Lentil Lectin Affinity Chromatography Research Articles

Induction of secondary cytotoxic T lymphocytes by liposomes containing HLA-DR antigens.

It is evident that the gene products of the major histocompatibility complex (MHC) have a crucial role in the induction of cytotoxic T lymphocytes (CTLs) and as target antigens for CTLs1–6. Assessment of the precise molecular requirements for CTL induction and recognition, however, is difficult when intact cells are used as targets. Recently, in several model systems, it has been demonstrated that, after detergent solubilisation, cell-surface antigens can be isolated that retain the ability to stimulate a secondary CTL response. CTL stimulating activity has been isolated by detergent solubilisation from murine tumour cell plasma membranes7,8 which co-purified with H–2 antigens through lentil lectin affinity chromatography and gel filtration9,10. Highly purified HLA-A and HLA-B antigens, from the human lymphoblastoid cell line JY, have been isolated and incorporated into well characterised liposomes11. Spleen cells from mice primed to JY cells developed cytolytic activity, specific for JY targets, after co-culture with these HLA-liposomes12. In these experiments it was observed that membranes which had been solubilised with deoxycholate and then dialysed to remove the detergent (reconstituted membranes) always stimulated greater cytolytic activity than did HLA-liposomes. At the time it was suggested that this disparity might reflect the loss of other antigens involved in CTL stimulation as a consequence of HLA-A and HLA-B purification. Data are presented here which demonstrate that other antigens are indeed able to induce secondary CTLs, independently of the HLA-A and HLA-B antigens. The results suggest that this activity is present on HLA-DR-associated molecules.

Identification of Ia glycoproteins in rat thymus and purification from rat spleen

AbstractA fraction of la‐like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody‐secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la‐like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I‐A subregion of the H‐2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti‐la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la‐positive B lymphocyte, and 45 000 molecules per la‐positive thymocyte.From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.

Purification with monoclonal antibody of a predominant leukocyte‐common antigen and glycoprotein from rat thymocytes

A leukocyte-common (L-C) antigen which can be dominant as an immunogen in rabbit anti-rat thoracic duct lymphocyte serum has been purified from rat thymocytes. Initially, an antigenic fragment of 100,000 apparent mol. wt. was prepared at 400 to 900-fold purification by lentil lectin affinity chromatography and gel filtration in deoxycholate. Mice were then immunized with this fraction, and a hybrid myeloma cell line secreting antibody to the L-C antigen was prepared by cell fusion. This antibody was used for affinity chromatography and gave pure L-C antigen at 1400-fold purification compared with thymocytes. The L-C antigen is a major membrane glyco-protein of rat thymocytes and has an apparent mol. wt. of 150,000 as determined by electrophoresis on polyacrylamide gels in sodium dodecyl sulfate. The antigen constitutes one of the three thymocyte glycoproteins which stain intensely for carbohydrate with periodic acid Schiff stain. It is present on greater than 95% of thymocytes, bone marrow cells and thoracic duct lymphocytes.

Lentil-Lectin Affinity Chromatography of Surface Glycoproteins and the Receptor for IgE from Rat Basophilic Leukemia Cells

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).

Trypanosoma brucei brucei: Isolation of the major surface coat glycoprotein by lectin affinity chromatography

We have purified soluble glycoproteins associated with Trypanosoma brucei brucei by lentil lectin affinity chromatography. The major surface coat glycoprotein (SCGp) accounts for 87% of the protein in the preparation with approximately 12 other glycoproteins accounting for the rest. This preparation can be further subdivided by DEAE chromatography. The nonabsorbed protein fraction is 95% SCGp and 1 to 2% each of three other glycoproteins. Elution of the DEAE column with a 0.05-0.5 M NaCl gradient yields a broad peak containing the remaining glycoproteins. The purification of the SCGp is 16- and 17.3-fold at each step out of a theoretical 18-fold possible purification. The preparation after lectin chromatography is stable, showing no degradation after standing 72 hr at 4 C. Additional purification procedures are discussed.

