Analysis of human lymphocyte protein expression. I. Identification of subpopulation markers by two-dimensional polyacrylamide gel electrophoresis

This study provides new knowledge on the changes in protein expression that differentiate the functionally and phenotypically different cells of the human immune system. Purification by flow cytometry of normal lymphocytes (both T and B cells), monocytes and granulocytes, combined with high-resolution two-dimensional polyacrylamide gel electrophoresis, revealed reproducible qualitative and quantitative changes between these cell populations. Characteristic profiles of marker proteins for each cell type were identified. Determination of markers for T lymphocyte subpopulations was achieved by the comparative analysis of normal T cells separated on the basis of CD4 and CD8 expression in combination with the analysis of cells from patients with T cell chronic lymphocyte leukemia. These results suggest that the modulation or regulation of proteins is very strictly controlled in lymphoid differentiation, and that several quantitative and a few qualitative differences can give rise to completely different phenotypes. Thus, instead of detecting numerous random differences among lymphocyte protein patterns, rather stringent regulation of protein expression in each subpopulation was found.     

Examination of membrane protein expression in Paracoccus denitrificans by two-dimensional gel electrophoresis

The well-known metabolic versatility of the soil bacterium Paracoccus denitrificans poses a challenge for modern proteomic approaches. We describe here improved preparation conditions that allow good separation and quantitative analyses of hundreds of membrane or periplasmic proteins. To illustrate this optimized procedure, the results of a screening for membrane proteins associated predominantly with aerobic or anaerobic (denitrifying) modes of growth are presented.     

Characterization of the B cell-specific adaptor SLP-65 and other protein tyrosine kinase substrates by two-dimensional gel electrophoresis.

The identification of substrates for protein tyrosine kinases in B cells is a critical step to a better understanding of the molecular mechanism(s) of lymphocyte activation through the antigen receptor. The substrate proteins were immunopurified from stimulated B cells and separated by two-dimensional gel electrophoresis techniques using either the isoelectric focussing (IEF)/SDS-PAGE or the non-equilibrium PH gradient electrophoresis (NEPHGE)/SDS-PAGE method. The biochemical characteristics of the proteins (isoelectric point and relative molecular mass) obtained and the subsequent use of antibodies that are specific for different cellular proteins confirmed the participation of HS1, Vav, Ig-alpha, Lyn and Btk in antigen receptor-mediated signal transduction. The heat shock cognate protein HSC70 was identified as a novel substrate protein in activated B cells. An important signaling function has previously been suggested for a 65-kDa protein (p65), whose phosphorylation can be detected before that of other substrate proteins. The analysis identified p65 as a so far unknown protein. Based on p65 peptide sequences, the full length cDNA was isolated and found to encode a B cell-specific adaptor protein, called SLP-65.     

Identification by two-dimensional gel electrophoresis of a 58-kilodalton tumor necrosis factor-inducing protein of Mycobacterium tuberculosis.

We have previously identified proteins in fractions of culture filtrate of Mycobacterium tuberculosis with the capacity to induce cytokine production in monocytes, by using a technique we defined as "monocyte Western blotting" (immunoblotting). In this series of experiments, we have extended this technique to two-dimensional gel electrophoresis and have identified a novel 58-kDa protein of M. tuberculosis which induces production of tumor necrosis factor by human monocytes. Nitrocellulose particles bearing this protein were used to develop murine monoclonal antibodies by the technique of intrasplenic immunization. The protein was purified by preparative isoelectric focusing and gel electrophoresis and subjected to N-terminal amino acid sequence analysis. As tumor necrosis factor is a mediator of both pulmonary necrosis and macrophage activation for intracellular killing, this 58-kDa protein may play an important role both in the immunopathogenesis of tuberculosis and in mycobacterial immunity.     

Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis.

Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate.     

Inhibition of cellular protein synthesis by vaccinia virus surface tubules

The infection of HEp-2 cells with vaccinia virus results in a rapid and selective inhibition of cellular protein synthesis. This effect appears to be due to a structural component(s) of the infecting virion. Experiments investigating the nature of the inhibitory principle showed that a vaccinia virion component, the surface tubule (ST), inhibits protein synthesis in HEp-2 cells without affecting either RNA or DNA synthesis. Furthermore, ST added to a rabbit reticulocyte cell-free system inhibited the incorporation of radiolabeled amino acids into acid-insoluble protein. ST inhibitory activity was destroyed by heat (56° for 60 min) and by prior incubation with specific anti-ST serum. A decrease in the polyribosome complement of cells exposed to purified ST, with a concomitant increase in the free ribosome pool, suggests that the main effect of tubules on cellular protein synthesis occurs at the level of polypeptide chain initiation.     

