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.

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.

Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier.

Effective immunization with short polypeptide antigens has typically only been possible when the peptide is conjugated to a large carrier substance, usually a protein. Such immunizations suffer from difficulties in producing conjugates of reliable composition, and from unwanted anti-carrier immune responses. When a chemically synthesized peptide, bearing hepatitis B virus a-determinant specificity, was conjugated to a dipalmityl-lysine moiety, a significant improvement in anti-hepatitis B surface antigen response was obtained, in comparison to the corresponding peptide-keyhole limpet hemocyanin conjugate. Dipalmityl lysyl peptide conjugates are readily made by standard Merrifield synthesis procedures, and are relatively free of byproducts that might cause unwanted immune responses. Gel filtration experiments suggest that the conjugates form large aggregates, possibly micelles, which may play a significant role in the enhancement of the anti-peptide response. These properties suggest that fatty acid conjugation may be a useful procedure for producing chemically synthesized peptide vaccines.

A computer program for predicting protein antigenic determinants.

A computerized method for predicting the locations of protein antigenic determinants is presented, which requires only the amino acid sequence of a protein, and no other information. This procedure has been used to predict the major antigenic determinant of the hepatitis B surface antigen, as well as antigenic sites on a series of test proteins of known antigenic structure [Hopp & Woods (1981) Proc. natn. Acad. Sci. U.S.A. 78, 3824-3828.] The method is suitable for use in smaller personal computers, and is written in the BASIC language, in order to make it available to investigators with limited computer experience and/or resources. A means of locating multiple antigenic sites on a homologous series of proteins is demonstrated using the influenza hemagglutinin as an example.

Immunochemical studies on alpha-lactalbumin.

The antigenic structural features of alpha-lactalbumin have been investigated using a radioimmunoassay, peptide inhibition of the quantitative precipitin reaction, and by immunodiffusion analysis after chemical modification of the molecule. Antigenic activity (in rabbits) was localized to several peptic fragments and the single arginine residue of bovine alpha-lactalbumin. Antigenic activity was also found to be associated with the single methionine residue. A peptic fragment containing a disulfide loop was found to possess antigenic activity in both bovine and goat alpha-lactalbumin. Radioimmunoassay cross-reactivity between the alpha-lactalbumins is correlated with amino acid sequence similarities; bovine alpha-lactalbumin antiserum cross-reacts with goat alpha-lactalbumin more extensively than with human alpha-lactalbumin, while the more distantly homologous protein, chicken lysozyme, does not cross-react at all. Nevertheless our data indicate that the alpha-lactalbumins and lysozyme share a similar distribution of antigenic determinants on their surfaces.

Metal-binding properties of a calcium-dependent monoclonal antibody.

The calcium-dependent mAb, M1 (also called anti-Flag or 4E11) was studied using a newly developed metal-sensitive enzyme-linked immunosorbent assay (ELISA). This antibody, specific for a calcium complex of the peptide antigen, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, has found widespread use as a mild purification reagent for Flag-epitope tagged recombinant proteins. Although M1 affinity columns release monovalent Flagged proteins in the absence of calcium, the antibody retains substantial affinity for the Flag sequence even in metal-free conditions, so that it has been impossible to use it to develop a metal-sensitive ELISA assay. This is due to the ability of the antibody to remain bound to polyvalent surface-coated antigen, for instance, when Flagged proteins are bound to ELISA plates or blotting filters. The resultant antigen polyvalence raises the avidity of the Flag antibody to a point where the reaction is essentially calcium-independent. However, when the antibody itself was made monovalent, by proteolytic cleavage to the Fab, this situation was reversed and the ELISA reaction became calcium-dependent. This new metal-dependent ELISA assay was used to explore the metal requirements of the antibody in detail. Among divalent metals, binding tapered off with increasing radius above that of calcium, or with decreasing radius below that of calcium. Several smaller metals, such as nickel, acted as inhibitors of the binding reaction. Substantial binding was demonstrated for heavy metals such as cadmium, lanthanum and samarium. Because it is of interest to use this antibody for the co-crystallization of recombinant Flag-fusion proteins, the ability to bind heavy metals was a significant finding.

