Identification of the naturally processed form of hen egg white lysozyme bound to the murine major histocompatibility complex class II molecule I-Ak.

Abstract

A murine B-cell lymphoma bearing the class II major histocompatibility complex molecule I-Ak was cultured with the protein antigen hen egg white lysozyme (HEL). The I-Ak molecules were purified, and their associated peptides were extracted for characterization. Five HEL peptides were identified. Four contained the 10 amino acid residues HEL 52-61 (DYGILQINSR) but were heterogeneous in length and flanking residues. This core sequence is known to confer a high binding affinity for I-Ak. ). An understanding of the mechanisms involved in determinant selection might be gained from an examination ofthe end products of antigen processing. Toward this end, the biochemical characterization of physiologically processed antigen in the form of peptides eluted from affinity-purified class I (8-10) and class II (11-14) MHC molecules has been initiated. The eluted peptides were found to be of low molecular weight and to have limited heterogeneity. This small size was in agreement with that of synthetic peptides, which substitute for processed antigen (15, 16). The naturally processed peptides isolated from class I molecules were 8 or 9 amino acids in length with fixed amino-and carboxylterminal ends and were derived from proteins synthesized in the cell cytosol. In contrast, peptides extracted from class II molecules were from 13 to 17 amino acids in length with variable carboxyl-terminal ends, and most were derived from cellular transmembrane glycoproteins. One peptide was recovered from bovine serum albumin, a protein found in the culture medium used to grow the cells (13).Studies in our laboratory have used hen egg white lysozyme (HEL) as a model antigen for processing and presentation by APC. HEL, a soluble nonglycosylated protein 129 amino acids in length, contains four intrachain disulfide bonds and requires intracellular processing for T-cell recognition. Analysis of the T-cell response to HEL in several mouse strains revealed that some regions were favored over others (17,18). We previously showed that the major component of the CD4 T-cell response to HEL in H-2k mice was directed toward the 10 amino acid residues 52-61 (19, 20). These residues lie partially buried inside the native molecule in a (3-sheet conformation near the active site of the enzyme. Peptides containing residues 52-61 of HEL bind with high affinity to I-Ak (5). The HEL peptide 52-61 contains amino acid residues that contact the I-Ak molecule interspersed with amino acid residues that contact the T-cell receptor (20).We are now using this well-characterized model antigen to investigate determinant selection and here report the sequences of physiologically presented HEL peptides eluted from I-Ak after processing by an APC line. MATERIALS AND METHODSIsolation of I-Ak-Associated Peptides. Peptide-loaded MHC class II molecules were purified from the I-Ak-expressing B-lymphoma line M12.C3.F6. This line was produced by DNA-mediated cotransfection of genomic clones for the Ak a and (3 chains into M12.C3, an Ia-negative B-lymphoma line (21). M12.C3.F...

Identification of naturally processed peptides bound to the class I MHC molecule H‐2Kd of normal and TAP‐deficient cells

“…The long peptides thus represent an alternative mode of processing and presentation of cytosolic proteins. Others and we have made a point that peptides derived from cytosolic proteins are found bound to class II MHC molecules [8,10,14,55]. The process that transports peptides from cytosol to either class I or II MHC processing is likely to be similar.…”

“…To understand the nature of peptides displayed by a particular MHC allele, it is important to identify the preferred peptide-binding motifs that allow interaction with the peptide-binding groove of the MHC molecule. Two broad approaches have been employed in this pursuit: synthesis of peptide libraries that contain motifs important for binding to MHC molecules (for example see [1][2][3][4]), and direct isolation and identification of naturally processed peptides selected by MHC molecules present on the cell surface of APC (examples in [5][6][7][8][9][10]). Although the second approach is more demanding, it has several advantages, most important of which is that the identified peptides are the physiological naturally presented peptides.…”

The Pathway of Antigen Uptake and Processing Dictates MHC Class II-Mediated B Cell Survival and Activation

“…Both 3A9 T cells and the C4H3 mAb recognize the peptide-MHC complex (16,31). Using a B cell hybridoma, four HEL peptides varying in length, but containing the core dominant sequence 52-61, were found complexed to I-A k , in addition to one peptide, HEL 48 -60 (32). The 3A9 T cell hybridomas appear to recognize less well epitope 52-61 compared with 48 -61, whereas other peptides from the core sequence were not recognized (31).…”

Specificity of peptide selection by antigen-presenting cells homozygous or heterozygous for expression of class II MHC molecules: The lack of competition

“…As described in previous studies, many of the peptides emerge as families in which all of the members share the same core sequence, along with varying lengths of flanking residues on the amino and carboxy termini (8,(26)(27)(28)(29). (We define as ''core'' the 9-aa stretch from the P1 to the P9 binding sites or pockets.…”