Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4.

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

HLA-DP2: self peptide sequences and binding properties.

Although self peptides bound to HLA-DQ and, especially, HLA-DR allotypes have been described in some detail, few ligands that bind to HLA-DP have been identified. Toward this aim, naturally processed peptides were isolated from immunoaffinity-purified HLA-DP2 molecules expressed in cultured B lymphocytes. The size distribution of the peptide repertoire is generally similar to those reported for self peptides bound to HLA-DR and HLA-DQ molecules. Twelve peptides representing individual sequences including two nested sets were sequenced by mass spectrometry and/or N-terminal Edman analysis. Source proteins included MHC molecules and other integral membrane proteins as well as secretory and serum proteins. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Peptide specificity and affinity were examined in binding assays using synthetic peptides and purified HLA-DP and HLA-DR molecules. Anchor residues were tentatively assigned using alanine-substituted analogues of two self peptides. Some structural features of HLA-DP2 that may relate to peptide binding are considered.

An interactive Web site providing major histocompatibility ligand predictions: application to HIV research.

EpiMatrix/HIV, a tool that is currently available on the World Wide Web, enables researchers to screen HIV proteins for potential MHC ligands. We have performed a comparison of EpiMatrix predictions to 158 published allotype-specific HLA-associated peptides (MHC ligands) derived from 133 proteins. The top 10 ranked EpiMatrix predictions for each of the 158 HLA allotype-protein pairs were selected for comparison with these published ligands. EpiMatrix correctly identified 134 of 158 published ligands (85%). The algorithm is now available for use by the HIV research community at the URL http:/(/)www.EpiMatrix.com/HIV.

The discovery and use of HLA-associated epitopes as drugs.

MHC receptors "display" peptide fragments to T cells. These peptides are predominantly derived from proteins expressed within or ingested by the presenting cell. Since empty MHC molecules are highly unstable, peptide ligands are bound prior to MHC surface expression and the ensuing t1/2 off rates are often on the order of days. It is the remarkable stability of MHC/peptide complexes, which provide us an opportunity to purify MHC molecules from infected, transfected, or antigen pulsed cells and subsequently identify the naturally processed peptides being presented. On the other hand, the stability of MHC/peptide complexes substantially reduces the potency of parenterally administered peptides in vivo. Using serial immuno-affinity chromatography and mass spectrometry, naturally processed peptides can be identified. When these peptides are then encoded into nucleic acid and delivered parenterally, they are highly immunogenic. Application of these techniques to induce vigorous CTL responses will be discussed.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Self-peptides bound to the type I diabetes associated class II MHC molecules HLA-DQ1 and HLA-DQ8.

Genetic susceptibility to several autoimmune disorders is associated with the expression of certain MHC class II alleles. Insight into the etiology of such diseases awaits the identification of the class II restriction elements and the possible pathogenic peptides. Towards these aims, self-peptides bound to HLA-DQ1 and HLA-DQ8, allotypes considered to be neutral and permissive respectively towards the development of insulin-dependent diabetes mellitus, are reported. These naturally processed peptides were isolated from immunoaffinity purified HLA-DQ molecules expressed in cultured B lymphocytes. The chromatographic profiles of the peptide repertoires are unique, whereas the size distributions exhibit general similarity to those reported for naturally processed self-peptides bound to HLA-DR. Twenty-eight individual peptides representing 10 nested sets were identified by combined Edman microsequencing and mass spectrometry. Peptide length varied from 13 to 74 amino acids. Source proteins included MHC molecules and other integral membrane proteins, as well as secretory, cytosolic and mitochondrial proteins. Promiscuous invariant chain peptides were identified among the self-peptides bound to HLA-DQ8. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Some structural features of DQ1 and DQ8 that may relate to the bound peptides are discussed. Peptide specificity was confirmed in binding assays with purified HLA-DQ and HLA-DR protein.

Selective release of some invariant chain-derived peptides from HLA-DR1 molecules at endosomal pH.

