Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line.

A novel basic heparin-binding monocyte chemotactic factor (MCF) was purified to homogeneity from the conditioned media of human myelomonocytic cell line THP-1 based on its in vitro monocyte chemotactic activity. The purified MCF was homogenous and estimated to be 15 kD on SDS-PAGE. Purified MCF stimulated normal human monocytes to be growth inhibitory in vitro at 2-3 d for several human tumor cell lines. This represents the first report of the identification and purification of a chemoattractant cytokine that also activates monocytes but is distinct from interferons and other known cytokines.

Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera.

The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes. A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function. The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV-III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo. Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts. The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product.

Immunogenic properties of soluble cytosol fractions on Meth A sarcoma cells.

Tumor-associated transplantation antigen (TATA) was found to be present in fractions derived from the cytosol of the Meth A cell. Meth A ascites cells were disrupted nuclei and membranes were removed by low- and high-speed centrifugation, and the soluble protein was fractionated by ammonium sulfate precipitation and gel filtration chromatography. The TATA of the soluble cytosol fractions appears to be identical with the TATA solubilized from plasma membranes. The TATA of the cytosol fractions was found to be associated with proteins of an approximate apparent molecular weight of 60,000, specific for the Meth A tumor, and as immunogenic as the membrane-derived TATA. In addition, the most enriched TATA cytosol fraction shows inhibition of an antiserum capable of detecting a tumor-specific surface antigen of Meth A. These results suggest that Meth A TATA is not an integral membrane protein and may be related to the tumor-specific surface antigen detected serologically.

Purification and characterization of mouse beta-2 microglobulin: allelic variants from two different strains.

Beta-2 microglobulin (beta 2M) is a 12,000 dalton protein associated with membrane-bound cell surface antigens. Variants of beta 2M, beta 2MA and beta 2MB, were first detected by Michaelson et al. (Immunogenetics 11, 93-95, 1980). An improved method was used to purify beta 2MA and beta 2MB from BALB/c and C57BL/6 mouse livers, respectively. Reproducible yields of 10% were obtained. The purifications were accomplished by a 3 M sodium thiocyanate (NaSCN) extraction of a crude membrane fraction, an acid precipitation step, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose and CM-cellulose in that order. The elution profile of beta 2MA and beta 2MB on the ion-exchange columns was found to be different, indicating the presence of structural changes. beta 2MA was found to be more acidic (pI = 7.35) than beta 2MB (pI = 7.68) by isoelectric focusing in gels. Complete sequence analysis of beta 2MA and partial sequence analysis of beta 2MB (61 of 99 residues) were performed by automated Edman degradation of the intact chain and of the overlapping peptides obtained by: (a) tryptic cleavage at arginines after acetimidation of lysine side chains, (b) BNPS-skatole cleavage at tryptophan residues and (c) hydroxylamine cleavage at asparagine-glycine linkages. A comparison of the primary structure of beta 2MA to the partial amino acid sequence obtained for beta 2MB revealed a single amino acid substitution (aspartic acid for alanine at position 85) that accounts for the differences in biochemical properties observed.

A novel growth stimulating activity from BRL-3A cell conditioned medium.

The R- cell line is a 3T3-like cell line originating from mouse embryos with a homozygous disruption of the type 1 insulin-like growth factor receptor (IGF-IR) genes. Although R- cells cannot grow at all in serum-free medium (SFM) supplemented by several known growth factors, either singly or in combination, they are able to grow in 10% serum, albeit at a reduced rate. These findings suggested that serum contains an unknown, or unidentified, growth factor that can promote cell growth even in cells devoid of IGF-IRs. In an effort to identify such growth factor, we searched, using R- cells, for a growth and DNA synthesis stimulating activity in SFM conditioned by different cell lines. We found that the BRL-3A cell line secreted an activity capable of stimulating DNA synthesis and cell proliferation in R- cells. This activity (which is concentration-dependent) can be collected and concentrated by ultrafiltration, it is heat-labile, proteinase K-sensitive and has a size larger than 10 kDa. Because of the resistance of R1 cells to stimulation by known growth factors, we believe that this activity is due to a novel polypeptide secreted by BRL-3A cells. Further characterization of the active component(s) is in progress.

Expression and purification of protein segments encoded by the envelope and 3'-orf genes of human immunodeficiency virus type 1.