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.

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.

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.

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.

Retrospective: 12 years of antigenic determinant predictions, and more.

The Hopp and Woods hydrophilicity method for locating antigenic determinants was published in 1981. In the years since then, the method has been used widely and has played a vital role in many antigenic structure studies. The method has been criticized occasionally and replacements for it have been proposed. However, at this time, the Hopp and Woods method remains a method of choice for identifying antigenic sites and other protein interaction sites, because it has a higher success rate than other similar methods. Key to the success of this method is its cautious approach to charge-charge interactions, giving equal weight to positively and negatively charged residues, whereas other methods tend to favor one or the other. It has become clear that sites chosen by our method tend to be highly exposed, charged regions of the protein's surface which project into the environment and therefore have ample opportunity to contact other proteins. We have been exploring new uses for the method, and have found some applications in locating sites for other types of interactions, including those with other macromolecules such as DNA and RNA. It seems likely that this simple and reliable procedure will continue to find use in predicting the locations of major antigenic epitopes, and may also find use as a general prediction method to identify interaction sites on proteins that make charge-dependent contacts with a variety of other biological macromolecules.

Primary structure of rabbit alpha-lactalbumin.

Rabbit alpha-lactalbumin was purified from the milk of New Zealand White rabbits. It was found to exist predominantly as a glycoprotein, containing 5 mol of glucosamine per mol of protein, as well as other sugars. The amino acid sequence of the protein was determined by sequenator analysis and carboxypeptidase digestion. There are 122 amino acids in the protein and a single carbohydrate moiety, probably attached to an asparagine residue at position 45. The C terminus of rabbit alpha-lactalbumin is one residue shorter than that of the other alpha-lactalbumins. Sequence comparisons indicate that the alpha-lactalbumin gene has been undergoing more frequent mutation than had previously been thought. A new method of preparative peptide mapping using 2,5-diphenyloxazole (PPO) fluor to detect peptides is described.

Prediction of protein antigenic determinants from amino acid sequences.

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinins, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.

Amino acid sequence and variant forms of favin, a lectin from Vicia faba.

We have determined the complete amino acid sequence (182 residues) of the beta chain of favin, the glucose-binding lectin from fava beans (Vicia faba), and have established that the carbohydrate moiety is attached to Asn 168. Together with the sequence of the alpha chain previously reported (Hemperly, J. J., Hopp, T. P., Becker, J. W., and Cunningham, B. A. (1979) J. Biol. Chem. 254, 6803-6810), these data complete the analysis of the primary structure of the lectin. We have also examined minor polypeptides that appear in all preparations of favin. Two lower molecular weight species (Mr = 9,500-11,600) appear to be fragments of the beta chain resulting from cleavage following Asn 76, whereas six high molecular weight forms (Mr = 25,000 or greater) appear to include aggregates of the beta chain and possibly some alternative products of chain processing.

Hepatitis B virus vaccine: identification of HBsAg/a and HBsAg/d but not HBsAg/y subtype antigenic determinants on a synthetic immunogenic peptide.

On the basis of theoretical considerations, a peptide (H peptide) was synthesized by Hopp and Woods [Hopp, T. P. & Woods, K. R. (1981) Proc. Natl. Acad. Sci. USA 78, 3824---3828]. This peptide contains a sequence of six amino acids postulated to represent the major epitope, or antibody-combining site, of hepatitis B virus surface antigen (HBsAg). We have used passive hemagglutination inhibition with monospecific antibodies against the a, d, and y subdeterminants of this antigen and against human serum albumin to investigate the antigenic specificities on this peptide, and we have found it to contain the HBsAg/a and HBsAg/d but not HBsAg/y or human serum albumin subdeterminants. When the peptide was conjugated onto human erythrocytes and injected into mice, it induced the formation of anti-HBsAg with and without the use of Freund's adjuvant. If anti-HBsAg/a confers immunity to infection with hepatitis B virus, as is generally thought, these findings may permit the development of a synthetic vaccine lacking all unnecessary antigenic determinants.