The predominant peptides bound to major histocompatibility complex class II molecules expressed on human B cells are derived from a relatively limited number of self proteins. To determine whether any of the prebound self peptides might be released in endosomes during recycling, water-soluble HLA-DR1 molecules were incubated with a high affinity synthetic peptide at pH 4.0 and 7.0 at 37 degrees C. The resulting bound peptide repertoire was then acid extracted, and separated by reversed-phase high performance liquid chromatography. Using a combination of mass spectrometry and ultraviolet spectroscopy, prebound self peptides and newly bound synthetic peptide were characterized. Most self peptides bound to HLA-DR1 were not appreciably released during extended exposure to pH 4.0. However, some invariant chain-derived peptides were uniquely released at this pH.

Analysis of MHC-presented peptides: applications in autoimmunity and vaccine development.

T cells recognize antigenic peptides presented on the cell surface by major histocompatibility complex (MHC) molecules. Here, Roman Chicz and Robert Urban describe the features of peptides bound to MHC molecules and the mechanism by which these surface proteins bind diverse peptide ligands with high affinity. In addition, they discuss the application of new technologies to the identification of MHC-associated peptides.

A subset of HLA-B27 molecules contains peptides much longer than nonamers.

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much longer than nonamers. Typical HLA-B27-binding peptides contain arginine in position 2. Further analysis by Edman sequencing of the pooled bound peptides revealed that the major population contained substantial amounts of arginine at positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected. The minor population of peptides also contained detectable amounts of arginine at these positions, but at the level of only approximately 10%; no marked enrichment at any position was observed. These long HLA-B27-bound peptides could represent either intermediates in the formation of nonamers or adventitiously bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface, indicating that the peptide transporter was required for delivery of the long peptides. Thus, during the folding of class I heavy chains, peptides of diverse lengths are available and participating.

Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.

Minute quantities of a single immunodominant foreign epitope are presented as large nested sets by major histocompatibility complex class II molecules.

The processing and presentation of immunogenetic peptides is an obligate event in the generation of an immune response. However, the degree of complexity with which an immunogenic foreign epitope is presented is still unclear. This question was addressed by analyzing the naturally processed peptides generated from exogenously-derived hen egg white lysozyme (HEL) bound to the murine major histocompatibility complex (MHC) class II molecule, H-2Ak. Using reversed-phase chromatography (RPC), T cell hybridomas and mass spectrometry, 16 peptides were identified that contain the minimal MHC binding epitope 52-61. These peptides exhibited substantial N- and C-terminal extensions and ranged from 13-28 amino acids in length. In contrast, MHC class I molecules present peptides of 8-11 residues and each foreign epitope appears to be represented by only a single peptide. The data here also show that only approximately 0.8% of the total bound peptide was derived from this single HEL epitope. These findings provide direct evidence that relatively small amounts of processed peptide are required to stimulate an effective T cell response.

Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.

Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.

Microenvironmental contributions to the chromatographic behavior of subtilisin in hydrophobic-interaction and reversed-phase chromatography.

Genetically engineered variants were used to examine how microenvironmental changes in the S1 substrate binding subsite of subtilisin contribute to chromatographic behavior of proteins on hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC) columns. Gradient elution studies over a wide pH range showed that conditions could be found where a HIC support could separate proteins varying by one amino acid. Although all single-site variants could not be separated by HIC, this chromatographic mode was found to be complementary to cation-exchange chromatography for the separation of such variants. RPC was found to be of much less utility in the resolution of variant proteins. Retention and resolution of subtilisin variants was found to vary on RPC with the concentration and type of mobile phase pairing agent.

Single amino acid contributions to protein retention in cation-exchange chromatography: resolution of genetically engineered subtilisin variants.

Genetically engineered proteins were used to determine the amino acid contributions of surface residues to subtilisin retention in cation-exchange chromatography. Crystallographic data were used to correlate the observed chromatographic behavior with enzymatic structure. Retention times of variants in gradient elution varied by as much as 33% compared to the wild type. The role of both charged and uncharged residues was investigated in isocratic separations and found to significantly influence protein retention in this electrostatically dominant separation method. This study demonstrates the ability of ion-exchange chromatography to discriminate between protein variants differing by a single residue in 275 amino acids.

Immobilized-metal affinity and hydroxyapatite chromatography of genetically engineered subtilisin.