The pJL6 expression vector and its derivatives, pJLA16 and pANH-1, have been used for the synthesis and high-level expression in Escherichia coli of restriction enzyme fragments derived from the envelope and 3'-orf genes of the BH10 and BH8 clones, respectively, of the human immunodeficiency virus (HIV-1). These bacterially expressed proteins have been purified to apparent homogeneity by sequential detergent extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The recombinant proteins have been used for the production of polyclonal and monoclonal antibodies, and the fusion proteins from the envelope gene are currently being evaluated for use as immunodiagnostic assay reagants.

Comparative analysis of gp41 antigens by enzyme-linked immunosorbent assays for detecting antibodies to human immunodeficiency virus type 1.

We have compared the antigenic qualities of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein with a synthetic oligopeptide (peptide R21S) and a bacterially synthesized protein (protein 566), which are homologous with the N-terminal region of gp41, in enzyme-linked immunosorbent assays (ELISA) for detecting antibodies to HIV-1 in sera of patients with the acquired immunodeficiency syndrome (AIDS) or the aids-related complex (ARC). Although the use of all three types of antigens readily allowed the detection of antibodies in human sera, ELISA employing purified gp41 glycoprotein and the protein 566 were more specific and sensitive than the peptide R21S ELISA.

Biochemical characterization and biologic activities of 82- and 86-kDa tumor antigens isolated from a methylcholanthrene-induced sarcoma, CII-7.

Two tumor-specific antigens, with molecular weights of approximately 82 and 86 kDa, have been isolated and purified to apparent homogeneity from the methylcholanthrene-induced sarcoma, CII-7. The method of purification used was essentially that previously employed for the isolation of the 82- and 86-kDa antigens from the Meth A sarcoma and the 86-kDa antigen from mKSA sarcoma. Cytosolic fractions were subjected to hexylamine agarose chromatography, Sepharose S-300 filtration and hydroxylapatite chromatography. A final step, HPLC-DEAE chromatography, was necessary for the purification of the 82-kDa protein. Both isolated antigens retained their specific immunogenicity for CII-7 as determined by in vivo tumor rejection assays, and failed to influence the growth of other syngeneic sarcomas, CI-4, CII-10 and mKSA, which have their own unique TATA. The 82- and 86-kDa antigens appear to be distinct proteins that are well conserved in nature and may represent members of distinct families of TATA.

Isolation of a tumor-associated transplantation antigen (TATA) from an SV40-induced sarcoma. Resemblance to the TATA of chemically induced neoplasms.

A tumor rejection antigen (TATA), obtained from the cytosol of a BALB/c mKSA sarcoma induced by SV40 virus, has been partially purified. This partially purified antigen is strikingly immunogenic against mKSA, providing more than 90% inhibition of growth at levels of 10-30 micrograms. This antigen preparation does not protect against challenge with another SV40-induced BALB/c sarcoma VLM which, however, shares a group-specific TATA with mKSA. The antigen also does not immunize against challenge with the methylcholanthrene-induced sarcomas of BALB/c mice, Meth A, CI-4, CII-7 and CII-10, each of which has its own unique TATA. Binding assays, using ELISA, failed to detect any SV40 antigen in the antigen preparation despite the fact that the large T antigen of SV40 (or fragments of it) constitutes the immunodominant TATA of mKSA.

Purification and biochemical properties of tumor-associated transplantation antigens from methylcholanthrene-induced murine sarcomas.

A tumor-associated transplantation antigen with an apparent molecular weight of 75,000 has been isolated from the cytosol of the BALB/c methylcholanthrene-induced sarcoma, Meth A. The antigen was purified either by preparative electrophoresis in the presence of NaDodSO4 or by immunoaffinity chromatography after hexylamine agarose chromatography, gel filtration, and hydroxylapatite chromatography. The 75-kilodalton (kDal) protein prepared by either of these methods effectively primed BALB/c mice to reject the Meth A tumor; such priming provided no protection against challenge by other independently derived sarcomas of BALB/c origin. A second protein, also 75 kDal, was isolated from the cytosol of the recently derived methylcholanthrene-induced sarcoma CI-4 by essentially the same chromatographic scheme. This protein also was immunogenic in the tumor rejection assay and provided protection only against CI-4 challenge. The antigens purified from the Meth A and CI-4 sarcomas appear to be closely related proteins. Both of them can be purified from the cytosol fraction and can be recognized by a rabbit antiserum prepared against the Meth A 75-kDal protein. The two proteins have approximately the same molecular weight, have similar but not identical amino acid compositions, and differ in their chromatographic behavior on hexylamine agarose and hydroxylapatite as well as in their isoelectric points. These results indicate that the individually specific transplantation antigens found in chemically induced sarcomas may be the products of a single multigene family or somatic derivatives of a single gene.

