The sites in the I-Ak histocompatibility molecule photoaffinity labeled by an immunogenic lysozyme peptide.
The class II histocompatibilty molecule I-Ak was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled alpha and beta chains were digested with protease from Staphylococcus aureus strain V-8 (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a major labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)-iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-Ak. The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicating that the photoaffinity labeling was site-specific. Conversely, the NH2-terminal conjugate iodo-4-azidosalicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-Ak. The labeling was also site-specific because reverse-phase HPLC of primary digests with protease V-8 and secondary digests with trypsin showed single major labeled components. The labeling of I-Ak by IASA-(46-61) was fully inhibitible by HEL-(46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment. The site of the I-Ak molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was determined. IASA-(46-61) labeled selectively at Pro-118 of a primary alpha chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary beta chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46-61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the sequence 52-61 and also competed for the binding of this smaller peptide to I-Ak. Thus, peptides that bind to the allele-specific binding site and are long enough to extend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both alpha and beta chains.
Luescher
I F
IF
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Crimmins
D L
DL
Schwartz
B D
BD
Unanue
E R
ER
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010777:Photochemistry; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D009113:Muramidase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin