Abstract
Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.
Highlights
Some HLA-B heavy chains and the HLA-DR heavy chain have a cysteine in the hydrophobic transmembrane region, pencillinase from Bacillus licheniforrnis as well as other bac- while other MHC antigen chains, such as some HLA-A antiterial membrane proteins
- but only the M, 44,000 band is labeled with ['H]palmitic acid. This same tritiated band is immunoprecipitated by an anti-P2-microglobulin xenoantiserum(alongwithanother band at theposition of /32-microglobulin;lanes 5 and 11) and from denatured lysates by antidenatured class I heavy chain xenoantiserum
This tritiated band is immunoprecipitated by the monoclonal antibody BB7.1,but not by PA2.1.The fact that B7 heavy chains but notA2 heavy chains are labeled by cysteine 123456 palmitate 7 8 9 10 11 12
Summary
0.1 mM EDTA was terminated by 10 pl of 100 mM PMSF in ethanol. Isolation and Analysis of Pronase Peptides-0.5-ml aliquots of Reagents-NP-40 (Particle Data, Elmhurst, IL), gel electrophore- detergent-soluble lysates corresponding to 5 x lo biosynthetically sis reagents including SDS (Bio-Rad, Richmond, CA), papain (Wor- labeled cells were proteolyzed with 5 ml of 1mg/ml trypsin in 20 mM thington, Freehold, NJ), a-chymotrypsin, and diphenyl carbamyl TrisC1, pH 8,O.l mM EDTA for 3 hat 37 "C and incubated with chloride-treated trypsin Immunoprecipitation and Gel Electrophoresis-Samples were precleared with normal rabbit serum and formalin-fixed Staphylococcus to various pH values (pH 5, 7, 9,or 11). 190 plof 100 mM TrisCI, pH 7, and 960 p1 of ch1oroform:methanol (2:l) were added, the samples were thoroughly vortexed, and the aqueous phase was removed. The plate was denatured material, 10 pI of 1 mg/ml ovalbumin was added to 50 pl scraped to give aliquots each corresponding to 1 cmwhichwere of lysate, the sample was precipitated with 0.5 mlof cold acetone, analyzed by scintillation counting using Aquasol. After incubation at were boiled in a modified Laemmli sample buffer and analyzed by room temperature for 1 h, 40 p1 of 10 mg/ml ovalbumin was added electrophoresis in 20-cm-long 7-15% polyacrylamide gradient gels and thesamples were precipitated with cold acetone. Digestionwas terminated by trichloroacetic acid precipitation and the samplewsere analyzed by SDS gel electrophoresis
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