Highly Purified Papain - Solubilized HL - A Antigens Contain # 2 - Microglobulin ( polypeptide chains / spleen - cell membranes / histocompatibility determinants ) PER

HL-A antigens comprising 11 different antigenic specificities were isolated after papain solubilization of spleen-cell membrane constituents. During the entire purification procedure, /32-microglolbulin appeared together with the HL-A antigens. The highly purified antigens were composed of two polypeptide chains. The large subunit carried the antigenic specificity whereas the small polypeptide chain was very similar, if not identical, to f32-nmicroglobulin. The two HL-A antigen polypeptide chains were held together by noncovalent interactions only, and 32-mnicroglobulin, isolated from urine, could replace the small subunit in forming a complex with the large polypeptide chain. The topographical relationship in the cell menibrane between 32-microglobulin and the large HL-A antigen polypeptide chain is unknown. The two polypeptide chains may be fortuitousdy bound as a result of the solubilization procedure. The major human histocompatibility determinants, HL-A antigens, are cell-surface markers present on lymphocytes and other nucleated cells. The great polymorphism exhibited by the histocompatibility antigens has severely impeded their isolation and chemical characterization. Various methods have been used to solubilize the cell surface-bound glycoproteins with serologic HL-A activity (1). The current methods used most successfully involve proteolytic treatment with papain, sonication, or extraction with high concentrations of salt (2-4), and apparently yield HL-A antigens differing in physical-chemical characteristics (4-6). Papain-solubilized HL-A antigens contain two polypeptide chains, one carrying the serologic activity and the other without serologically detectable HL-A antigenic determinants (6, 7). The latter of the two polypeptide chains has an estimated molecular weight of 10,000-12,000 (6, 7) and occurs in serum and urine (7, 8). We earlier demonstrated that. the lowmolecular-weight plasma proteinB2-microglobulin, with molecular weight 11,800, is present on the surface of leukocytes (9). Amino-acid sequence analysis of this protein has revealed extensive homology with heavy and light chains of immunoglobulin G (9, 10). It was suggested that 02-microgiobulin is a free immunoglobulin domain that evolved before the gene duplication events giving rise to regular immunoglobulin polypeptide chains (9). Later work is compatible with this idea, since 02-microglobulin is manufactured by various mesenchymal and epithelial human cell lines (11). Furthermore, no correlation between immunoglobulin synthesis and 02-microglobulin production has been found (11-13). The occurrence of 02rmicroglobulin on the cell membrane and the recently reported subunit structure of HL-A antigens warranted a study of the possible relationship between HIA antigens and #2-microglobulin. In this communication data are presented that demonstrate that ,B2-microglobulin constitutes one of the two polypeptide chains that occur in highly purified papain-solubilized HL-A antigens. MATERIALS AND METHODS Spleens were surgically removed from three male and one female donors due to hemolytic anemia or esophageal cancer. Assay of HL-A Specificities and [r2Microglobulin. Soluble HL-A antigens were assayed by their ability to inhibit immune cytolysis measured by trypan blue uptake (14). #2-Microglobulin was determined by a solid-phase radioimmunoassay (15). The specific antisera directed against HL-A were obtained from multiparous women. Antiserum against 02microglobulin was raised in rabbits (16). Isolation of HL-A Antigens. Four preparations of HL-A antigens were made. Briefly, a crude spleen-cell membrane fraction was suspended in 0.01 M Tris HCl buffer, pH 8.1, containing 5 mM cysteine and the protein concentration was adjusted to 10 mg/ml. To this membrane fraction, 2 mg of twice crystallized papain (20 units/mg, Sigma) were added per ml and proteolysis proceeded for 30 min at 370. The supernatant recovered after centrifugation of the papain-digested membrane fraction was subsequently dialyzed against 4 mM sodium phosphate buffer, pH 6.0, and fractionated on a carboxymethyl-cellulose column. The column was eluted in steps with (i) the equilibrating buffer, (ii) the equilibrating buffer containing 0.15 AM NaCl, and (iii) the equilibrating buffer containing 1.0 M NaCl. All the recovered HLA antigenic activity as well as all f2rmicroglobulin were obtained in the first fraction. The material was further purified on a column of Sephadex G-200 equilibrated with 0.02M Trise HCI buffer, pH 7.4, containing 0.15 M NaCl. The material exhibiting HL-A antigenic activity was eluted in the molecular weight region of 40,000-50,000 as a singlp peak coincident with the elution position for the first of two #2rmicroglobulincontaining peaks. The second 02-microglobulin peak, comprising less than one-third of the total, appeared in the same position as urinary /32-microglobulin. Rechromatography of the HLA antigenic material on a column of Sephadex G-100 gave rise to two protein peaks, the latter one of which contained all 02-microglobulin and HL-A serologic activity. Various analyses performed on the material constituting the second protein peak indicated that it contained EL-A antigenic material of about $0-9Q% purity. 35 Abbreviation: SDS, sodium dodecyl sulfate. 36 Immunology: Peterson et al.