is to monitor changes of proteins in perturbed systems, a type of study referred to as differential expression analysis. To perform these studies requires the ability to execute some type of differential comparison of a given protein state in reference to some type of a control. The iTRAQ reagents are a set of isobaric reagents which are amine specific and allow for the identification and quantitation of up to four different samples simultaneously. The amine specificity of these reagents makes most peptides in a sample amenable to this labeling strategy with no loss of information from samples involving post-translational modifications, such as the scrutiny of signal transduction pathways that often involve phosphorylation phenomena. In addition, the multiplexing capacity of these reagents allows for information replication within certain LC-MS/MS experimental regimes, providing additional statistical validation within any given experiment. The results presented herein demonstrate a few examples of the wide variety of quantitative information that can be realized when undertaking such experimental approaches. These include temporal analysis of drug-induced-protein expression, discovery and elucidation of disease markers, and protein-protein interactions in multi-protein complexes.     

An unbiased genome-wide analysis of zinc-finger nuclease specificity

Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.     

Structural determinants of sphingolipid recognition by commercially available anti-ceramide antibodies

The sphingolipid mediator ceramide is involved in cellular processes such as apoptosis, differentiation, responses to cytokines, and stress responses. Experimental evidence suggests that the intracellular location of ceramide may be a key factor in determining its ultimate cellular effects. One approach to ceramide localization is the use of recently developed anti-ceramide antibodies for immunocytochemical studies. Two such commercial preparations are now available; we sought to compare and contrast their specificity for ceramide and/or other cellular lipids. By using lipid overlay assays and a diverse panel of sphingolipids, we were able to delineate the specificity and thus, the utility of these reagents. Our results indicate that one of these anti-ceramide preparations is quite specific for ceramide and dihydroceramide, whereas the other preparation recognizes dihydroceramide, phosphatidylcholine, and sphingomyelin. Furthermore, through the use of chemically modified ceramides in similar assays, we were able to determine some structural determinants of lipid recognition by both of these reagents.     

The synthesis and properties of peptidylmethylsulphonium salts with two cationic residues as potential inhibitors of prohormone processing.

Peptidylmethylsulphonium salts incorporating consecutive basic residues at the C-terminus of the peptidyl portion such as -Arg-Arg-, -Arg-Lys-, -Lys-Lys- and -Lys-Arg- were synthesized and examined as proteinase inhibitors. Serine proteinases with a specificity directed towards hydrolysis at cationic residues were found to be unaffected by these derivatives. On the other hand, cysteine proteinases, cathepsin B and, in particular, clostripain were readily inactivated by affinity labelling. The reagents thus are of promise for the study of prohormone processing promoted by cysteine proteinases.     

Anti-idiotypic regulation of an insulin-reactive T cell clone

A series of MHC-restricted, bovine-insulin-(BI) reactive T cell clones were generated. The specificity of one group was shown to be for an insulin A-chain loop determinant; the other group apparently demonstrated specificity of a B-chain determinant and/or amino acid residue A4. Guinea pig anti-idiotypic antisera were prepared against two idiotypically related BI monoclonal antibodies of similar A-chain loop specificity. These reagents were able to modulate the antigen-specific proliferation of an insulin-reactive, A-chain loop-specific T cell clone. Because the monoclonal antibodies and the T cell clone recognize a similar molecular domain of the insulin molecule, these data suggest that the anti-idiotypic sera mimic an insulin-like determinant, perhaps by bearing an "internal image" of the antigen and thereby interfering with T cell antigen recognition. Further, these results suggest that such reagents may be useful in characterization of T cell antigen receptor specificity and lend further credence to the concept of idiotypic-anti-idiotypic regulation of the immune response.     

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.     

Specificity of Rho insert-mediated activation of phospholipase D1

Members of the Rho subfamily of GTP-binding proteins regulate phospholipase D1 (PLD1) activity and signaling. In previous work, we demonstrated that binding of the Rho family member Cdc42 to PLD1 and the subsequent stimulation of its enzymatic activity are distinct events. Deletion of the insert helix from Cdc42 does not interfere with its switch I-mediated, GTP-dependent binding to PLD1 but inhibits Cdc42-stimulated PLD1 activity. To understand the mechanism of the insert-mediated activation of PLD1 by Cdc42 and to develop reagents to study Cdc42-activated PLD1 in cellular signaling events, we have undertaken a mutational analysis of the Rho insert region of Cdc42 and examined the specificity of the insert helix requirement in the other Rho family members, RhoA and Rac1. Here, we identify a critical residue, serine 124, in the Cdc42 insert helix central to its activation mechanism. Further, we examine this activation mechanism with respect to other members of the Rho family and demonstrate that each Rho protein activates PLD by distinct mechanisms, potentially allowing for unique signaling outcomes in the cell.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles

