Expression levels of FUT6 gene transfected into human colon carcinoma cells switch two sialyl-Lewis X-related carbohydrate antigens with distinct properties in cell adhesion

A human colon carcinoma cell line KM12-LX, expressing low levels of monoclonal antibody (mAb) FH6 epitope, was transfected with alpha 1,3-fucosyltransferase VI cDNA. Clonal populations with high or intermediate expression levels of the mRNA, shown by RT-PCR (FT6hi and FT6in cells, respectively) were obtained. FT6hi cells were found to express both mAb FH6 and KM93 epitopes by flow-cytometric analysis, whereas FT6in cells expressed mAb FH6 epitopes but not mAb KM93 epitopes. The mAb FH6-binding was abrogated by endo-beta-galactosidase treatment of FT6in, but not FT6hi, cells. FT6hi but not FT6in cells adhered to Chinese-hamster-ovary cells expressing human E-selectin. FT6in cells adhered to sections of mouse liver and the adhesion was blocked by treatment of the cells with endo-beta-galactosidase. The results indicate that endo-beta-galactosidase-sensitive and mAb FH6-reactive carbohydrate chains are generated under the control of expression levels of FUT6 and involved in the adhesion of colon carcinoma cells to liver sections.     

Binding of sialyl Lewis X antigen to lectin-like receptors on NK cells induces cytotoxicity and tyrosine phosphorylation of a 17-kDa protein

BACKGROUND: Natural killer (NK) cells mediate cytotoxicity through cell-surface receptors including lectin-like receptors. We have investigated whether sialyl Lewis X (sLe(X)) antigen, Neu5Acalpha2,3Galbeta1,4(Fucalpha1,3) GlcNAc-R, can bind to the lectin-like receptors on human NK-derived KHYG cells, using transferrin secreted by human hepatoma-derived HepG2 cells (Hep-TF), whose N-glycans are rich in alpha1,3-fucosylated bi-, tri-, and tetra-antennary type complexes, and commercially available human transferrin (Nor-TF), which is comprised of bi-antennary N-glycans without alpha1,3-fucosylation. RESULTS: High sLeX-expressing erythroleukemia-derived K562 cells isolated from fucosyltransferase-3-transfected cells were 2.5-fold more susceptible than wild-type K562 cells to KHYG cells. Fluorescein isothiocyanate (FITC)-labeled Hep-TF bound 1.8-fold more strongly to KHYG cells than did FITC-labeled Nor-TF; the binding was suppressed by treatment with anti-NKG2D, anti-NKG2C, anti-CD94 and anti-CD161 antibodies. FITC-labeled Hep-TF bound more strongly to human monocyte-derived U937 cells transfected with NKG2D and CD94 than to wild-type U937 cells. Moreover, tyrosine phosphorylation of a 17-kDa protein in the KHYG cells was enhanced by incubation on a Hep-TF coated plate and treatment with an anti-NKG2D antibody, but not by a Nor-TF coated plate and an anti-CD94 antibody. CONCLUSION: The interaction of sLe(X) antigen with lectin-like receptors on NK cells induces cytotoxicity that is mediated through a tyrosine-phosphorylated 17-kDa protein.     

The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivo functional sublocalization and stability in the Golgi

