Harry Towbin

Characterization of Histone H2A and H2B Variants and Their Post-translational Modifications by Mass Spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M. For all core histones, the modifications in the G1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G2/M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G2/M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Glycosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography

A method for detecting glycosphingolipids (GSL) in situ after thin layer chromatography is described. The separated GSL are transferred by diffusion to nitrocellulose. The replica is incubated with poly- or monoclonal antibodies and bound antibodies are detected with second antibodies coupled to peroxidase. Advantages of the procedure are its speed, the non-radioactive detection method, and its suitability for screening applications. In addition, small scale affinity purification of antibodies from the replicas is possible. The presence of Forssman antigen in mouse tissues and the reaction of monoclonal antibodies with human GSL is demonstrated.

Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin.

Protein phosphorylation represents a ubiquitous control mechanism in living cells. The structural prerequisites and consequences of this important post‐translational modification, however, are poorly understood. Oncoprotein 18/stathmin (Op18) is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Here we document that phosphorylation of Ser63, which is located within a helix initiation site in Op18, disrupts the transiently formed amphipathic helix. The phosphoryl group reduces tubulin binding 10‐fold and suppresses the MT polymerization inhibition activity of Op18s C‐terminal domain. Op18 represents an example where phosphorylation occurs within a regular secondary structural element. Together, our findings have implications for the prediction of phosphorylation sites and give insights into the molecular behavior of a globally disordered protein.

Sandwich immunoassay for the hapten angiotensin II a novel assay principle based on antibodies against immune complexes

Immunoassays for haptens such as short peptides or drugs are usually based on the principle of competition for a limited number of binding sites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 751-759) that bind to hapten-mAb complexes in a reaction where conformational changes on the primary antibody are important. Here, we report on monoclonal antibody pairs able to form ternary complexes with the octapeptide angiotensin II. The first mAb (mAb1) is conventional and binds angiotensin II with high affinity (Kd 10(-11) M). The secondary (anti-metatypic) mAbs (mAbs2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. An immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAbs2s were able to bind angiotensin II by themselves but all efficiently bound the complex of angiotensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 complex and one mAb2 distinctly improved the specificity of the assay for angiotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml angiotensin II have been established. The kinetics of the complex formation was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the liganded conformation of mAb1 was higher than towards the free mAb1. By contrast, the mAb2s dissociated at similar rates from complexed and uncomplexed mAb1.

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients

Post‐translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)‐immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS), and was designed to match those used in two‐dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14‐3‐3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow‐range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post‐translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin – which contains four phosphorylation sites – as an example, the effects on the pI‐shifts were well predicted. This sensitive and widely applicable IEF‐blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two‐dimensional electrophoresis.

Monoclonal antibodies in analysis of cathepsin G-digested proteolytic fragments of human plasma fibronectin

Proteolytic fragments of fibronectin, obtained by digestion with cathepsin G, were transferred electrophoretically from sodium dodecyl sulphate (NaDodSO4) polyacrylamide gels to nitrocellulose sheets and used as antigens for monoclonal antibodies. All 9 monoclonal antibodies tested reacted with undenatured intact fibronectin or its fragments applied directly to nitrocellulose sheets. Two of the clones did not react with the NaDodSO4-treated transferred material suggesting reactivity with conformational determinants. Distinct fragments of fibronectin could be detected by several of the antibodies. None of the monoclonal or the polyclonal antibodies used reacted with the Mr = 40,000 or Mr = 30,000 gelatin-binding fragments of fibronectin. However, one of the monoclonal antibodies reacted specifically with their precursor Mr = 64,000 fragment, but apparently with its gelatin-nonbinding segment. The apparent non-immunogenicity of the gelatin-binding domain is conspicuous, suggesting that it may be highly conserved in evolution. The present method, combination of controlled proteolytic cleavage with electrophoretic transfer, provides an effective means for characterization of monoclonal antibodies raised against proteins.

Chemiluminescent and Enzyme-Linked Immuno Assays for Sensitive Detection of Human IFN-γ

We have produced and characterized 4 mAbs to human IFN-gamma and established sensitive, non-radioactive immuno-assays. The first two assays use microtiter plates as the solid phase and enzymes or chemiluminescence (acridinium ester) for development. The use of chemiluminescence instead of peroxidase increased the sensitivity of the assay by a factor of about 75. The third and the fourth assays utilize polystyrene beads as the solid phase and enzymes or acridinium ester for development. The use of beads also increased the sensitivity of detection. The most sensitive IFN-gamma detection was achieved by the combination of bead with acridinium ester. In this configuration we were able to detect about 0.2 pg/ml IFN-gamma (1/250th of a unit). These chemiluminescent immunoassays (CLIA) appear to be more sensitive than existing ELISAs or radioimmunoassays and may find new application areas.

Quantitation of ineterferon-induced Mx protein in whole blood lysates by an immunochemiluminsescent assay: elimination of protease activity of cell lysates in toto

Due to the rapidly expanding usage of interferons and its costliness of therapy, it is important to evaluate the clinical efficacy of the various interferons. Directly assaying circulating interferon is technically quite difficult. Here, we present an alternate method to evaluate interferon therapy by assaying a unique protein, called Mx protein, which is a 78 kDa cytoplasmic protein selectively induced by type-1 interferon in human leukocytes. The current assay is a two-site chemiluminescent immunoassay, designed to detect Mx protein in whole blood lysates. Since the Mx protein once solubilized, is highly susceptible to proteolysis in whole blood lysates, we have devised a new procedure both to maximize its solubility and virtually eliminate its proteolytic degradation. A mouse monoclonal antibody conjugated to the derivatized-paramagnetic particles and an acridinium ester-labeled antibody serve as the solid phase capture and detector antibodies, respectively. This assay is applicable to both manual and automated modes with a detection limit of Mx protein at 20 ng/ml whole blood. Availability of a reliable assay for Mx protein should facilitate the clinical evaluation of many of the newly constructed type-1 interferons.