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.     

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.     

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.     

Transfection of a human alpha-(1,3)fucosyltransferase gene into Chinese hamster ovary cells. Complications arise from activation of endogenous alpha-(1,3)fucosyltransferases

In order to isolate a human gene encoding an alpha-(1,3)fucosyltransferase (alpha-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing alpha-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of alpha-(1,3)Fuc-T action. CHO cells do not express alpha-(1,3)Fuc-T activity but contain at least two, silent alpha-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the alpha-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an alpha-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like alpha-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human alpha-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed alpha-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO alpha-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human alpha-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.     

Molecular cloning, genomic mapping, and expression of two secretor blood group alpha (1,2)fucosyltransferase genes differentially regulated in mouse uterine epithelium and gastrointestinal tract

Fucosylated oligosaccharides have been proposed to be involved in multiple cell-cell interactions, including mouse blastocyst adhesion and intestine-microbe interactions. To begin to define the regulation and function of terminal alpha(1,2)fucosylated carbohydrates in these and other tissues, we isolated and characterized a 85-kilobase (kb) genomic region of mouse chromosome 7, 23.2 centimorgans analogous to human chromosome 19q13.3 that encodes three alpha(1,2)fucosyltransferases. Gene-specific DNA probes from the open reading frames of the mouse fucosyltransferase genes corresponding to human FUT1, FUT2, and SEC1 demonstrate distinct tissue-specific expression patterns by Northern blot analyses. Flow cytometry profiles of cultured cells transfected with DNA segments containing the open reading frames of the mouse genes confirm that each encodes an alpha(1,2)fucosyltransferase. In uterus and colon, a 3.3-kb FUT2 mRNA represents the major fucosyltransferase gene expressed. Steady-state FUT2 mRNA levels are cyclically regulated during the estrus cycle, increasing 10-fold from early diestrus to a relative maximum in proestrus. In contrast, SEC1 and FUT1 do not show prominently regulated expression in uterus. FUT2 expression localizes to luminal uterine epithelium by in situ hybridization, implying that this gene determines expression of cell surface Fucalpha1-->2Galbeta epitopes proposed to mediate blastocyst adhesion.     

Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes.

We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.     

Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion.

We have used the human Lewis blood group fucosyltransferase cDNA and cross-hybridization procedures to isolate a human gene that encodes a distinct fucosyltransferase. Its DNA sequence predicts a type II transmembrane protein whose sequence is identical to 133 of 231 amino acids at corresponding positions within the catalytic domain of the Lewis fucosyltransferase. When expressed by transfection in cultured cell lines, this gene determines expression of a fucosyltransferase capable of efficiently utilizing N-acetyllactosamine to form the Lewis x determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc). By contrast, biochemical and flow cytometry analyses suggest that the enzyme cannot efficiently utilize the type II acceptor NeuNAc alpha 2----3Gal beta 1----4GlcNAc, to form the sialyl Lewis x determinant. In Chinese hamster ovary cells, however, the enzyme can determine expression of the alpha 2----3-sialylated, alpha 1----3-fucosylated structure known as VIM-2, a putative oligosaccharide ligand for ELAM-1. Cell adhesion assays using VIM-2-positive, sialyl Lewis x-negative transfected Chinese hamster ovary cells indicate that surface expression of the VIM-2 determinant is not sufficient to confer ELAM-1-dependent adhesive properties upon the cells. These results demonstrate that substantial structural similarities can exist between mammalian glycosyltransferases with closely related enzymatic properties, thus facilitating isolation of their cognate genes by cross-hybridization methods. The results further suggest that cell surface expression of the VIM-2 determinant is not necessarily sufficient to mediate ELAM-1-dependent cell adhesion.     

