Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Menard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue

The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/)

A Novel Ganglioside Isolated from Renal Cell Carcinoma

In renal cell carcinoma (RCC), the level of higher gangliosides is correlated with degree of metastatic potential, and cell lines derived from metastatic deposits of RCC are characterized by high expression of disialogangliosides (Saito, S., Orikasa, S., Ohyama, C., Satoh, M., and Fukushi, Y. (1991) Int. J. Cancer 49, 329–334 and Saito, S., Orikasa, S., Satoh, M., Ohyama, C., Ito, A., and Takahashi, T. (1997) Jpn. J. Cancer Res. (Gann) 88, 652–659). We now report two disialogangliosides, G1 and G2, found in the RCC cell line TOS-1. G1 from TOS-1 cells was characterized as having a novel hybrid structure between ganglio-series (region I as in Structure FTI; same as the terminal structure of ganglioside GM2), and the lacto-series type 1 (region II). The characterization was based on reactivity with various monoclonal antibodies (mAbs) with defined epitope specificity, as well as monosaccharide and fatty acid component analysis, ^1H NMR spectroscopy, and electrospray ionization mass spectrometry of the intact compound. G1 showed strong reactivity with mAb RM2, raised originally against TOS-1 cells, and weak cross-reactivity with anti-GM2 mAb MK-1–8. The antigen is hereby termed GalNAc disialosyl Lc_4Cer (IV^4GalNAcIV^3NeuAcIII^6NeuAcLc_4; abbreviated GalNAcDSLc_4). G2 was identified by^1H NMR and mass spectrometry as having a structure similar to Structure FTI but without the GalNAcβ1→4 substitution and showed strong reactivity with mAb FH9 reported previously to be specific for disialosyl lacto-series type 1 (disialosyl Lc_4) having vicinal α2→3 and α2→6 sialosyl residues, an antigen associated with human colonic cancer. Clinicopathological studies indicate that expression of these disialoganglioside antigens in RCC tissue is correlated with the metastatic potential of RCC

The structural and functional role of myelin fast-migrating cerebrosides: pathological importance in multiple sclerosis

A family of neutral glycosphingolipids containing a 3-O-acetyl-sphingosine galactosylceramide (3-SAG) has been characterized. Seven new derivatives of galactosylceramide (GalCer), designated as fast-migrating cerebrosides (FMCs) by TLC retention factor, have been identified. The simplest compounds - FMC-1 and FMC-2 of this series have been characterized as the 3-SAG containing nonhydroxy and hydroxy fatty acyl, respectively. The next two -FMC-3 and FMC-4 - add 6-O-acetyl-galactose and the most complex glycosphingolipids, FMC-5, -6 and -7, are 2,3,4,6-tetra-O-acetyl-3-SAG. These hydrophobic myelin lipid biomarkers coappear with GalCer during myelinogenesis and disappear along with GalCer in de- or dys-myelinating disorders. Myelin lipid antigens, including FMCs, are keys to myelin biology, opening the possibility of new and novel immune modulatory tools for treatment of autoimmune diseases including multiple sclerosis

The structural and functional role of myelin fast-migrating cerebrosides: pathological importance in multiple sclerosis

A family of neutral glycosphingolipids containing a 3-O-acetyl-sphingosine galactosylceramide (3-SAG) has been characterized. Seven new derivatives of galactosylceramide (GalCer), designated as fast-migrating cerebrosides (FMCs) by TLC retention factor, have been identified. The simplest compounds - FMC-1 and FMC-2 of this series have been characterized as the 3-SAG containing nonhydroxy and hydroxy fatty acyl, respectively. The next two -FMC-3 and FMC-4 - add 6-O-acetyl-galactose and the most complex glycosphingolipids, FMC-5, -6 and -7, are 2,3,4,6-tetra-O-acetyl-3-SAG. These hydrophobic myelin lipid biomarkers coappear with GalCer during myelinogenesis and disappear along with GalCer in de- or dys-myelinating disorders. Myelin lipid antigens, including FMCs, are keys to myelin biology, opening the possibility of new and novel immune modulatory tools for treatment of autoimmune diseases including multiple sclerosis

Stress-Induced Cell Death Is Mediated by Ceramide Synthesis in Neurospora crassa▿

The combined stresses of moderate heat shock (45°C) and analog-induced glucose deprivation constitute a lethal stress for Neurospora crassa. We found that this cell death requires fatty acid synthesis and the cofactor biotin. In the absence of the cofactor, the stressed cells are particularly sensitive to exogenous ceramide, which is lethal at low concentrations. When we extracted endogenous sphingolipids, we found that unique ceramides were induced (i) by the inhibitory glucose analog 2-deoxyglucose and (ii) by combined heat shock and 2-deoxyglucose. We determined that the former is a 2-deoxyglucose-modified ceramide. By structural analysis, we identified the latter, induced by dual stress, as C18(OH)-phytoceramide. We also identified C24(OH)-phytoceramide as a constitutive ceramide that continues to be produced during the combined stresses. The unusual C18(OH)-phytoceramide is not made by germinating asexual spores subjected to the same heat and carbon stress. Since these spores, unlike growing cells, do not die from the stresses, this suggests a possible connection between synthesis of the dual-stress-induced ceramide and cell death. This connection is supported by the finding that a (dihydro)ceramide synthase inhibitor, australifungin, renders cells resistant to death from these stresses. The OS-2 mitogen-activated protein kinase, homologous to mammalian p38, may be involved in the cell death signaling pathway. Strains lacking OS-2 survived the combined stresses better than the wild type, and phosphorylated OS-2 increased in wild-type cells in response to heat shock and combined heat and carbon stress