Sen-Itiroh Hakomori

Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.

Two monoclonal antibodies (MC631 and MC813‐70) raised against 4‐ to 8‐cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage‐specific embryonic antigens, the previously defined SSEA‐3 and SSEA‐4, described herein. These antibodies were both reactive with a unique globo‐series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813‐70 recognizes the terminal ‘a’ structure whereas antibody MC631 recognizes the internal ‘b’ structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo‐series glycolipids defined by these antibodies decrease and the lacto‐series glycolipids, reacting with the SSEA‐1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo‐series to lacto‐series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre‐implantation mouse embryos.

Tumor-associated glycolipid antigens, their metabolism and organization

A number of experimental animal tumors as well as human cancers have been characterized by dramatic changes of glycolipid composition and metabolism. This review focuses on the chemical and enzymatic basis of the appearance of tumor-associated glycolipid antigens belonging to four major structural classes, i.e., globo, ganglio, lacto type 1, and lacto type 2 series. Some antigens represent the accumulation of precursors with deletion of more complex glycolipids, and others are the result of enhanced synthesis of new structures, most of which are aberrant fucosylation or sialylation or their combination; thus, novel structures such as di- or trimeric Le chi, trifucosyl Le gamma, sialyl Le chi, sialyl dimeric Le chi and disialyl Le alpha A have been isolated and characterized. Many monoclonal antibodies are capable of recognizing antigens in high density but are not capable of reacting with the same antigen in low density. Therefore, the expression of novel structures in high densities at the cell surface is important for recognition of tumor-association antigens. Molecular models of a typical tumor-associated antigen and its organization in membranes are also presented.

Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities.

Abstract A pure straight chain ceramide hexasaccharide (“lacto-N- nor hexaosylceramide” Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc→Ceramide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Galβ1→4GlcNAc residues and GlcNAcβ1→3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-I antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues.

Carbohydrate-carbohydrate interaction as an initial step in cell recognition

Specific interaction between specific carbohydrate moieties of glyco- sphingolipids (GSLs) has been demonstrated based on interaction of GSL- containing liposomes with GSL-coated solid phase, and affinity adsorption of multivalent GSL oligosaccharide on solid-phase GSL column. Evidence is presented that such GSL-GSL interaction is an initial step in the cell recognition process. Examples are LeX-LeX interaction at the morula stage of mouse embryogenesis, Gg3-GM3 interaction in melanoma/lymphoma cell interaction, and galactosylceramide-sulfatide interaction in myelin sheath membrane formation. The molecular basis of such interactions is discussed.

A Novel Sphingosine-dependent Protein Kinase (SDK1) Specifically Phosphorylates Certain Isoforms of 14-3-3 Protein

Protein kinases activated by sphingosine or N,N′-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739–747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50–60 kDa that is strongly activated byN,N′-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the α and ζ isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (η, β, ζ) but not others (ς, τ). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 β. The ς and τ isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N,N′-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent phosphorylation of specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or induce conformational changes that alter 14-3-3 association with other kinases involved in signal transduction.

Coordinate expression of X and Y haptens during murine embryogenesis

The X hapten (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) may play an important role in the adhesion of blastomeres during compaction. Therefore, we have investigated more thoroughly developmental changes in the fucosylation of lactoseries carbohydrate chains and the enzymatic basis of these fucosylation changes using well-characterized monoclonal antibodies. The Y hapten (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and polymeric X haptens were detected by fluorescence-activated flow cytometry on murine embryonal carcinoma cells. In paraffin sections of postimplantation mouse embryos, the Y hapten was detected in the embryonic ectoderm and visceral endoderm on Days 5.5-7.5; this pattern of antigen expression is identical to that previously reported for the X hapten (SSEA-1). Thus, the Gal:alpha 1----2 (H) and GlcNAc:alpha 1----3 (X) fucosyltransferases appear to be co-regulated during embryogenesis. Reciprocal changes in X and Y hapten expression were observed, however, during preimplantation development. Unlike the X hapten, the Y hapten is expressed maximally on 16-cell morulae and 32- to 64-cell blastocysts. Eight-cell embryos cultured to the blastocyst stage in vitro did not acquire the Y hapten, however, suggesting a role for the uterine environment in carbohydrate antigen expression. Homogenates of F9 embryonal carcinoma cells were found to possess a potent GlcNAc:alpha 1----3 fucosyltransferase activity, as well as a weaker Gal:alpha 1----2 fucosyltransferase activity, using paragloboside as a substrate. The results suggest that embryonic cell surface carbohydrate phenotypes represent a balance in the competition between glycosyltransferases for available substrates. Rapid changes in carbohydrate expression during development may reflect intermediate states of cellular commitment and determination that are critical for lineage formation and morphogenesis.