Biosynthesis of the sialyl-Lex determinant carried by type 2 chain glycosphingolipids (IV3NeuAcIII3FucnLc4, VI3NeuAcV3FucnLc6, and VI3NeuAcIII3V3Fuc2nLc6) in human lung carcinoma PC9 cells

The pathway for synthesis of three glycosphingolipids bearing a common sialyl-Lex determinant (NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNac beta 1----R) from their type 2 lactoseries precursors has been studied using the 0.2% Triton X-100-soluble fraction from human lung carcinoma PC9 cells. Two enzymes were found to be required for their synthesis: (i) an alpha 1----3 fucosyltransferase, the properties of which have been characterized as being similar to the enzyme from human small cell lung carcinoma NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627); and (ii) an alpha 2----3 sialyltransferase that was efficiently solubilized by 0.2% Triton X-100 and required divalent metal ions and 0.3% Triton CF-54 for optimal activity at pH 5.9 in cacodylate buffer. Biosynthesis of the sialyl-Lex determinant was shown to proceed via sialylation of nLc6 and nLc4, followed by alpha 1----3 fucosylation at the penultimate GlcNAc residues, based on the following: (i) transfer of NeuAc by PC9 cell sialyltransferase was found only when the nonfucosylated acceptors nLc4 and nLc6 were added, and none of the glycolipids with Lex structure (III3FucnLc4; V3FucnLc6; III3V3Fuc2nLc6) were sialylated; and (ii) the PC9 cell fucosyltransferase was active with both neutral and ganglioside neolacto (type 2 chain) acceptors. Transfer of fucose to VI3NeuAcnLc6 yielded mono- and difucosyl derivatives, whereas only a monofucosyl derivative was obtained when VI6NeuAcnLc6 was the acceptor. This is most probably due to different conformations at the terminus of the two acceptor gangliosides. The fucosyltransferase was incapable of transferring fucose to sialyl 2----3 lactotetraosylceramide (IV3NeuAcLc4).     

Transfer and co-amplification of a gene encoding a 96-kDa immune IFN-inducible human melanoma-associated antigen. Preferential expression by mouse melanoma host cells

An immune IFN-inducible human melanoma-associated glycoprotein Ag, 96-kDa MAA, having preferential distribution on metastases has been defined by mouse mAb CL203.4. To initiate molecular genetic analysis of 96-kDa MAA, the gene encoding the Ag was transfected into mouse B16 melanoma cell clone B78H1. Formation of B78H1-transfectant colonies expressing a surface Ag reactive with mAb CL203.4 in an immunorosetting assay was dependent on addition of chromosomal DNA from human melanoma cells [primary (1 degree) transfer] or from Ag-expressing transfectant cells (2 degrees, 3 degrees, 4 degrees transfer). The mAb CL203.4-reactive species expressed by the transfectant cells is a glycoprotein with a molecular mass 93-kDa, within the range of 93 to 96-kDa observed for the endogenous human Ag. In the presence of tunicamycin, an inhibitor of N-linked glycosylation, both mouse melanoma transfectants and human melanoma cells express a 50- to 51-kDa antigenic species. Human alu family repeat sequences (h-alu) are present in the genomes of 3 degrees transfectant cells. Continued presence of these h-alu after dilution of extraneous human DNA by three cycles of transfection suggests their association with the transferred 96-kDa MAA gene. Use of a selective co-amplification procedure led to transfectant cells' increased expression of 96-kDa MAA and to commensurate increases in their content of presumed 96-kDa MAA gene-associated h-alu. Preferential DNA-mediated transferability of the 96-kDa MAA+ phenotype into B78H1 cells as compared with LMTK- mouse fibroblasts suggests host cell specificity of 96-kDa MAA gene expression.     

Fucosyltransferases in Schistosoma mansoni development

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.     

A promising genomic transfectant into Xeroderma pigmentosum group A with highly amplified mouse DNA and intermediate UV resistance turns revertant

Following transfection of genomic mouse DNA into an SV40 transformed fibroblast cell line from a patient with Xeroderma pigmentosum (complementation group A, XPA), a single UV resistant cell clone was isolated out of a total of 10(4) independent transfectants. The recipient XPA cell line has as yet not produced spontaneous revertants among 2.2 x 10(8) cells. The isolated cell clone contains 50-70 kb of mouse sequences which are heavily amplified (500-fold), and has acquired both intermediate resistance to UV killing and intermediate unscheduled DNA synthesis (UDS) capacity. By continued passage without selective pressure, cells were generated, which had lost both the dominant marker gene and repetitive mouse sequences. Single colonies of these cells were still intermediately resistant to UV suggesting that either undetected unique mouse DNA had segregated from the bulk of repetitive DNA, or, more likely, that the initially isolated transfectant was a spontaneous revertant. This documents that a persuasive clone isolated can still be a false positive (spontaneous revertant) and that an extremely laborious approach may lead into a dead end.     

