Colorectal cancer-associated nuclear antigen

By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with M(r) of 35-40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix.     

Biosynthesis of the cancer-associated sialyl-Lex determinant in human amniotic fluid

Biosynthesis of the sialyl-Lex determinant (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)-GlcNAc beta 1-3-R) in human amniotic fluid has been shown to proceed via the same sequence of glycosylation steps established previously for lung carcinoma PC 9 cells (Holmes, E. H., Ostrander, G.K. & Hakomori, S. (1986) J. Biol. Chem. 261, 3737-3743): sialylation of type-2-chain-precursor substrates (paragloboside) by an amniotic alpha 2-3-sialyltransferase precedes fucosylation of sialylated intermediates (sialosyl paragloboside) by an organ-characteristic alpha 1-3-L-fucosyltransferase.     

Biosynthesis and glycosylation of the carcinoembryonic antigen.

Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.     

Biosynthesis of enterobacterial common antigen.

Cultures of Salmonella typhimurium pulse-labeled with N-acetyl-D-[3H]glucosamine ([3H]GlcNAc) incorporated isotope into a GlcNAc-linked lipid that was tentatively identified as GlcNAc-pyrophosphorylundecaprenol. The incorporation of [3H]GlcNAc into this compound was abolished when cells were pulse-labeled in the presence of the antibiotic tunicamycin. Tunicamycin also abolished the in vivo synthesis of the haptenic form of enterobacterial common antigen (ECA) in S. typhimurium as determined by the passive hemagglutination test. These data indicated that the synthesis of the GlcNAc-linked lipid is related to ECA synthesis. Support for this conclusion was provided by the following observations. Cultures of Escherichia coli and S. typhimurium incorporated [3H]GlcNAc into cell envelope components that migrated as a homologous series of polymers when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The [3H]GlcNAc-labeled polymers were not detected in mutants of E. coli and S. typhimurium defective in ECA synthesis due to lesions in either the rfe or rff gene clusters. These polymers were identified as ECA based on Western blot analyses employing anti-ECA monoclonal antibody. The incorporation of [3H]GlcNAc into ECA polymers was abolished by tunicamycin when the drug was added to cultures to give a minimum concentration of 3 micrograms/ml. In addition, pulse-chase experiments provided evidence for a precursor-product relationship between the GlcNAc-linked lipid and ECA. These results strongly suggest that the GlcNAc-linked lipid is involved in the biosynthesis of ECA in a manner analogous to the role of carrier lipid in the biosynthesis of O-antigen and peptidoglycan.     

Biosynthesis of mouse Thy-1 antigen

The biosynthesis and the maturation of Thy-1 antigen of mouse thymocytes have been studied by using a xenogeneic rabbit anti-mouse Thy-1 antibody. The earliest form of Thy-1 detected after a 5-min pulse with [35S]methionine and [35S]cysteine had an apparent m.w. of 26,500. During chase, this band converted to a molecular ratio (Mr) = 25,000 polypeptide, probably derived from the latter by trimming of glucose or mannose residues from the three high-mannose glycan units of Thy-1. Mature Thy-1 molecules were detected at the cell surface after a 15-min chase. At least one of the three N-linked oligosaccharide units was shown to be in the high mannose form at the cell surface, as indicated by its susceptibility to endo-beta-N-acetylglucosaminidase H digestion. Treatment of the early and late forms of Thy-1 antigen with endo-beta-N-acetylglucosaminidase F generated a single polypeptide of Mr = 13,500. The same precursor was obtained when cells were labeled in the presence of tunicamycin. This indicates the absence of O-linked glycan in the mature cell surface antigen. Finally, the resistance of Thy-1 antigen to trypsin digestion when associated with membranes confirmed that this molecule has no cytoplasmically oriented portion.     

Novel oligosaccharides with the sialyl-Lea structure in human milk

We have isolated four novel oligosaccharides with the sialyl-Lea structure from human milk using a monoclonal antibody, MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-performance liquid chromatography. The results of structural analyses, i.e., 500-MHz 1H NMR spectroscopy, fast atom bombardment mass spectrometry, and binding to specific anticarbohydrate antibodies, are consistent with the following structures. (formula; see text)     

Biosynthesis and processing of murine T-cell antigen receptor

The antigen-specific receptor of C6VL T-lymphoma cells is a disulfide-linked heterodimer composed of 39 kd alpha chain and a 41 kd beta chain, both of which exhibit charge microheterogeneity. Pulse-chase labeling experiments indicate that epitopes reactive with the anti-receptor xenoantiserum #8177 were detectable by 2 min, while the clonotypic epitope reactive with monoclonal antibody 124-40 was not detectable until 10 min. Digestion with endoglycosidases H and F revealed that both subunits have at least three N-linked oligosaccharide side chains. The deglycosylated alpha and beta subunits were 27 and 32 kd, respectively. These data suggest that the dimeric receptor is formed shortly after translation, followed by extensive glycosylation. Emergence of the C6VL clonotypic epitope, and perhaps the antigen binding site, may therefore be dependent on post-assembly events.     

Synthesis of cancer-associated glycoantigens: stage-specific embryonic antigen 3 (SSEA-3)

The synthesis of the tumor-associated carbohydrate antigens SSEA-3 and Gb3 in a semi-convergent fashion using building blocks bearing a S-thiazolinyl (STaz) moiety is reported. Complete stereoselective control of a difficult alpha-(1-->4)-galactosylation and high overall yields were achieved.     