Mapping the domain(s) critical for the binding of human tumor necrosis factor-alpha to its two receptors.

The extracellular domains of the two human tumor necrosis factor (TNF) receptors critical for binding TNF-alpha were examined by deletion mapping. The ligand binding capability of full-length and truncated recombinant soluble TNF receptors (TNFRs) was assessed by ligand blot analysis and their binding affinity determined by Scatchard analysis. The results showed that deletion of the fourth cysteine-rich domain of the p55 receptor (TNFR-1) did not alter ligand binding affinity significantly. Deletion of domains 3 and 4 of TNFR-1 resulted in no ligand binding, suggesting that domain 3, but not 4, of TNFR-1 binds directly to ligand. Deletion of domain 4 of TNFR-2 resulted in drastically reduced protein yield and 3-fold reduction in ligand binding affinity, while deletion of both domains 4 and 3 yielded no protein. Thus, the domain 4 of TNFR-2, but not that of TNFR-1, appears to be involved directly in binding TNF, although it is also possible that the domain 4 of TNFR-2 is involved in the correct folding of other domains. These results suggest that the modes of interaction between TNF-alpha and its dual receptors are different, providing opportunity to modulate each receptor specifically for research and therapeutic purposes.

Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein.

CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

Expression and characterization of a protein encoded by the human c-myc exon 1 in Escherichia coli.

Our previously reported data from DNA sequence studies of the c-myc locus show that the human c-myc exon 1 has an open reading frame capable of encoding a protein of 188 amino acid residues. To confirm the presence of the open reading frame, we constructed a recombinant vector (pMCP60) that contains a segment of the lambda cII translational initiation region, a portion of the N-terminus of the v-mos gene, and 639 base pairs of the first exon of the human c-myc gene. pMCP60 expresses a 38 kilodalton tripartate protein (cII-mos-myc), which was purified by high-pressure liquid chromatography. The presence of myc exon 1 sequences in the cII-mos-myc fusion protein was confirmed by partial amino acid sequence analysis. These experiments further establish that the first exon of the human c-myc gene contains an open reading frame capable of expressing a protein in Escherichia coli.

Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism.

The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.

FKBP46, a novel Sf9 insect cell nuclear immunophilin that forms a protein-kinase complex.

Recently, we identified a 59-kDa nuclear phosphoprotein that is associated with a recombinant mouse FKBP-52 (Alnemri, E. S., Fernandes-Alnemri, T., Nelki, D. S., Dudley, K., DuBois, G. C., and Litwack, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6839-6843). Here we describe the cloning, overexpression, and characterization of this protein from Spodoptera frugiperda insect cells (Sf9 cells). The cloned cDNA codes for an acidic protein of 412 amino acids with distinct structural domains. Starting with the N terminus, the first 218 amino acids contain two highly acidic domains separated by a short basic domain. Following the second large acidic domain is another basic domain of 87 amino acids with significant sequence and structural homology to HMG1 and HMG2 DNA binding proteins. The two basic domains contain several nuclear targeting signals. The last 108 C-terminal amino acids contain a binding domain for immunosuppressive drugs FK506 and rapamycin, which makes this protein a new member of the immunophilin family. We provide evidence that the new immunophilin (FKBP46) is a DNA binding protein that can bind immunosuppressive drug FK506 and possesses peptidylprolyl isomerase activity. FKBP46 is localized in the nucleus and is associated with a nuclear kinase that specifically phosphorylates it in the presence of Mg2+ and ATP. Upon subsequent sequence analysis of the mouse FKBP52 cDNA used in our previous study, it was observed that a spermatid nuclear transition protein 2 (TP2) sequence is fused in frame with the C terminus of the recombinant FKBP52 probably as a result of a cloning artifact. We demonstrate that the FKBP46 does not form a complex with the FKBP52 but rather with the highly basic nuclear protein TP2. Our data suggest that interaction of FKBP46 with TP2 is mediated by the N-terminal acidic domains of FKBP46. This implies that the acidic domains of FKBP46 are involved in protein-protein interaction between nuclear FKBP46 and other basic chromatin proteins.