Transfection of metastatic capability with total genomic DNA from human and mouse metastatic tumour cell lines

Abstract. From a large series of experiments involving transfer of high molecular weight total genomic DNA from highly metastatic human and mouse tumour cell lines to other mouse tumour cell lines we have derived a few cell lines with greatly augmented metastatic properties. In one of these experiments the transfected cell line (designated AH8 Test) not only colonised the lungs but also formed secondary tumour colonies in several extrapulmonary sites including the skin, skeletal muscles, bone, liver diaphragm, spleen and heart, There were no qualitative and quantitative effects of this magnitude when we used DNA from several non-metastatic or non-tumourigenic sources.
Secondary transfection of metastatic capability with DNA obtained from a metastasis formed by one of the primary transfectant lines (AH8 Test) has also been accomplished. Concomitant transfer of human DNA through both transfection cycles in this experiment was confirmed by a variety of methods including Southern blot analysis, in situ hybridisation and polymerase chain reaction (PCR) amplification of DNA using primers recognising human-specific Alu repeat sequences.
The findings offer opportunities for the isolation of sequences programming metastatic behaviour and we have cloned and sequenced a fragment of human DNA, which has not been previously characterised, from the transfected cells.     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.     

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 (Ménard, 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.     

Y and blood group B type 2 glycolipid antigens accumulate in a human gastric carcinoma cell line as detected by monoclonal antibody. Isolation and characterization by mass spectrometry and NMR spectroscopy

A monoclonal antibody (mAb), BR55-2, was generated from mice immunized with MCF-7 human breast carcinoma cells. This mAb specifically detected glycolipids with the Y determinant Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)-beta 1----3Gal beta 1----4Glc beta 1----1 Cer and the Y-related B-active difucosylated determinant Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4GlcNAc(3----1 alpha Fuc) beta 1----3Gal beta 1----4Glc beta 1----1 Cer, but was not reactive with related monofucosylated glycolipids of type 2 chain (X-antigen, blood group H), type 1 chain (Lea antigen, blood group H and B) or with difucosylated type 2 and type 1 chain structures (A blood group antigen or blood group B and Leb, respectively). A series of glycolipids with Y and blood group B type 2 determinants were detected in human gastric adenocarcinoma cell line KATO III with mAb BR55-2 and with a previously characterized anti-blood group B mAb PA83-52 (Hansson, G. C., Karlsson, K.-A., Larson, G., McKibbin, J. M., Blaszczyk, M., Herlyn, M., Steplewski, Z., and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097). The isolated antigens were structurally characterized by mass spectrometry of permethylated and permethylated-reduced derivatives and by proton NMR spectroscopy. In a chromatogram binding assay, mAb BR55-2 and mAb PA83-52 detected minor components with slower mobility than the Y-6 and blood group B-7-type 2 structures. The detection of a B type 2 determinant is the first chemical evidence for the presence of an autologous difucosyl blood group B type 2 antigen in human adenocarcinoma cells.     

Oncofetal expression of Lex carbohydrate antigens in human colonic adenocarcinomas. Regulation through type 2 core chain synthesis rather than fucosylation

The mechanism of expression of a series of glycolipid antigens carrying the Lex determinant structure, Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----, and characterized by oncofetal expression in fetal colon and colonic adenocarcinomas has been studied in human fetal and adult proximal colon tissue. Results presented from TLC immunostain analysis of neutral glycolipids isolated from normal adult colonic mucosa have indicated the presence of only barely detectable quantities of both an Lex-active glycolipid that co-migrated with III3V3Fuc2nLc6 and its precursor nLc6. These structures were found in large quantities in glycolipid fractions from human adenocarcinoma tumors and human small cell lung carcinoma NCI-H69 cells. In contrast, type 1 chain-based Lea antigen structures were found in both normal mucosa and adenocarcinomas. Analysis of gangliosides of normal colonic mucosa by TLC immunostain indicated the presence of a series of type 2 chain-based gangliosides; however, sialyl-Lex was not detected. The ability of normal colonic mucosa to synthesize type 2 chain core structures was demonstrated by the presence of a beta 1----4 galactosyltransferase activity with Lc3 as an acceptor in an amount equivalent to 60-65% of the total galactosyltransferase activity. An alpha 1----3 fucosyltransferase was also found to be expressed in significant quantity in adult colonic mucosa. Kinetic studies indicated that this is most probably the alpha 1----3/4 fucosyltransferase suggested to be a product of the Lewis gene (Le). Thus, although normal adult colonic mucosa contained the enzymes to synthesize Lex and sialyl-Lex structures, these antigens were not found. Tissue immunofluorescence studies indicated that type 2 chain precursors and the alpha 1----3/4 fucosyltransferase were found in different cell populations in adult proximal colonic mucosa. However, both type 2 chain core structures and their fucosylated derivatives were found to be associated with epithelial cells of fetal colon. These results indicate that oncofetal expression of Lex antigens in fetal colonic epithelium and in adenocarcinomas but not in normal adult mucosa is due to the retrogenetic expression of type 2 chain precursors which are not found in normal adult colonic epithelial cells.     