Identification of normal rat organs by two-dimensional protein electrophoresis

Proteins from nine normal rat organs, including heart, lung thymus, liver, spleen, kidney, prostate, abdominal muscle, and brain, were solubilized, separated by electrophoresis according to their different isoelectric points and molecular weights, and stained with Coomassie blue. Patterns of major proteins unique to each organ were identified. Nine "unknown" samples, chosen from the same sources and submitted for analysis in a single blind study, were easily identified by comparing their protein profiles against the nine "reference" patterns. The ability to identify the origin of a tissue sample without recourse to microscopy, by comparing the pattern of its electrophoresed proteins with a "catalogue" of identified protein profiles, provides a prototype for the identification of histologically indeterminate normal and abnormal cells, tissues, and organs. Application of this technique to problems in human pathology and forensic medicine could prove to be very useful.     

Human influenza A virus: comparative analysis of the structural polypeptides by two-dimensional polyacrylamide gel electrophoresis.

The biochemical properties of the major virion polypeptides (HA₁, HA₂, M, NP, NA) of 19 influenza A virus strains have been compared by two-dimensional polyacrylamide gel electrophoresis using nonequilibrium pH gradient electrophoresis in the first dimension. The highly variable surface antigen of the virus, the hemagglutinin (HA), exhibited multiple polypeptide subspecies varying extensively in charge. Comparison of the HA of the different influenza A strains demonstrated that most strains exhibit a unique hemagglutinin with distinguishable electrophoresic properties. Differences in charge and/or molecular weight of at least one of the two HA subunits (HA₁ or HA₂) were found for strains within the same subtype and for serologically indistinguishable strains such as A/USSR/90/77 and A/FW/l/50. Differences in the matrix (M) and neuraminidase (NA) proteins also were observed between strains. The results of this study indicate that the comparative examination of the two-dimensional polypeptide patterns of a particular virus isolate may be useful for the purposes of strain identification, determination of strain purity, homogeneity, and determination of gene origin following experimental or natural recombination events.     

Direct blotting with viable cells of protein mixtures separated by two-dimensional gel electrophoresis

A procedure is described which combines the high resolution power of two-dimensional (2D) gel electrophoresis with the advantage of direct probing with viable cells. This device permits the transfer by electroelution of 480 distinct fractions from a 2D gel into soluble phase. Transferred fractions are virtually nontoxic, thus allowing direct probing with viable cells. Using this procedure it was shown that T cells from normal healthy individuals recognized a multitude of Mycobacterium tuberculosis antigens and that the fine antigen recognition pattern of T cells changed after short-term culture in vitro. The application of this procedure to the verification of antigen purity at the T cell level and to the identification of antigens within crude bacterial lysates which are recognized by cloned T cells is described. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen, for example from important pathogens against which a subunit vaccine is desirable. Moreover, it could be helpful for the analysis of interactions between soluble ligands and their target cells.     

Identification of proteins from colorectal cancer tissue by two-dimensional gel electrophoresis and SELDI mass spectrometry

We investigated protein profiles obtained from colorectal tumor tissue and adjacent normal mucosa to identify tumor specific changes. Protein extracts of biopsis were separated by two-dimensional gel electrophoresis and >40 low-molecular mass proteins were identified by peptide fingerprinting using surface-enhanced laser desoption/ionization mass spectrometry (SELDI-MS). Among these, PACAP protein, hnrnp A1, flavin reductase, calgizzarin, NDK B (NM23-H2), cyclophilin A and smooth muscle protein 22-alpha showed significantly differential abundancy in the analyzed specimens. In addition, immunohistochemical analysis of tissue distribution and subcellular localization of some of the differentially expressed proteins demonstrated alterations in subcellular protein distribution. Further investigations are in progress to assess whether these differentially expressed proteins are associated with tumor development and tumor progression.     

Interaction of plasma proteins with heparinized gel particles studied by high-resolution two-dimensional gel electrophoresis.

In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.     

Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase

When mouse L cells are infected by vaccinia virus, a specific kinase inhibitory factor is produced which inhibits the interferon-induced, double-stranded RNA-dependent protein kinase (P. Whitaker-Dowling and J. S. Youngner (1983) Virology 131, 128-136). This inhibitory factor appears early in vaccinia infection (90 min) and its production requires protein synthesis. It inhibits the phosphorylation of the alpha subunit of protein synthesis initiation factor eIF-2 and it is active in mixed extracts of IFN-treated cells and vaccinia-infected cells. The vaccinia-mediated inhibition of the IFN-induced protein kinase is not due to a specific phosphatase or a specific protease and can be reversed by the addition of excess double-stranded RNA. Evidence is presented which suggests that the specific kinase inhibitory factor interacts in a stoichiometric manner with the double-stranded RNA which is required for the activation of the interferon-induced protein kinase.     