Serum levels of carcinoembryonic antigen, gastrointestinal cancer-associated antigen and alphafetoprotein in staging and management of patients with advanced carcinoma of the stomach

Serum levels of carcinoembryonic antigen (CEA), gastrointestinal cancer-associated antigen (GICA or CA 19-9), and alphafetoprotein (AFP) were concurrently determined in patients with carcinoma of the stomach: in 84 preoperatively, and in 67 serially postoperatively. Before surgery, serum CEA gave information about the tumor load analogous to serum GICA in 69% of the patients: true-positive in 25% and false-negative in 43%; less information in 18% and more in 14%. The sensitivity of the test tended to be better in the more advanced stages, and was higher for CEA with GICA than for CEA alone or GICA alone. During follow-up, serum CEA gave information about the presence or absence of active disease analogous to serum GICA in 78% of the patients: true-positive in 30%, true-negative in 36% and false-negative in 12%; less information in 9% and more in 13%. Neither test gave any false-positive indications. Sensitivity of the test rose from 67% for CEA alone and 60% for GICA alone to 81% for CEA with GICA. Serum AFP was elevated only preoperatively in 2% of patients. We conclude that joint application of CEA and GICA tests gave only slightly better preoperative sensitivity than CEA alone or GICA alone but proved fairly sensitive for postoperative follow-up of the patients. AFP was of little value for either purpose.     

Biosynthesis and molecular nature of the T3 antigen of human T lymphocytes

Immunoprecipitates of the T3 antigen prepared from HPB-ALL cells by using the monoclonal antibody UCH-T1 were analysed by SDS-polyacrylamide gel electrophoresis. Cells which had been biosynthetically labelled for up to 4 h gave a major polypeptide of mol. wt. 19 000 plus two weaker, more diffuse bands of mol. wts. 21 000 and 23 000, whereas surface labelled cells gave a prominent band of mol. wt. 19 000, a major band of 21 000 and a weaker diffuse band of approximately 26 000. As judged from their sensitivity to proteinase-K digestion, all the above polypeptides possess a transmembrane orientation. Digestion with endoglycosidases H and F (endo-H and endo-F), and tunicamycin treatment indicate that all the polypeptides, except that of 19 000 mol. wt. are N-glycosylated. The 21 000 and 23 000 mol. wt. chains possess both immature and mature oligosaccharide units, whereas the 26 000 mol. wt. band apparently has mature units only. Pulse chase experiments combined with digestion by endo-F and endo-H suggest that the N-glycosylated polypeptides are derived from two polypeptides of mol. wts. 14 000 and 16 000. It is concluded that the T3 antigen is derived from three different non-glycosylated polypeptides two of which are subsequently N-glycosylated to give the 21 000, 23 000 and 26 000 forms. The cell surface T3 antigen most probably comprises at least two distinct, non-covalently associated polypeptides, but the number and types of polypeptides giving rise to the whole molecule and whether different complexes exist is at present unclear.     

Biosynthesis of the Pseudomonas aeruginosa common polysaccharide antigen by D-Rhamnosyltransferases WbpX and WbpY

Glycoconjugate Journal - The Gram-negative bacterium Pseudomonas aeruginosa simultaneously expresses two O-antigenic glycoforms. While the O-specific antigen (OSA) is variable in composition, the...     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

Three novel oligosaccharides with the sialyl-Lea structure in human milk: isolation by immunoaffinity chromatography

We have determined the structures of three novel oligosaccharides isolated from human milk using the monoclonal antibody MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-performance liquid chromatography. From the results of 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, their structures were deduced to be (formula; see text) These oligosaccharides bound to MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but bound to another sialyl-Lea structure-directed monoclonal antibody, NS 19-9, only weakly.     

Biosynthesis of 117 antigen: a cell cohesion molecule in Dictyostelium discoideum

117 antigen is involved in the process of intercellular cohesion in Dictyostelium discoideum [Brodie et al., 1983]. The antigen, a 69- and 72-kDa doublet, was found to arise from a 60- and 62-kDa precursor. The mature antigen contains N-linked oligosaccharides that are sulfated and fucosylated [Sadeghi et al., 1987]. These oligosaccharide chains are resistant to endoglycosidase H digestion. 117 antigen also contains a post-translationally added carbohydrate-containing modification(s). Unlike the N-linked oligosaccharide, this carbohydrate moiety is sensitive to periodate oxidation. 117 antigen is developmentally regulated, and the changes in rate of 117 antigen synthesis reflect changes in the cellular levels of its mRNA. 117 mRNA accumulates in starving cells and reaches its maximum when cells become aggregation competent. The mRNA levels then decline, and by the time the slug structure is formed, no 117 mRNA is present. 117 mRNA reaccumulates for a brief period during early culmination and then returns to an undetectable level.     

Proteomic maps of the cancer-associated infectious agents

The number of infectious agents associated with cancer is increasing. There is a need to develop approaches for the early detection of the infected host which might lead to tumor development. Recent advances in proteomic approaches provide that opportunity, and it is now possible to generate proteomic maps of cancer-associated infectious agents. Protein arrays, interaction maps, data archives, and biological assays are being developed to enable efficient and reliable protein identification and functional analysis. Herein, we discuss the current technologies and challenges in the field, and application of protein signatures in cancer detection and prevention.