Chicken major histocompatibility complex class II molecules of the B haplotype present self and foreign peptides.

The chicken major histocompatibility complex (MHC), or B-complex, mediates genetic resistance and susceptibility to infectious disease. For example, the B19 haplotype is associated with susceptibility to Marek's disease. Here, we describe the sequencing and analysis of peptides presented by B19 MHC class II molecules. A B19/B19 B-cell line was used for the immunoaffinity purification of MHC class II molecules, which was followed by acid elution of the bound peptides. The eluted peptides were then analysed using tandem mass spectrometry. Thirty peptide sequences were obtained, ranging from 11 to 25 amino acids in length. Source protein cellular localization included the plasma membrane, cytosol and endosomal pathway. In addition, five peptides from the envelope glycoprotein of chicken syncytial virus (CSV) were identified. Chicken syncytial virus had been used as a helper virus along with reticuloendotheliosis virus strain T for transformation of B19/B19B cells. Alignment and analysis of the peptide sequence pool provided a putative peptide-binding motif for the B19 MHC class II.

Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide antigen.

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.

Hb(64-76) epitope binds in different registers and lengths to I-Ek and I-Ak.

The nature of peptide binding to MHC molecules is intrinsically degenerate, in what, one given MHC molecule can accommodate numerous peptides which are structurally diverse, and one given peptide can bind to different alleles. The structure of the MHC class II molecules allows peptides to extend out of the binding groove at both ends and these residues can potentially influence the stability and persistence of peptide/class II complexes. We have previously shown that both I-E(k) and I-A(k)-restricted T cell hybridomas could be generated against the Hb(64-76) epitope. In this study, we characterized the binding register of the Hb(64-76) epitope to I-A(k), and showed that it was shifted by one residue in comparison to its binding to I-E(k), and did not use a dominant anchor residue at P1. This conclusion was further supported by the modeling of the Hb(64-76) epitope bound to I-A(k), which revealed that all of its putative anchor residues fit into their corresponding pockets. We identified the naturally processed Hb epitopes presented by both I-E(k) and I-A(k), and found that they consisted of different species. Those associated with I-A(k) being 20-22 residues long, whereas, those found to I-E(k) contained 14-16 residues. These findings suggested that the lack of a dominant P1 anchor could be compensated by the selection of longer peptides. Overall, these studies revealed the Hb(64-76) epitope bound to I-E(k) and I-A(k) in distinct registers and lengths, demonstrating the plasticity MHC molecules have in generating distinct TCR ligands from the same amino acid sequence.

Isolation and quantitation of a minor determinant of hen egg white lysozyme bound to I-Ak by using peptide-specific immunoaffinity.

We report here the identification and quantitation of a minor epitope from hen egg white lysozyme (HEL) isolated from the class II MHC molecule I-Ak of APCs. We isolated and concentrated the peptides from the I-Ak extracts by a peptide-specific mAba, followed by their examination by electrospray mass spectrometry. This initial step improved the isolation, recovery, and quantitation and allowed us to identify 13 different minor peptides using the Ab specific for the HEL tryptic fragment 34-45. The HEL peptides varied on both the amino and carboxy termini. The shortest peptide was a 13-mer (residues 33-45), and the longest peptide was a 19-mer (residues 31-49). The two most abundant were 31-47 (1.3 pmol) and 31-46 (1 pmol), while the least abundant were 31-45 (40 fmol) and 32-45 (4 fmol). Only 0.3% of the total class II molecules were occupied by this family of HEL peptides. The amount of the 31-47 peptide, the predominant member of this series, was 22 times lower than that of 48-62, the major epitope of HEL. The 31-47 peptide bound about 20-fold weaker to I-Ak compared with the dominant 48-62 peptide. Thus, the lower abundance of the minor epitope correlated with its weaker binding strength.

Structures and characteristics of novel siderophores from plant deleterious Pseudomonas fluorescens A225 and Pseudomonas putida ATCC 39167.

When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH. P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH. Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH. All four of the pseudobactins contain a dihydroxyquinoline-based chromophore. The amino acid sequence for the octapeptide in case of pseudobactins from P. putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr. In case of pseudobactins from P. fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr. For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond. Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I. For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore. The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine. The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different. The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms. The receptor proteins in the two species are similar but not identical.

Presentation on class II MHC molecules of endogenous lysozyme targeted to the endocytic pathway.

We compared the processing and presentation of the model Ag, hen-egg white lysozyme (HEL) expressed in C3.F6 APC as a fusion protein to three different acid hydrolases: cathepsin D, to an unglycosylated form of cathepsin D, and to pepsinogen. As expected from the biology of mannose 6-phosphate (Man-6-P)-containing enzyme, cathepsin D-HEL was delivered to the endosomal/lysosomal system. In contrast, the unglycosylated cathepsin D-HEL was retained in ER/Golgi and some was found in lysosomes. Most of pepsinogen-HEL was rapidly secreted from the APC. All transfectants presented HEL epitopes to T cell hybridomas. Regardless of the main route of traffic of the proteins, the strong I-Ak binding epitope HEL 48-62 was well presented by all. The biochemical forms of this epitope were identical for all. Three other epitopes of HEL that bind I-Ak with less affinity were processed equally well by unglycosylated cathepsin D-HEL and HEL-Ld. The glycosylated cathepsin D-HEL was less efficient in generating the 114-129 epitope. Pepsinogen-HEL was the less efficient of all transfectants in presenting these subdominant epitopes. Soluble cathepsin D-HEL recovered from culture supernatant was strongly immunogenic when added to C3.F6. The uptake was inhibited by free Man-6-P, indicating that the surface Man-6-P receptor can effectively deliver proteins to the class II MHC system.

Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC) class II molecules bind antigenic peptides for display to T lymphocytes. Although the enzymes involved remain to be identified, it is commonly believed that class II associated peptides are released from intact antigens through a series of proteolytic steps carried out inside antigen presenting cells. We have examined the effect of amino acid substitutions on proteolytic processing of the model antigen hen-egg lysozyme (HEL). Altered HEL molecules, engineered by site-directed mutagenesis of a HEL cDNA, were expressed as separate stable transfectants in a B cell lymphoma line. Each transfectant processed a different mutant HEL protein for presentation on MHC class II. We purified the resulting class II-associated peptides and analyzed them by mass spectrometry. Our results strongly support the hypothesis that antigen processing continues after peptide binding to the MHC class II molecule and are most consistent with a scenario in which long peptides first bind to MHC class II and are then trimmed by exopeptidase.