The Biochemical characterization of syrian hamster cell-surface alloantigen

The first alloantiserum to be described in Syrian hamsters has been characterized for its ability to react with externally and internally radiolabeled antigens derived from normal hamster lymphoid cells. Utilizing conventional biochemical techniques, radioiodinated and(3)H-leucine labeled cellular extracts have been prepared, partially purified by lentil lectin affinity chromatography, and indirectly immunoprecipitated with experimental alloantisera. Analysis of the precipitated radiolabeled antigens by polyacrylamide gel electrophoresis in SDS has identified two prominent cell-surface proteins at 39,000 (p39) and 29,000 daltons (p29) on 2-mercaptoethanol reduced gels. Further analysis of the radiolabeled extract has demonstrated the existence of hamster cell-surface proteins at 43,000 and 12,000 daltons which are immunoprecipitated by a xenoantiserum directed against human β(2) microglobulin. Coelectrophoretic studies indicate the independent identity of these four species of hamster cellsurface proteins. These results suggest that between two hamster lines, derived from animals caught 40 years apart from different geographic locations in Syria, polymorphism of cell-surface antigens is restricted to p39 and p29 molecular species.

Purification and characterization of a murine tumor cell surface glycoprotein of 75,000 daltons that is related to the major envelope glycoprotein of murine leukemia virus

An antigen which competes with murine C-type viral glycoprotein in an interspecies radioimmunocompetition assay has been purified from the surface of EL-4 tumor cells. The EL-4 tumor cell is virus particle negative. The amount of p30 was extremely low and there was no detectable p12 or p15, confirming that the tumor cell was not expressing a murine oncornavirus. In homologous competition radioimmunoassays, the tumor cell was negative for Rauscher and AKR gp71, but in an interspecies assay employing 125I-Rauscher gp71 and anti-feline leukemia virus serum, there was a reactive antigen. The gp71-like antigen was on the surface of the tumor cell since, by immunofluorescence and immunoelectronmicroscopy, the EL-4 cell could be labeled with antiserum against Rauscher virus gp71. The gp71 cross-reactive antigen was purified by lithium diiodosalycilate extraction, DEAF; chromatography, and lentil lectin affinity chromatography. It is a glycoprotein of about 75,000 daltons which could be precipitated by various broadly reactive antisera to murine gp71s and antisera to murine virus. Based on radioimmunocompetition assays, the purified gp75 was not related to AKR or Rauscher gp71, but did compete in an assay using BALB/2 xenotropic gp71 and anti-C57/L virus serum. It also competed in an assay using 125I-Moloney virus gp71 and anti-Moloney virus serum, but not in a more type-specific assay assay 125I-Moloney virus gp71 and anti-Moloney gp71 serum. Tryptic peptide maps of iodinated proteins were prepared and the cell surface antigen was compared with AKR gp71, Moloney gp71, Rauscher gp71, and xenotropic BALB/2 and NZB gp71s. The EL-4 gp75 was not identical with or very similar structurally to any of the viral gp71s. The differences in the structures of the various viral gp71s shown here and also in other laboratories are consistent with the idea that the env genes of murine viruses which code for gp71s are sites of frequent recombinational events. Recombination between different endogenous viral sequences or between viral and host allelic genomes could have resulted in many immunologically related proteins on viruses, cell surfaces and, in sera of mice, which are immunologically related, about 70,000-dalton molecular weight, but have divergent structure.

Labeling Characteristics and Separation of Ia Antigen Subunits

Experiments with biosynthetic incorporation of 3H-amino acids into murine and guinea pig Ia antigens have indicated that these antigens consist of two polypeptide chains of 33,000 and 25,000 daltons, respectively, occasionally linked by disulfide bonds into a 58,000 dalton molecule. In contrast, studies with lactoperoxidase-catalyzed radioiodination have indicated that these Ia antigens consist of only a single chain of 25,000 daltons. We therefore undertook a study to explore the basis of these discrepant results. Since 3H-tyrosine labeled both chains well, the lack of tyrosine residues in the 33,000 dalton chain could not be the explanation for the lack of radioiodination. However, by partially purifying the Ia antigen preparation with Lens culinaris (lentil) lectin affinity chromatography before immunoprecipitation and by increasing the resolution of analysis by using discontinuous-SDS polyacrylamide gel electrophoresis, it was possible to show that the 33,000 dalton chain was in fact radioiodinated, though still poorly so relative to the 25,000 dalton chain, and that a radioiodinated 58,000 dalton molecule could be detected. These experiments suggest that the 25,000 dalton chain is more exposed to the external cellular environment, and thus more readily iodinated by lactoperoxidase. In addition, the studies indicate that the choice of labeling method, purification procedures, and analytical methods must be taken into account when interpreting experimental results.