Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galß1-4GlcNAcß1-6(Galß1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucß1-3 GalNAc and Fucα-1-2 D-Fucß-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc ß-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II.

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galß1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.

Mammalian sialyltransferase ST3Gal-II: its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated glycoproteins and specific gangliosides.

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galß1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galß1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galß1,3GalNAcß1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galß1,3GalNAcß1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin ß subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins.

Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50muM per-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins.

The first chemical synthesis of novel MeO-3-GlcUA derivative of hyaluronan-based disaccharide to elucidate the catalytic mechanism of hyaluronic acid synthases (HASs).

The first chemical synthesis of MeO-3-GlcUAß(1→3)GlcNAc-UDP to elucidate the catalytic mechanism of hyaluronic acid synthases (HASs) is described. Construction of the desired ß(1→3)-linked disaccharide 10 was achieved very efficiently by coupling MeO-3-GlcUA donor 3 with the suitable protected GlcNTroc acceptor 4 using BF(3(.) )Et(2)O as Lewis acid. Chemoselective removal of anomeric NAP, phosphorylation, hydrogenation, coupling with UMP-morpholidate and finally complete deprotection gave the target compound 1 in good yield.

Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products.

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.

Potential tumor markers for human gastric cancer: an elevation of glycan:sulfotransferases and a concomitant loss of alpha1,2-fucosyltransferase activities.

PURPOSE: Several reports indicate a complexity in glycosyltransferase activities which lead to several tumor associated carbohydrate structures in gastric carcinoma. The present study was aimed to identify the carbohydrate associated transferases which exhibit the most marked and consistent change of activity in gastric tumorigenesis. METHODS: We examined the levels of fucosyl, beta-galactosyl-, beta-N-acetylgalactosaminyl, sialyl- and glycan:sulfotransferase activities, which generate the outer ends of oligosaccharide chains in tumorous and adjacent normal gastric tissues of the same patient in ten gastric carcinoma cases by using well defined specific synthetic acceptors utilized in our several earlier published studies as referenced in the text (e.g. Chandrasekaran et al. in J Biol Chem 279:10032-10041, 2004; Biochemistry 44:15619-15635, 2005; Carbohydr Res 341:983-994, 2006). RESULTS: Among glycosyltransferases only alpha1,2-fucosyltransferase (FT) was unique in showing a remarkable 40-90% decrease of activity in seven cases. Uniquely several fold elevation of Gal3Sulfo-T(2) (1.9 --> 156.7 fold) and Gal3Sulfo-T(4) (2.4 --> 149.0 fold) activities in all ten cases and moderate elevation of GlcNAc6Sulfo-T (1.3 --> 37.5 fold) activities in nine cases were identified. Poorly differentiated Signet ring cell carcinoma expresses mainly Gal3Sulfo-T(2) activity whereas poorly differentiated adenocarcinoma express predominantly Gal3Sulfo-T(4) activity and also GlcNAc6Sulfo-T activity. But, very low level of these sulfotransferase activities were identified in moderately differentiated gastric carcinomas as well as non-epithelial gastric stromal sarcoma. CONCLUSION: Up regulation of glycan:sulfotransferase activities and down regulation of alpha1,2-fucosyltransferase activity are apparently associated with human gastric tumorigenesis.

Synthesis of fluorinated mucin core 2 branched oligosaccharides with the potential of novel substrates and enzyme inhibitors for glycosyltransferases and sulfotransferases.

Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C-3 or C-4 position of the pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases and 3-O-sulfotransferases involved in the biosynthesis of O-glycans. Our acceptor substrate specificity studies on three cloned sialyltransferases (Sia-Ts) revealed that a 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for alpha2,6(N)Sia-T and alpha2,3(N)Sia-T, whereas 4-fluoro-Galbeta1,3GalNAcalpha was a good acceptor for alpha2,3(O)Sia-T. Uniquely, 4-F-Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-OBn was an inhibitor of alpha2,6(N)Sia-T activity but not alpha2,3(N)Sia-T activity. Further we found that the activities of only Gal 3-O-sulfotransferases and not sialyltransferases were adversely affected by a C-3 fluoro substituent at the other Gal terminal of mucin core 2. The strategy of building branched mucin core 2 structures by three glycosidation sequence coupling three classes of glycosyl donors with the reactivity-matching acceptors proved to be successful in syntheses of modified mucin-type core structures of O-glycan. The relative poor yields of the glycosylations using fluorinated galactosyl donors indicated that the fluorine modification dramatically decreased the donor reactivity due to electron-withdrawing effect.

Analysis of the specificity of sialyltransferases toward mucin core 2, globo, and related structures. identification of the sialylation sequence and the effects of sulfate, fucose, methyl, and fluoro substituents of the carbohydrate chain in the biosynthesis of selectin and siglec ligands, and novel sialylation by cloned alpha2,3(O)sialyltransferase.