Detection of mutations within the thymidine kinase gene of herpes simplex virus type 1 by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis has been applied widely for the detection of mutation(s) and polymorphisms of various genes. It is shown that this system is also feasible for analyzing mutations in the thymidine kinase (tk) gene of herpes simplex virus type 1 (HSV-1). Thus, this system is applicable for examining whether a drug-resistant HSV-1 contains a tk mutation(s) or otherwise. This system can also be useful for detecting heterogeneity and the emergence of drug-resistant mutants in clinical samples.     

Immunogenicity of the Plasmodium falciparum glutamate-rich protein expressed by vaccinia virus.

The glurp gene of Plasmodium falciparum F32 has been inserted into a vaccinia virus, and the recombinant virus was designated VVG4. Expression of glurp in VVG4-infected Vero cells was analyzed by immunoprecipitation and revealed a primary GLURP product of approximately 220,000 Da; GLURP was detected both intracellularly and in culture supernatants. To study the immunogenicity of vaccinia virus-expressed GLURP, mice were immunized with VVG4 and serum samples were analyzed for antibody reactivity with three polypeptides, covering almost the entire GLURP molecule; these three polypeptides were produced in recombinant form in Escherichia coli. The immune response was primarily directed against a carboxy-terminal repeat region. The mouse anti-GLURP serum recognized authentic GLURP by immunoprecipitation analysis from P. falciparum grown in vitro. These results demonstrate that vaccinia virus-expressed glurp product can induce a humoral immune response against GLURP derived from blood-stage parasites.     

Comparison of synaptic plasma membrane and synaptic vesicle polypeptides by two-dimensional polyacrylamide gel electrophoresis.

Extracts of chick brain synaptic plasma membranes, synaptic vesicles, and mixtures of membranes and vesicles were examined by electrophoresis on two-dimensional polyacrylamide gels by a modification of the O'Farrell technique. Synaptic plasma membranes had twenty-one major polypeptides; synaptic vesicles had seventeen. Thirteen major polypeptides were common to both fractions. The similarities between the synaptic vesicle and synaptic plasma membrane patterns are unlikely to be due to contamination of one fraction by the other or to contamination of both fractions by microsomes, synaptoplasm or mitochondria. Our findings are consistent with mixing of membrane proteins occurring during exocytosis but it remains to be shown that these synaptic subfractions are not contaminated by a type of membrane for which markers are not yet available.     

Identification of leptospiral flagellar antigens by gel electrophoresis and immunoblotting

Flagella extracted from five serovars, representative of the pathogenic and saprophytic species of the Leptospiraceae, were morphologically similar. Analysis of Leptospira interrogans flagellar preparations by polyacrylamide gel electrophoresis revealed three common major bands in the (30-40) x 10(3)-mol. wt region, and serovar-specific bands in the lower region of the gels. Although some differences were observed, flagella extracted from L. biflexa serovar patoc and Leptonema illini revealed similar electrophoretic profiles to those seen in L. interrogans flagella. Immunoblot analysis showed that while flagellar components in the (20-30) x 10(3)-mol. wt region were recognised only by homologous rabbit antisera, a major protein doublet of (33-34) X 10(3)-mol. wt, depending on the species, was also demonstrated by heterologous antisera. The serovar-specific bands in the (20-30) x 10(3)-mol. wt region were composed of lipopolysaccharide (LPS). These results show that leptospiral flagella are immunogenic and contain antigens which are conserved among the different genera of the family Leptospiraceae.     

Comparative analysis of Trichinella spiralis and Trichinella nativa proteins by two-dimensional gel electrophoresis

Trichinella spiralis and Trichinella nativa are both common wildlife parasites in Finland. However, they differ substantially in their resistance to below 0°C temperatures in their natural hosts. T. nativa can live in frozen fox meat for years, whereas T. spiralis dies when frozen. In mouse muscle, the difference is not as evident; even T. nativa cannot maintain infectivity when kept at −20°C for 1 week. Crude larval protein extracts of these two parasite species were analyzed by two-dimensional gel electrophoresis (2DE). The protein patterns showed clear differences, but matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide mass fingerprint followed by database searches failed to identify these proteins, suggesting that they may still be uncharacterized. The patterns compared after freezing treatment at −20°C revealed changes in the intensity of some protein spots. The antigenic differences of the species were analyzed with two-dimensional Western blots, which showed T. spiralis-specific proteins.     

Identification of Campylobacter jejuni and C. coli by gel electrophoresis of the outer membrane proteins.

Analysis of the electrophoretic profiles of the outer membrane proteins could be used to differentiate Campylobacter jejuni (16 strains) from Campylobacter coli (10 strains). This observation was confirmed by the study of DNA homology obtained by a quantitative filter hybridization method. The hippurate hydrolysis test gave a poor correlation with the results of differentiation obtained by DNA homology studies and outer membrane protein profile.