Many host cell surface proteins, including viral receptors, are incorporated into enveloped viruses. To address the functional significance of these host proteins, murine leukemia viruses containing the cellular receptors for Rous sarcoma virus (Tva) or ecotropic murine leukemia virus (MCAT-1) were produced. These receptor-pseudotyped viruses efficiently infect cells expressing the cognate viral envelope glycoproteins, with titers of up to 10⁵ infectious units per milliliter for the Tva pseudotypes. Receptor and viral glycoprotein specificity and functional requirements are maintained, suggesting that receptor pseudotype infection recapitulates events of normal viral entry. The ability of the Tva and MCAT-1 pseudotypes to infect cells efficiently suggests that, in contrast to human immunodeficiency virus type 1 entry, neither of these retroviral receptors requires a coreceptor for membrane fusion. In addition, the ability of receptor pseudotypes to target infected cells suggests that they may be useful therapeutic reagents for directing infection of viral vectors. Receptor-pseudotyped viruses may be useful for identifying new viral receptors or for defining functional requirements of known receptors. Moreover, this work suggests that the production of receptor pseudotypes in vivo could provide a mechanism for expanded viral tropism with potential effects on the pathogenesis and evolution of the virus.     

Use of coagglutination for serotyping Shigella dysenteriae and Shigella boydii

The coagglutination test was used to identify Shigella boydii and Shigella dysenteriae. A trial was carried out with 13 native rabbit antisera to S. boydii and 10 antisera to S. dysenteriae, as well as with coagglutinating reagents prepared from these antisera. The use of coagglutinating reagents was shown to ensure the complete specificity of the results, to prevent the adsorption of diagnostic antisera and to decrease their consumption 50 times. The importance of the coagglutination test for the identification of shigellae is discussed.     

Detection of intracellular signal transduction molecules in PBMC from rhesus macaques and sooty mangabeys

Abstract: One of the manifestations of human HIV-1 and nonhuman primate SIV infection that lead to disease is reasoned to be secondary to generalized T-cell dysfunction. The molecular mechanisms associated with the T-cell dysfunction remain to be elucidated. To address this issue, we sought to utilize the nonhuman primate model to study intracellular signaling events in cells from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Because relatively little is known about these events in nonhuman primates, our laboratory defined optimal conditions, reagents, and assays for the study of signal transduction events in cells from nonhuman primates. The protein phosphorylation patterns in the two monkeys exhibited quantitative, qualitative, and kinetic differences. Antibodies to Stat6 detected a unique band in macaque cell lysates. This band is markedly decreased human cell lysates and never seen in mangabey cell lysates. Detection of various other intracellular signaling proteins is also described.     

IgD allotypic determinants. I. Determinants expressed on murine IgD of the a allotype

Allotypes of membrane IgD of 27 strains of mice were determined with a series of anti-delta reagents, each capable of binding to the IgD of BALB/c spleen cells. One of these reagents is a newly defined allospecific rabbit anti-mouse-delta a. Typing with these reagents reveals the strain distribution of the previously described IgD.36 (Ig5.1) and IgD.43 (Ig5.4) specificities and demonstrates the existence of a new specificity, IgD.45 (Ig5.5). The results confirm the previous subdivision of the Igh-Ce haplotype into Igh-Ce, to which A mice are assigned, and Igh-Cn, to which NZB and NZW mice are assigned. In addition, it is shown that the Igh-Cd haplotype must also be subdivided. AKR mice, which express the a allele at the Igh-5(delta) locus, are designated as possessing the Igh-Cd haplotype while AL/N and C.AL20 mice, which have the e allele at the Igh-5(delta) locus, are assigned a new Igh-C haplotype designation, o.     

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.     

Chlamydomonas reinhardtii chloroplasts as protein factories

Protein-based therapeutics are the fastest growing sector of drug development, mainly because of the high sensitivity and specificity of these molecules. Their high specificity leads to few side effects and excellent success rates in drug development. However, the inherent complexity of these molecules restricts their synthesis to living cells, making recombinant proteins expensive to produce. In addition to therapeutic uses, recombinant proteins also have a variety of industrial applications and are important research reagents. Eukaryotic algae offer the potential to produce high yields of recombinant proteins more rapidly and at much lower cost than traditional cell culture. Additionally, transgenic algae can be grown in complete containment, reducing any risk of environmental contamination. This system might also be used for the oral delivery of therapeutic proteins, as green algae are edible and do not contain endotoxins or human viral or prion contaminants.