Sialic acids are key determinants in many carbohydrates involved in biological recognition. We studied the acceptor specificities of three cloned sialyltransferases (STs) [alpha2,3(N)ST, alpha2,3(O)ST, and alpha2,6(N)ST] and another alpha2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2 tetrasaccharide [Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn] and Globo [Galbeta1,3GalNAcbeta1,3Galalpha-O-Me] structures containing sialyl, fucosyl, sulfo, methyl, or fluoro substituents by identifying the products by electrospray ionization tandem mass spectral analysis and other biochemical methods. The Globo precursor was an efficient acceptor for both alpha2,3(N)ST and alpha2,3(O)ST, whereas only alpha2,3(O)ST used its deoxy analogue (d-Fucbeta1,3GalNAcbeta1,3-Gal-alpha-O-Me); 2-O-MeGalbeta1,3GlcNAc and 4-OMeGalbeta1,4GlcNAc were specific acceptors for alpha2,3(N)ST. Other major findings of this study include: (i) alpha2,3 sialylation of beta1,3Gal in mucin core 2 can proceed even after alpha1,3 fucosylation of beta1,6-linked LacNAc. (ii) Sialylation of beta1,3Gal must precede the sialylation of beta1,4Gal for favorable biosynthesis of mucin core 2 compounds. (iii) alpha2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when beta1,3Gal has already been alpha2,3 sialylated. (iv) alpha2,6(N)ST was absolutely specific for the beta1,4Gal in mucin core 2. Either alpha1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reduced the activity. Sialylation of beta1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity. (v) Prior alpha2,3 sialylation or 3-O-sulfation of beta1,3Gal would not affect alpha2,6 sialylation of Galbeta1,4GlcNAc of mucin core 2. (vi) A 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for the cloned alpha2,6(N)ST and alpha2,3(N)ST, whereas 4-fluoro- or 4-OMe-Galbeta1,3GalNAcalpha was a good acceptor for cloned alpha2,3(O)ST. (vii) 4-O-Methylation of beta1,4Gal abolished the acceptor ability toward alpha2,6(N)ST but increased the acceptor efficiency considerably toward alpha2,3(N)ST. (viii) Just like LNCaPalpha1,2-FT and Gal-3-O-sulfotransferase T2, the cloned alpha2,3(N)ST which modifies terminal Gal in Galbeta1,4GlcNAc also efficiently utilizes the terminal beta1,3Gal in the Globo backbone. Utilization of C-3 blocked compounds such as 3-O-sulfo-Galbeta1,3GalNAcbeta1,3Galalpha-OMe as acceptors by cloned alpha2,3(O)ST and analyses of the resulting products by lectin chromatography and mass spectrometry indicate that alpha2,3(O)ST is capable of attaching NeuAc to another position in C-3-substituted beta1,3Gal.

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry.

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.

Specificity of a soluble UDP-galactose: fucoside alpha1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium.

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

The binding characteristics and utilization of Aleuria aurantia, Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells.

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.

Complex oligosaccharide investigations: synthesis of an octasaccharide incorporating the dimeric Le(x) structure of PSGL-1.

The synthesis of an octasaccharide containing the dimeric Le(x) oligosaccharide structure found in PSGL-1 carbohydrate chains is reported. Several approaches were investigated employing regioselective and stereoselective glycosylation procedures, and a novel Lewis(x) trisaccharide donor, 7, was prepared and utilized as a key intermediate building block in the scheme developed for the construction of octasaccharide 3. Toward the preparation of 7, investigations into the influence of different protecting groups upon the relative reactivities of disaccharide acceptor moieties, 25 or 26, and the fucosyl donors, 10 and 11, were conducted using similar glycosylating conditions. Dramatic differences were noted between the effects of electron-donating and electron-withdrawing groups upon the reactivity of the acceptor hydroxyl. A similar effect upon the glycosylating capability of the donor molecule was, likewise, observed. The repeat use of donor 7 was instrumental in the synthesis of the desired dimeric octasaccharide structure 3. The structure and purity of 3 and important intermediates were fully characterized by DQF-COSY, TOCSY, ROESY, and ESI mass spectroscopy.

Biosynthesis of the carbohydrate antigenic determinants, Globo H, blood group H, and Lewis b: a role for prostate cancer cell alpha1,2-L-fucosyltransferase.

Prostate carcinoma LNCaP cells were unique among several human cancer cell lines which include two other prostate cancer cell lines, PC-3 and DU-145, in expressing alpha1,2-L-fucosyltransferase (FT) as an exclusive FT activity. Affinity gel-GDP and Sephacryl S100 HR columns were used for a partial purification of this enzyme from 3.9 x 10(9) LNCaP cells (approximately 200-fold; 40% yield). The K(m) value (2.7 mM) for the LacNAc type 2 acceptor was quite similar to the one reported for the cloned blood group H gene-specified alpha1,2-FT [Chandrasekaran et al. (1996) Biochemistry 35, 8914-8924]. N-Ethylmaleimide was a potent inhibitor (K(i ) 12.5 microM). The enzyme showed four-fold acceptor preference for the LacNAc type 2 unit in comparison to the T-hapten in mucin core 2 structure. Its main features were similar to those of the cloned enzyme: (1) C-6 sulfation of terminal Gal in the LacNAc unit increased the acceptor efficiency, whereas C-6 sialylation abolished acceptor ability; (2) C-6 sulfation of GlcNAc in LacNAc type 2 decreased by 80% the acceptor ability, whereas LacNAc type 1 was unaffected; (3) Lewis x did not serve as an acceptor; (4) the C-4 hydroxyl rather than the C-6 hydroxyl group of the GlcNAc moiety in LacNAc type1 was essential for activity; and (5) the acrylamide copolymer of Galbeta1,3GlcNAcbeta-O-Al was the best acceptor among the acrylamide copolymers. Additionally, highly significant biological features of alpha1,2FT were identified in the present study. The synthesis of Globo H and Lewis b determinants became evident from the fact that Galbeta1,3GalNAcbeta1,3Galalpha-O-Me and Galbeta1,3(Fucalpha1,4)Glc-NAcbeta1,3Galbeta-O-Me served as high-affinity acceptors for this enzyme. Further, D-Fucbeta1,3Gal-NAcbeta1,3Galalpha-O-Me was a very efficient acceptor, indicating that the C-6 hydroxyl group of the terminal Gal moiety in Globo H is not essential for the enzyme activity. Thus, the present study was able to demonstrate three different catalytic roles of LNCaP alpha1,2-FT, namely, the expressions of blood group H, Lewis b from Lewis a, and Globo H.