Glycosphingolipids as differentiation-dependent, tumor-associated markers and as regulators of cell proliferation

Abstract Glycosphingolipids alter their synthesis and membrane organization in association with cellular differentiation and oncogenic transformation. Many, if not all, tumor cells express aberrant glycosphingolipid markers which are defined by their monoclonal antibodies. Examples of human cancer markers are presented, which show typical oncofetal expression. In addition, a possible role of gangliosides in regulation of growth factor receptor function is discussed.

Glycosphingolipids in detergent-insoluble substrate attachment matrix (DISAM) prepared from substrate attachment material (SAM): Their possible role in regulating cell adhesion☆

The glycosphingolipids isolated from the detergent-insoluble material (DIM) of whole cells as well as from a similar detergent-insoluble substrate attachment matrix (DISAM) have been investigated in comparison with the glycosphingolipids of whole cells. The proportion of glycolipids in the total lipid extract was enriched in the DISAM as well as DIM fractions as compared to whole cells. The ratio of ganglioside (GM3) to neutral glycolipids was also higher in the DISAM fractions than in whole cells. The radioactivity incorporated into DISAM glycolipids of BHK cells, metabolically labeled with radioactive glucosamine, was greater in confluent cells than in sparsely growing cells; however, label incorporation into glycolipids of the DISAM fraction of BHKpy cells was 2-3-fold higher than that of confluent BHK cells, although the chemical quantity of GM3 in whole cells was much lower in BHKpy cells than in BHK cells. In order to confirm the enhanced label in DISAM glycolipids of BHKpy cells by other procedures, the labeled cells were detached by EGTA, washed, and reattached on plates. The amount of label in DISAM glycolipids of the reattached matrix of BHKpy cells was much higher than that of BHK cells. Cell spreading and cell attachment on plastic plate were inhibited by inclusion of GM3 in the medium. These data suggest that: (i) glycolipids, particularly GM3, at the cell attachment site have different metabolic activity from those of whole cells; the label in glycolipids goes preferentially into cell attachment sites, and may have some functional role in regulating cell attachment of BHK cells; (ii) metabolic activity and turnover of GM3 in cell attachment sites of confluent cells are higher than actively growing cells, yet those of transformed cells are much higher than any state of non-transformed cells.

“Ganglioprotein and globoprotein”: The glycoproteins reacting with anti-ganglioside and anti-globoside antibodies and the ganglioprotein change associated with transformation☆

Abstract The cell surface-labeled glycoproteins that are reactive to anti-glycolipid antibodies were solubilized by zwitterionic or non-ionic detergents and separated by specific anti-glycolipid antibodies through double immune precipitation or specific absorption on Staphylococcus aureus . The patterns of these glycoproteins (“ganglioprotein” or “globoprotein”) as compared to glycolipids were examined on SDS-acrylamide gel electrophoresis. The major “globoprotein” of human erythrocytes was not in “glycophorin” or “PAS 1” but in the “PAS 2 or 3” region. The GM 1 -ganglioproteins of 3T3 cells were all deleted in KiMSV transformants, whereas the intense protein peaks reactive to anti-asialo GM 2 (ganglio-N-triosylceramide) appeared in these transformants.

Sulfated lipids represent common antigens on the surface of Trypanosoma cruzi and mammalian tissues.

Cross-reacting lipid antigens were isolated from Trypanosoma cruzi and the mammalian brain with the monoclonal antibody VESP 6.2. Chemical reactions indicated that the sulfate group of the lipids is an important part of the epitope recognized by the monoclonal antibody. Lipid extracts of mouse brain contained all the antigenic species present in the parasite. One of the antigens was demonstrated by three different methods: (i) high performance thin layer chromatography immunostaining, (ii) solid phase radioimmunoassay, (iii) lysis of artificial liposomes. The T. cruzi sulfated lipid antigens were shown to be of parasite origin rather than scavenged from the culture medium. They could be radiolabelled with [35S]sulfate. Furthermore, lipid extracts from two T. cruzi strains grown in different media contained the same antigens while the media contained either no antigens or different species.