We provide evidence for the presence of targeting signals in the cytoplasmic, transmembrane, and stem (CTS) regions of Golgi glycosyltransferases that mediate sorting of their intracellular catalytic activity into different functional subcompartmental areas of the Golgi. We have constructed chimeras of human alpha1, 3-fucosyltransferase VI (FT6) by replacement of its CTS region with those of late and early acting Golgi glycosyltransferases and have stably coexpressed these constructs in BHK-21 cells together with the secretory reporter glycoprotein human beta-trace protein. The sialyl Lewis X:Lewis X ratios detected in beta-trace protein indicate that the CTS regions of the early acting GlcNAc-transferases I (GnT-I) and III (GnT-III) specify backward targeting of the FT6 catalytic domain, whereas the CTS region of the late acting human alpha1,3-fucosyltransferase VII (FT7) causes forward targeting of the FT6 in vivo activity in the biosynthetic glycosylation pathway. The analysis of the in vivo functional activity of nine different CTS chimeras toward beta-trace protein allowed for a mapping of the CTS donor glycosyltransferases within the Golgi/trans-Golgi network: GnT-I < (ST6Gal I, ST3Gal III) < GnT-III < ST8Sia IV < GalT-I < (FT3, FT6) < ST3Gal IV < FT7. The sensitivity or resistance of the donor glycosyltransferases toward intracellular proteolysis is transferred to the chimeric enzymes together with their CTS regions. Apparently, there are at least three different signals contained in the CTS regions of glycosyltransferases mediating: first, their Golgi retention; second, their targeting to specific in vivo functional areas; and third, their susceptibility toward intracellular proteolysis as a tool for the regulation of the intracellular turnover.     

N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells

Human alpha3/4fucosyltransferase (FT3) catalyses the synthesis of fucosylated glycoconjugates involved in cell-cell interactions. FT3 has two potential N-glycosylation sites at Asn(154) and Asn(185). Soluble secretory forms of the enzyme (SFT3) and mutant forms with the first, second and both glycosylation sites (SFT3DN1, SFT3DN2, SFT3DN) mutated have been expressed in baby hamster kidney (BHK) and Spodoptera frugiperda (Sf9) cells. Deletion of the first or both sites caused total enzyme inactivation. Deletion of the second site caused 99% and 75% decrease of secretory enzyme expression in BHK and Sf9 cells, respectively. Sf9 cells produced 1 mg/l SFT3 and 0.3 mg/l SFT3DN2; these values were 175- and 3750-fold higher, respectively, than those observed for BHK cells. A significant amount of protein was accumulated intracellularly in Sf9 cells which for SFT3 was active and for SFT3DN2 was inactive, indicating the importance of the glycans from the second glycosylation site for protein folding. The corresponding full-length forms FT3, FT3DN1 and FT3DN2 associated with calnexin as observed by immunoprecipitation studies, which indicated the possible role of this chaperon in the folding of glycosylated glycosyltransferases.     

Generation of serum-stabilized retroviruses: reduction of alpha1,3gal-epitope synthesis in a murine NIH3T3-derived packaging cell line by expression of chimeric glycosyltransferases

Retroviral vectors released from mouse-derived packaging cell lines are inactivated in human sera by naturally occurring antibodies due to the recognition of Galalpha1,3Galbeta1,4GlcNAc (alphagal-epitope) decorated surface proteins. In this study, an extensive analysis of the glycosylation potential of NIH3T3-derived PA317 packaging cells using combined MALDI/TOF-MS and HPAE-PAD reveals that 34% of the N-glycan moiety represents alphagal-epitope containing structures. Stable expression of glycosyltransferases and transport signal chimeras has been demonstrated to represent an efficient tool to alter cell- and species-specific glycosylation (Grabenhorst and Conradt, 1999. J. Biol. Chem. 274, 36107-36116). In order to reduce alphagal-epitope synthesis selected chimeric glycosyltransferases were constructed by fusing Golgi-signal sequences for compartment-specific localization with the catalytic domain of alpha2,3-sialyltransferase (ST3). Stable expression of these constructs in these cells resulted in a significant reduced alphagal-epitope synthesis, and moreover, a release of retroviral vectors showing an up to 3.5-fold increase in serum stability. Thus, our results suggest that the stably transfected cells stably transfected with chimeric glycosyltransferases compete efficiently with endogenous alpha1,3-galactosyltransferase. This approach allows favored glycodesign and we anticipate the applicability of such improved retroviral vectors produced by glycosylation engineered host cells for in vivo gene therapy and, furthermore, suggest the therapeutic benefit of this technology for xenotransplantation.     

