Search PubMed⌕ Search

Biomedical subjects

T P Hopp

Publications and source records attributed to T P Hopp.

At least 19 recordsLinked to original sources
PubMed1995

Evidence from sequence information that the interleukin-1 receptor is a transmembrane GTPase.

Evidence is presented that the cytoplasmic domain of the type I interleukin-1 receptor (IL-1R) may be a GTPase. This domain conserves segments of hydrophobic amino acids that suggest a structural relatedness to the ras protooncogene protein and other members of the GTPase superfamily, despite a lack of significant detectable sequence homology. When the hydrophobic segments of the IL-1R were aligned with similar segments of the GTPases, it became apparent that the IL-1Rs possess a number of conserved amino acids that represent plausible functional residues for base-specific binding of GTP, magnesium chelation, and phosphate ester hydrolysis. Furthermore, a segment of five contiguous residues were found that is identical between ras and the IL-1R, and which is positioned to form part of the guanine base binding pocket. If this model is correct, then the IL-1Rs possess a highly conserved effector protein binding region, but one that is entirely unrelated to the effector regions of other superfamily members. Therefore, if the IL-1R is indeed a GTPase, then its activation function may be directed to as-yet unrecognized effector target proteins, as part of a unique cellular signal transduction pathway.

Amino Acid Sequence↗
PubMed1995

Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: identification of potent calcium-independent antigens.

The detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the 'FLAG' sequence DYKDDDDK-NH2, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52 x 10(6) and 4 x 10(12) different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif-DYK_K_(-), and were recognized by mAb M1 in the absence of calcium with 50- to 100-fold higher affinity than the FLAG octapeptide (IC50 = 273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.

Amino Acid Sequence↗
PubMed1989

A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta.

Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.

Amino Acid Sequence↗
PubMed1989

Use of hydrophilicity plotting procedures to identify protein antigenic segments and other interaction sites.

Hydrophilicity analysis of the surface properties of proteins continues to be an important means for understanding the interactions that occur between proteins and other macromolecules. We have shown that the procedure of Hopp and Woods is useful in developing synthetic peptide immunogens and for understanding the relationship of protein sequence and folding to the interactions between macromolecules. Using a standardized procedure and the optimized hydrophilicity scale of amino acid values, it is possible to display the surface-exposed and buried portions of a polypeptide chain, as well as such features as membrane-spanning segments. Finally, an example was provided to show that hydrophilicity analysis has a place in protein engineering, allowing the creation of new surface segments with predictable properties.

Amino Acid Sequence↗
PubMed1989

A calcium-dependent antibody for identification and purification of recombinant proteins.

We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase.

Antibodies, Monoclonal↗
PubMed1988

Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli.

A cDNA encoding the viral protease from the 3C region of human rhinovirus type 14 was expressed in Escherichia coli through the use of a periplasmic secretion vector. The recombinant protease contained an eight amino acid N-terminal extension that enabled its detection by a specific antibody. It was expressed at a level of approximately 1 mg/L of E. coli culture. Biological activity of the protease was assessed in vitro by using a chemically synthesized peptide consisting of a consensus picornavirus protease cleavage site, Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu. The peptide was cleaved by the recombinant protease at the Gln-Gly bond, generating the product peptides Arg-Ala-Glu-Leu-Gln and Gly-Pro-Tyr-Asp-Glu, which could be separated from the substrate peptide by reversed-phase HPLC. An in vitro assay for the rhinovirus 3C protease was developed by observing the rate of disappearance of the substrate peak from chromatograms of the supernatants of digestion mixtures.

Amino Acid Sequence↗
PubMed1988

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor.

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.

Base Sequence↗
PubMed1987

Localization of human mononuclear cell interleukin 1.

The detection and localization of interleukin (IL) 1 in human monocytes was carried out by flow cytometry using monoclonal antibodies to IL-1 alpha and IL-1 beta proteins. IL-1 alpha was detected on the surface of monocytes and the surface expression increased following lipopolysaccharide activation. No demonstrable IL-1 beta protein could be observed on the cell surface by antibody staining, while both IL-1 alpha and IL-1 beta could be visualized intracellularly by the appropriate monoclonal antibodies following acetone permeabilization of the monocytes. Further experiments with cell associated IL-1 revealed that most of the biological activity of human monocytes could be inhibited by affinity purified polyclonal antibodies to IL-1 alpha protein, whereas no inhibitory activity was observed with IL-1 beta specific antibodies. These data support the hypothesis that a differential localization of IL-1 alpha and IL-1 beta exists within human blood-derived monocytes.

Antibodies, Monoclonal↗
PubMed1987

Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.

Amino Acid Sequence↗
PubMed1986

Protein surface analysis. Methods for identifying antigenic determinants and other interaction sites.

