John Smart

Acute lymphoblastic leukaemia associated antigen—II. Isolation and partial characterisation

Abstract An acute lymphoblastic leukaemia (ALL) associated antigen has been isolated and purified from leukaemic cells and established leukaemic cell lines. Under reducing conditions the antigenic determinants are identified on a single glycosylated polypeptide with an apparent molecular weight in 10% SDS polyacrylamide gels of 100,000 daltons. The cellular specificity of this molecular species parallels that of the ALL membrane antigen previously defined by antibody binding criteria on whole cells (see previous paper). Leukaemic cell lines release the 100K molecule into the culture medium in a soluble form and one line (MOLT-4) produces these molecules but has no detectable cell surface expression.

Three species of polyoma virus tumor antigens share common peptides probably near the amino termini of the proteins

Detergent extracts of polyoma virus-infected mouse cells contain three major proteins of approximately 100,000--108,000 (100K), 55,000 (55K) and 21,500 (22K) daltons, which react with sera obtained from rats carrying tumors induced by the virus. A comparison of the 35S-methionine-, 3H-leucine- and 3H-proline-labeled tryptic peptides of each of these proteins by cation-exchange chromatography followed by descending paper chromatography has shown that: at least five peptides are shared by all three T-reactive proteins; at least three peptides are shared by the 55K and 22K proteins, but not by the 100K protein; at least three peptides are found only in the 22K protein; at least six peptides are found only in the 55K protein; and at least sixteen peptides are found only in the 100K protein. The results are consistent with the hypothesis that the polypeptide chains of the 100K, 55K and 22K dalton tumor antigens of polyoma virus share a common virus-coded amino terminal region. The data also suggest that there is a portion of the polypeptide chains (probably immediately adjacent to the common amino terminal region of the molecules) that is shared by the 55K and 22K proteins, but not by the 100K protein (perhaps because this portion of the genetic information is spliced out of the messenger RNA coding for the 100K protein). The facts that all the peptides common to the 100K and 55K proteins are also found in the 22K protein and are thus assigned to the common amino terminal region of the molecules, and that there are several peptides unique to the 100K protein, as well as several peptides unique to the 55K protein, suggest that the presumed carboxy terminal portion of the polypeptide chain of the 100K protein is considerably, if not entirely, different from that of the 55K protein.

Polypeptides of feline leukemia virus: a glycosylated gag-related protein is released into culture fluids

Cells infected with feline leukemia virus (FeLV) shed viral antigens into cell culture fluids. Immunoprecipitation and SDS-PAGE analyses of radiolabeled antigens show that the major virus-specific protein released is a molecule of 40,000 molecular weight which carries gag gene product antigenic determinants. The 40,000 molecular weight (40K) protein was found to be glycosylated and was precipitable with antisera to FeLV gag proteins p27, p12, and pl0 but not p15. Tryptic peptide mapping confirmed the relatedness of FeLV p27 and the 40K glycosylated protein, which was found to be released from all FeLV-infected cells, including untransformed embryonic fibroblasts and transformed lymphoblastoid cells. These results suggest that the FeLV gag gene proteins, like those of murine leukemia viruses, can be processed in two distinct pathways and that this is independent of cell type.

Alteration in membrane glycoproteins after type-C virus infection of murine fibroblasts.

NEW proteins and glycoproteins appear on cell membranes after infection of the cells by type-C viruses1. Some of these new proteins are virus coded and are positioned in the membrane away from the sites of virus budding2. GP70, the major murine glycoprotein has been identified serologically on the membranes of virus-shedding cells3, transformed but non-virus-producing cells4, and on some chemically induced sarcomas5. The internal core protein p30 has been identified as a common antigen on membranes of type-C virus-infected cells of many species6. It has been suggested that this protein, possibly a product of degraded virus, can bind nonspecifically to virus-shedding cells5. Non-virion virus-coded antigens have also been described. The best characterised tumour-specific surface antigen (TSSA) is that found on avian sarcoma cells7. It consists of a major fucose-containing glycoprotein of molecular weight 100,000 and possibly a minor glycoprotein of 32,000. These glycoproteins do not cross react antigenically with the avian virion glycoproteins. A similar antigen, the feline oncornavirus membrane antigen (FOCMA), has been described on virus-infected feline cells8. This antigen has not been well characterised but it is also thought not to be related to the virion proteins. Evidence has been sought largely unsuccessfully for a comparable TSSA in murine model systems9. There have, however, been a few reports of sarcoma specific antigens10. In this paper we describe alterations which occur in membrane glycoproteins after infection of murine fibroblasts by Moloney leukaemia–sarcoma virus (MSV–MLV-M) and by an N-tropic virus isolated from leukaemic AKR mice (MLV–AKR). The technique of lectin chromatography is used to fractionate glycoproteins from other membrane proteins enabling better characterisation of these important surface molecules.

Structural similarities of proteins encoded by three classes of avian sarcoma viruses

The structure and location of the phosphorylation sites of a number of avian sarcoma virus polyproteins have been examined by protease cleavage analysis. The PRCIIp and PRCII polyproteins, P170gag-fps and P105gag-fps yield indistinguishable cleavage fragments from an N-terminal region of 65,000 molecular weight, including the gag/non-gagjunction. This provides strong support for the view that PRCII arose directly from PRCIIp by a genomic deletion. For P909agag-yes, P800gag-yes, and P105gag-fps the major tyrosine phosphorylation sites are on C-terminal fragments of 27,000, 26,500, and 36,000 molecular weight, respectively. Further similarities have been shown by partial sequence analysis of the tyrosine phosphorylation sites; the positions of trypsin and staphylococcal V8 protease cleavage sites largely correspond in the src, fps, and yes gene products. The homology between the src and yes products is particularly striking. They yield C-terminal V8-resistant fragments of similar size, containing the major tyrosine phosphorylation sites which are indistinguishable after further cleavage with several proteases. These results suggest structural and functional relatedness between the src, fps, and yes gene products despite the lack of hybridization between their DNA sequences.

The detection of glycoproteins immunologically related to RSV gp85 in uninfected avian cells and in sera from uninfected birds.

Abstract The experiments described in this study deal with the detection of components immunologically related to oncornaviral glycoprotein (gp85) in uninfected cell extracts and sera. Cell extracts from chick helper factor- (chf) negative fibroblasts contain components detected by two of the three antisera used. Chf(+) chicken cell extracts contain in addition an antigenic determinant, presumably viral gp85, recognized by the third antiserum. The sera from uninfected chickens may contain two components immunologically related to gp85: One component recognized in a radioimmuno-precipitation assay (RIA) employing anti-AMV gp85 serum is present in high amounts in the sera of chf(+) and chf(-) birds, while a second component detected in a RIA employing anti-Prague B RSV serum is present only in the sera of chf(+) birds. Some biochemical studies dealing with the properties and size of the cross-reacting components are described. It is postulated that the components which immunologically cross-react with, but are not identical to, endogenous viral glycoprotein are products of genes related to the env gene of RAV-0 which, however, are nonfunctional as viral glycoprotein.