Sialyl-Lewis(x) on P-selectin glycoprotein ligand-1 is regulated during differentiation and maturation of dendritic cells: a mechanism involving the glycosyltransferases C2GnT1 and ST3Gal I

To fulfil their function as APCs, dendritic cells (DC) and their precursors need to travel from blood to the peripheral tissues and, upon activation, migrate from tissues to draining lymph nodes. Because O-glycans play a role in T cell trafficking, we investigated the O-glycosylation profile of human monocyte-derived DC. Sialyl-Lewis(x) (sLe(x)), a glycan involved in extravasation via selectin binding, was found to be expressed exclusively on P-selectin glycoprotein ligand-1 in monocytes and immature DC. However, sLe(x) was lost from mature DC even though these cells retained expression of P-selectin glycoprotein ligand-1. Maturation of DC led to a rapid change in the expression of glycosyltransferases involved in O-linked glycosylation. A down-regulation of C2GnT1 mRNA and enzymatic activity was observed with a concurrent up-regulation of ST3Gal I and ST6GalNAc II mRNA resulting in a loss of the core 2 structures required for sLe(x) expression as a P-selectin ligand. Interestingly, the early regulation of these glycosyltransferases was mediated by PGE(2), which is known to be required for human DC migration. The pattern of O-glycosylation seen in mature cells was very similar to that expressed by naive T cells, which home to lymph nodes. Our data show that the regulation of O-glycosylation controls sLe(x) expression, and also suggest that O-glycans may have a function in DC migration.     

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.     

Affinity and kinetics of sialyl Lewis-X and core-2 based oligosaccharides binding to L- and P-selectin

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.     

Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.     

Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells.

Murine bone marrow cells expressing the cell surface Ag RB6-8C5 were identified by fluorescence-activated cell-sorting analysis using a rat IgG mAb. The fluorescent intensity of RB6-8C5 was variable on bone marrow cells. This made it possible to separate bone marrow cells into distinct subpopulations, RB6-8C5neg, RB6-8C5lo, and RB6-8C5hi cells. Morphologic analysis of the sorted populations demonstrated that the Ag was expressed on myeloid cells. The expression of RB6-8C5 increases with granulocyte maturation, whereas expression is transient on cells in the monocytic lineage. The RB6-8C5hi sorted cells were enriched for end-stage neutrophils (75%), whereas the RB6-8C5lo sorted cells contained more immature myeloid cells and myelocytes (75%). Lymphocytes and macrophages were less than 5% in any RB6-8C5+ population, whereas the erythroid precursors were RB6-8C5neg. The colony forming unit culture (CFU-C) (greater than 90%) were found in the RB6-8C5neg and RB6-8C5lo populations, and all the CFU-granulocyte, erythroid, megakaryocyte, and macrophage (CFU-GEMM) and burst-forming units-erythroid (BFU-E) were in the RB6-8C5neg population. Granulocyte-macrophage-CSFR (GM-CSFR) and IL-1 alpha R were expressed on RB6-8C5hi bone marrow cells, whereas no receptors could be detected on RB6-8C5neg and RB6-8C5lo cells. The expression of the RB6-8C5 Ag can be induced on RB6-8C5neg cells in liquid culture by IL-3 and granulocyte-macrophage CSF. Thus, RB6-8C5 is a myeloid differentiation Ag whose expression can be regulated by cytokines.     

Development of a sensitive separation and quantification method for sialyl Lewis X and Lewis X involving anion-exchange chromatography: biochemical characterization of alpha1-3 fucosyltransferase-VII