A variety of methods are currently employed in attempts to identify antigenic determinants and other features of proteins. Most of these involve calculations based on scales of values for the 20 amino acids that reflect their likelihood of occurrence at the surface of proteins or as part of secondary structures such as helices or beta-bends. The most successful of these procedures all use scales related to the water solubility of the individual amino acids. In particular, the highest success rates are obtained using hydrophilicity scales that emphasize the charged and polar amino acids, but are not overly selective for either positive or negative charges. Such a method can correctly pinpoint major antigenic sites on the proteins of most well characterized infectious organisms. The hydrophilicity profiles also contain information concerning other types of protein interaction sites and membrane spanning segments.

Amino Acid Sequence↗
PubMed1985

Detection and characterization of high affinity plasma membrane receptors for human interleukin 1.

Interleukin 1 (IL-1) is a polypeptide hormone that acts as a central mediator of inflammation. Since IL-1 action is presumably mediated by specific cell surface receptor(s), we have characterized the binding of this hormone to cells. Purified human IL-1 was labeled to high specific activity with 125I, using Bolton-Hunter reagent. The labeled protein binds specifically to LBRM-33-1A5 (a murine T lymphoma line previously shown to produce IL-2 in response to phytohemagglutinin and IL-1) with an affinity of approximately 0.2-2 X 10(10)/M and, at saturation, to approximately 500 receptors per cell, on intact cells at 8 degrees C in the presence of sodium azide. The affinity of unmodified IL-1 for the murine plasma membrane receptor is 0.9-2 X 10(10)/M, as measured by the inhibition of 125I-IL-1 binding. The murine receptor specificity has been confirmed by demonstrating that, among a series of 12 polypeptide hormones, only IL-1 inhibits 125I-IL-1 binding to LBRM-33-1A5 cells. Treatment of surface-bound 125I-IL-1 with bivalent water-soluble crosslinkers identified a membrane polypeptide of Mr 79,500 to which IL-1 is crosslinked. A variety of cell types have been surveyed for the capacity to bind 125I-IL-1 specifically. The presence of specific binding correlates with the capacity of the cells tested to respond to IL-1. Our results indicate that the biological effects of the polypeptide hormone IL-1 are mediated by high affinity plasma membrane receptors. The identification of these receptors should provide valuable insight into the apparently diverse biological activities of IL-1.

Animals↗
PubMed1985

Human interleukin 1. Purification to homogeneity.

We have purified human interleukin 1 (IL-1) to homogeneity by a simplified procedure that results in excellent yields of pure material that retains a high level of biological activity. IL-1, secreted by human peripheral blood macrophages that have been stimulated with Staphylococcus aureus, was purified by ion exchange chromatography and affinity chromatography on Procion Red agarose. The pure protein has a specific activity of 3.2 X 10(8) U/mg in the thymocyte mitogenesis assay, and is pyrogenic. No molecular weight heterogeneity was observed, in contrast to findings for mouse IL-1 and earlier reports of human IL-1. Purified IL-1, as analyzed by two-dimensional electrophoresis/electrofocusing gels, exhibited a series of charged species with isoelectric points ranging from 6.0 to 4.9, all with a molecular weight of approximately 17,500. Amino acid analysis indicated an abundance of acidic residues, in agreement with the low isoelectric points. There is little or no cysteine in the molecule. No evidence was found for the presence of carbohydrate moieties. The overall yield for this procedure was approximately 31% of the activity contained in the initial culture supernatant.

Animals↗
PubMed1985

Generation of anti-peptide monoclonal antibodies which recognize mature CSF-2 alpha (IL 3) protein.

The colony-stimulating factor CSF-2 alpha (IL 3) has been purified to homogeneity, the protein sequenced, and the gene encoding this lymphokine cloned. Knowledge of the protein sequence permitted the synthesis of peptides corresponding to the amino terminus of the molecule. These peptides, after conjugation to palmitic acid, were used to immunize mice. Spleen cells from mice immunized with one of these peptides (CSF-2 alpha 1-14) were fused with the myeloma cell line NS-1. The fusion resulted in the isolation of two hybridoma cell lines, designated 6A5 and 4D4, that secreted antibodies that were specific for the immunizing peptide. The antibodies did not react with a closely related peptide CSF-2 alpha 7-16. The antibodies were capable, however, of recognized CSF-2 alpha protein as judged by the ability of the antibodies to remove CSF-2 alpha activity from culture medium of PHA-stimulated LBRM-33-5A4 cells, to immunoprecipitate radiolabeled CSF-2 alpha protein, and to detect CSF-2 alpha protein bound to nitrocellulose membranes.

Animals↗