High-performance immobilized-metal affinity and hydroxyapatite chromatography were employed to investigate the engineered subtilisin S1 binding site microenvironment. Although these methods are classified as affinity techniques, unlike traditional affinity columns, both are capable of probing the entire surface of a molecule. The metal chelate study employed gradient elution to assemble retention maps for a wide range of mobile-phase pH. Resolution of single substitution variants was achieved at the optimum mobile-phase pH. A total of four metals were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. Hydroxyapatite chromatography, albeit an established technique, has only recently been developed as a high-performance chromatographic method. Gradient elution separations were performed to determine selectivity. Immobilized-metal affinity chromatography was found to be the more effective method for the separation of site-specific variants.

Surface-mediated retention effects of subtilisin site-specific variants in cation-exchange chromatography.

Wild-type subtilisin and several site-specific variants were resolved on a strong cation-exchange column by isocratic elution and a series of sodium chloride concentrations. Changes in primary sequence at the protein surface have an observable effect on the chromatographic behavior of subtilisin. This supports the concept that three-dimensional structure determines which biopolymer surface residues are in position to interact with the stationary phase surface. The retention data fit the stoichiometric displacement model (SDM) of retention. Plots of ln k' vs. ln 1/[NaCl] yield values for the average number of ionic groups (Z) on the protein that interact with the support matrix. Application of the SDM to the chromatographic retention of the variants has uncovered an unusual phenomenon at the protein surface at low ionic strength. A SDM plot normally provides a linear relationship between ln k' and ln 1/[NaCl] with the slope corresponding to the Z number. This study revealed two lines differing in slope and intercept, indicating that the Z number of subtilisin changes at some intermediate ionic strength of the eluent. These results are attributed to some salt-induced protein surface event that triggers a change in structure. Chromatographic detection of this occurrence reflects the connection between the surface-mediated event and mobile phase ionic strength.

Preparation and evaluation of inorganic anion-exchange sorbents not based on silica.

The use of alternate macroporous (greater than 200 A) inorganic support materials in the preparation of pellicular anion-exchange packings was explored. Alumina, magnesia, titania, and a zirconia-coated silica were chosen for comparison with silica. The stationary phase attached to the support surfaces was an adsorbed polyethyleneimine, crosslinked with 1,4-butanediol diglycidyl ether. Packing materials were characterized by static elemental analyses, chromatographic retention, static loading capacity and pH stability. Titania, alumina, and zirconyl-clad silica packings were found to be substantially more stable under alkaline conditions than silica-based materials. The data show that the stationary phase was successfully bonded in all cases and functioned in anion-exchange chromatography. When the surface area and pore diameter of these alternate materials is equivalent to silica, there is little difference in chromatographic properties.

Follicle regulatory protein noncompetitively inhibits microsomal 3 beta-hydroxysteroid dehydrogenase activity.

A heat- and trypsin-labile follicular fluid protein (FRP) extracted from both human and porcine follicular fluid has been shown to modulate ovarian steroidogenesis. To further investigate the effects of FRP, its effect on the kinetics of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) was evaluated in cell-free microsomal preparations from human placenta. Test fractions of follicular fluid protein were preincubated with placental microsomes followed by the addition of various substrate concentrations (pregnenolone + NAD). Subsequent progesterone formation was interpreted as the velocity of the reaction. The 50% inhibitory dose (ID50) of FRP for 3 beta-HSD for the three substrate concentrations was 300 micrograms/ml. Although a clear decrease in 3 beta-HSD activity typically occurred after pre-incubation with 730 micrograms/ml of FRP, a paradoxical augmentation in 3 beta-HSD activity was present with the lower concentrations of FRP (10-30 micrograms/ml) and the more concentrated microsomal preparations. Double reciprocal plots of these reactions demonstrated a Km for 3 beta-HSD of 1.8-2.1 X 10(-6) M. Analysis of all reactions was found to be consistent with a noncompetitive mode of enzyme inhibition with an apparent Ki of 120 ng/ml or approximately 10(-8) M assuming a mol. wt of 16,000 Daltons for FRP. This derived Ki for FRP is within the biological concentration of FRP in follicular fluid.