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) in human leukocytes is mediated by alpha1-3 fucosyltransferase-VII (FucT-VII), which catalyzes the transfer of fucose from GDP-beta-fucose to the 3-OH of alpha2-3 sialyl N-acetyllactosamine (SA-LN). We developed a simple method for quantitating the reaction product of FucT-VII involving Anion-Exchange Chromatography (AEC). The AEC assay involved the separation of a radio-labeled acceptor from the unreacted nucleotide sugars with 0-0.5 M NH(4)OAc (pH9.0) on QAE-Toyopearl 550C. Furthermore, this assay enabled the separation of the fucosylated products of sialylated and non-sialylated oligosaccharides with this column. Analysis of the FucT-VI reaction mixture showed that Lewis X (Le(x)) was eluted in the flow-through fraction and sLe(x) was eluted with 0.1 M NH(4)OAc, and these products were clearly separated from the fraction of unreacted GDP-[(3)H]fucose. Therefore, this method could be a powerful tool for the characterization of recombinant FucT-VII and for establishing a high-throughput screening system for FucT-VII inhibitors. Beside FucT-VII, this method will be applicable to the assaying of many different glycosyltransferases, including sialyltransferases and glucosaminyltransferases, which are reactive to alpha2-3 SA-LN or N-acetyllactosamine sequences.     

The "early' CD4+CD8+ stage of thymocte differentiation hallmarks the end of a strong positive correlation between extracellular matrix receptor expression and protein tyrosine phosphorylation.

We used irradiation-induced thymic regression/reconstitution to study phosphotyrosine (PTyr) levels and expression of extracellular matrix receptors in thymocyte subsets by flow cytometry. High PTyr levels (PTyr(hi)) characterized cells from the CD4-CD8-(DN)CD25in/hi to the "early" CD4-CD8+(DP)CD25- stage. Correlation indexes (R) between the percentages of these PTyrhi cells and cells with up-regulated expression of alpha4 integrin (alpha4hi) were strongly positive (R= 0.91, P= 0.002, for DN; R= 0.98, P= 0.0001 for DP). At the "early" DP stage, R between PTyrhi cells and cells with up-regulated expression of alpha5 integrin and L-selectin (alpha5hi and L-sel(hi)) also rendered strongly positive (R>0.95, p<0.0003). "Late" expanding DP cells exhibited intermediate PTyr levels (PTyr(in)), associated with a down-regulation of the adhesion receptors assessed. Triple-labeling suggested that in most early CD3-/lo cells, alpha4hi and alpha5hi, but not L-sel(hi) expression preceded a PTyr(hi) content. CD3in/hi-enriched CD8+ cells were also PTyr(hi), but conversely to the immature ones exhibited a tendency for a negative R between PTyr(hi) and alpha4hi (R = -0.93, P = 0.067, n= 4) or alpha5hi cells (R = -0.77, P = 0.23, n = 4). CD4+ cells were either PTyr(hi) or PTyr(in), exhibiting a tendency for a positive R (R = 0.59, P = 0.124, n= 8) between PTyr(hi) and L-sel(hi) cells only. In conclusion, our results associate an up-regulation of alpha4 and alpha5 chains expression with PTyr(hi) levels and, as elsewhere published, with increased adhesion to fibronectin up to the "early" DP stage, but not afterwards.     

Alpha1,3fucosyltransferases in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a glycophenotype of aberrant sialylation and/or fucosylation. The CF glycophenotype is expressed on membrane glycoconjugates of CF airway epithelial cells as increased fucosyl residues in alpha1,3/4 linkage to N-acetyl glucosamine, decreased fucosyl residues in alpha1,2 linkage to galactose and decreased sialic acid. To define the cause of this phenotype, the enzyme activity of alpha1,3fucosyltransferase (FucT) was examined in extracts of CF airway epithelial cells with a variety of low molecular weight substrates. Using Galbeta1,4GlcNAc as substrate, the activity was divided into 66% alpha1,3FucT and 34% alpha1,2FucT. mRNA expression examined with probes to FucTIII, IV, and VII showed that the highest expression of two CF cell lines was for FucTIV. Only one CF cell line expressed mRNA for FucTIII. The non CF airway epithelial cells had significant enzyme activity for alpha1,3FucT and strong mRNA expression for FucTIV. Thus as reported previously for alpha1,2FucT, the biochemical capacity for alpha1,3FucT was present in both the CF and non CF cells and can not be the cause of the CF glycophenotype. These results support the hypothesis that wild type CFTR acts in the Golgi and when mutated as in CF, faulty compartmentalization of terminal glycosyltransferases results, yielding the CF glycophenotype.     

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3- galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.