Evolutionary relationship between immunoglobulins and transplantation antigens.

154

Cell surface proteins of cultured cells are disulfide bonded to a greater degree than are total cellular proteins. In particular, the "large external transformation-sensitive" (LETS) protein, a major surface protein, is present almost exclusively in disulfide-bonded complexes including homodimers and also higher aggregates held together bydisulfide bonds or noncovalent interactions. Other cell surface proteins also appear to be involved in disulfide bonding, both intramolecular and intermolecular. In virally transformed cells, LETS protein and its disulfide complexes are absent and certain other disulfidebonded proteins are also not observed. Much recent research suggests that the surface of mammalian cells undergoes a change in organization on oncogenic transformation. Electron microscopic studies show altered surface morphology (1, 2). Studies with lectins indicate that the surface proteins of many transformed cells have greater freedom of lateral mobility than do those of their normal counterparts (3). It has been suggested frequently that these changes in surface properties are related to changes in cytoskeleton (4-8). Alterations in the organization of surface proteins also could be due to interactions between surface molecules (9). Whichever is the case, it is necessary to learn more about the molecular nature of the cell surface and the changes in its structure that occur on transformation. Numerous investigations have been made of cell surface proteins in normal and transformed cells (reviewed in ref. 10). The most prominent alteration in cell surface proteins is loss of a large external transformation-sensitive (LETS) glycoprotein (10-14). Few investigations have dealt with interactions between cell surface proteins except in erythrocytes (15-18). We report here that the surface of normal mammalian cells has extensive disulfide bonding, particularly involving the LETS protein. MATERIALS AND METHODSCells and Culture Conditions. The normal hamster cell line, NIL8, and a derivative transformed by hamster sarcoma-virus (NIL8-HSV) were cultured as described (11). Cultures were used as confluent monolayers. For labeling, cells were allowed to incorporate [35S]methionine (22 Ci/mmol) or [3H]glucosamine (10 Ci/mol; New England Nuclear) at 50 and 25 taCi/ml.The medium was modified by decreasing the methionine or glucose concentrations to 10% of the usual levels to improve incorporation.Lactoperoxidase-Catalyzed lodination. This was carried out on monolayers as previously described (11); this procedure labels only surface proteins in these cells.Oxidation of Sulfhydryl Groups. Oxidation to disulfide bonds was carried out with o-phenanthroline and copper sulfate (17,18) (18,20), samples were run in 3-mm-diameter cylindrical gels. The first-dimension cells were placed at the top of a slab gel and sealed in place with 0.5% (vol/vol) agarose in electrophoresis buffer. For reduction prior to the second dimension, the agarose contained 10% 2-mercaptoethanol. Markers for the second dimension were applied polymer...

Section: Extraction Of Surface Proteins Is Promoted By Reducingmentioning

confidence: 99%

Extensive disulfide bonding at the mammalian cell surface.

Hynes¹,

Destree²

1977

154

“…Indeed, the V (variable) and C (constant) regions of immunoglobulins demonstrate no primary sequence homologies, but they have very similar three-dimensional structures. In this regard, if the transplantation antigens actually turn out to consist of "heavy" (45,000 dalton) and "light" (12,000 dalton) chains (17) that fold into immunoglobulin-like domains (25), perhaps the contact residues between the two chains would be structurally homologous to their immunoglobulin counterparts from the VL and VH doThis simple calculation assumes that any of the twenty different amino acids are equally likely to appear at any of the residue positions. Hence the probability that any two random sequences are as closely related as indicated in Fig.…”

Section: )mentioning

confidence: 99%

“…We have recently suggested that alleles (or allotypes) can be divided into two categories (27). Alternative forms of simple allotypes segregate in a Mendelian fashion in mating studies and differ by one or a few amino-acid substitutions [e.g., the Inv marker of the human K chain (25)]. In contrast, alternative forms of complex allotypes differ by multiple amino-acid residues and generally segregate in a Mendelian fashion [e.g., the group a and group b allotypes of the rabbit (see ref.…”

Section: )mentioning

confidence: 99%

See 1 more Smart Citation

Structure and evolution of transplantation antigens: partial amino-acid sequences of H-2K and H-2D alloantigens.

Silver¹,

Hood²

1976

“…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)(7)(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.…”

mentioning

confidence: 99%

Evidence for tryosine peptide homologies in the HLA antigens system.

Cunningham‐Rundles¹,

Jersild²,

Svejgaard³

et al. 1975

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...