Acetolysis and 1H NMR studies on mannans isolated from very flocculent and weakly flocculent cells of Pichia pastoris IFP 206

Growth of the yeast Pichia pastoris IFP 206 in methanol- and glucose-containing media led respectively to very and weakly flocculent cells. Mannans from both kinds of cells were extracted and compared. Chemical analysis and molecular mass estimation showed some differences between the mannans from very and weakly flocculent cells, especially in quantitative amino acid content. 1H NMR analysis showed that both kinds of mannan contained alpha-1,2 and beta-1,2 linkages. Two acetolysis conditions, combined with 1H NMR analysis, revealed that mannans from both kinds of cells were composed of mannose, mannobiose, mannotriose, mannotetraose and mannopentaose side-chains with the following respective structures: Man; Man alpha 1---2Man; Man alpha 1----2Man alpha 1----2Man; Man beta 1----2Man alpha 1----2Man; Man beta 1----2Man beta 1----2Man alpha 1----2Man; Man alpha 1----2Man beta 1----2Man beta 1----2Man alpha 1----2Man. Additionally the beta-1,2 linkages of the non-reducing terminal residues of the mannotetraose were shown to be acetolysis-labile. The mannans from very flocculent cells were richer in mannopentaose than the mannans from weakly flocculent cells. According to these results, the extended conformations in the branching moieties of the mannan could be the basis of the higher degree of flocculation of the methanol-grown cells.     

Chromosome mediated gene transfer of six DNA markers linked to the cystic fibrosis locus on human chromosome seven.

The DNA probes met and pJ3.11 are derived from loci on chromosome seven that are closely linked to, and probably flanking, the gene mutation causing cystic fibrosis (CF). We have shown that mitotic chromosomes from the cell line MNNG-HOS, which contains an activated met oncogene, can induce morphological transformation of mouse NIH-3T3 cells. Southern analysis of isolated transfectant cell lines with cloned dispersed repetitive human DNA sequences as probes demonstrated that several lines of transformed NIH 3T3 cells had stabley incorporated large segments of chromosome seven DNA. Southern blot analysis also demonstrated the presence of met, pJ3.11 and several other single copy sequences that had been previously localised to chromosome 7 within the transgenomes. In this way a further four genetic markers were shown to be physically linked to met, and thus to CF. These probes may prove useful in confirming the order of loci around CF and in the prenatal diagnosis of this common autosomal recessive disease.     

Secretion of glycosylated human interleukin-2 by recombinant mammalian cell lines

The production of glycosylated forms of the human T cell growth factor (interleukin-2, IL-2) has been studied after transfection of a mouse L cell line and a chinese hamster ovary cell line with a plasmid containing the human chromosomal interleukin-2 gene. Both cell lines produced IL-2 constitutively. Based on their behavior in reversed-phase l.c. and their sodium dodecyl sulfate-gel-electrophoresis pattern, human IL-2 protein secreted by L cells showed a similar distribution of glycosylated (Mr 16 500) and nonglycosylated (Mr 14 500) forms as the natural protein secreted by human peripheral lymphocytes, whereas the hamster cell line secreted preponderantly the glycosylated forms. Exoglycosidase digestion of the 16 500 Mr IL-2 protein shifted the gel electrophoretic mobility towards the low-molecular weight form as is true for the natural glycosylated IL-2, which contains the usual tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-[alpha-NeuAc-(2----6)]-D-GalNAc (IL-2 N2) and the trisaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-D-GalNAc (IL-2 N1) as the major carbohydrate constituents. These results support the applicability of recombinant DNA technology as a tool for studying glycoprotein biosynthesis in mammalian cells.