Evidence for tryosine peptide homologies in the HLA antigens system.

Abstract

The tetrameric HLA antigens are composed of two heavier chains which carry the alloantigenic determinants and two lighter chains identified as #2-microglebulin.Although at least 40 different antisera are required to define the varying HLA specificities, it appears that these antigens may be closely related to each other and to the immunoglobulins. Through the use of a new electrophoretic technique, which is able to compare simultaneously the tyrosine peptides produced from radioiod nated cell surface proteins, this report gives evidence that HLA antigens of the three chromosomal loci may have similar amino-acid sequences. Since the retention of homologous tyrosine residues and a tendency for sequence preservation surrounding these residues are features of immunoglobulin structure, this may indicate that similarly conservative evolutionary mechanisms have been operative in the HLA allelelic proteins or that immunoglobulins and HLA antigens may indeed have a common evolutionary origin.The serologically determined histocompatibility antigens are tetrameric structures composed of two polypeptide chains of molecular weight 45,000, and two light chains of molecular weight 11,500, now identified as , 32-microglobulin (,62-m) (1-4). The larger component carries the serologically identified alloantigenic determinants (5). Although at least 40 different antisera are required to define the varying specificities of the HLA antigens, accumulating evidence from several sources has suggested that a rather limited degree of chemical heterogeneity may actually be present (6-8). This investigation was undertaken to compare HLA antigens of different specificities, using a newly devised method for the chemical analysis of microgram amounts of lactoperoxidase radioiodinated cell surface antigens. Since lactoperoxidase binds [125I]iodine to tyrosine residues (9), the electrophoretic technique described here permits direct comparison of radioiodinated cell surface proteins, based upon the number and mobility of tyrosine peptides. In particular, this report presents evidence that antigenic products of the first, second, and third HLA chromosomal locit may have substantial homology of primary amino-acid sequence (10). High Voltage Electrophoresis. The radiolabeled HLA immune complexes were washed three times in phosphatebuffered saline, dissolved in 5 M urea-0.1 M mercaptoethanol, and incubated at 370 for 30 min. After dialysis against 5% formic acid overnight (using viscose dialysis tubing which retains proteins exceeding 10,000 daltons), the complexes were digested with pepsin (EC 3.4.23.1), 2% by weight (twice crystallized, Wprthington) at 370 for 16 hr, and then dried under reduced pressure over NaOH. After reconstitution in 15% NH4HCO3, L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone trypsin (EC 3.4.21.4), 2% by weight (Worthington), was added, and digestion was continued for 4 hr at 370. The digested proteins were then dried under reduced pressure over P205. Radioactivity was determined in a Packard Auto Gamma Spect...