RESUMO
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.
Assuntos
Arachis/química , Glicopeptídeos/química , Lectinas de Plantas/química , Polissacarídeos/química , Ricinus/química , Sambucus nigra/química , Triticum/químicaRESUMO
Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyltransferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galß1-3GlcNAc), LacNAc Type-II (Galß1-4GlcNAc), and mucin core-1/Type-III (Galß1-3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated ß1,3GalT and α2,3SialylT activity that can form α2,3SialylatedType-IIIglycans (Siaα2-3Galß1-3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4FucT activity was higher in breast, but not in colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galß1-3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not in normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylatedType-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict truncated O-glycan structures in tumors. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis.
Assuntos
Antígenos de Neoplasias/química , Neoplasias da Mama/imunologia , Glucosiltransferases/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Miniaturização , Análise Serial de TecidosRESUMO
Glycosyltransferases (glycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate-, lipid-, and protein-based acceptors. We examined strategies to scale down and increase the throughput of glycoT enzymatic assays because traditional methods require large reaction volumes and complex chromatography. Approaches tested used (i) microarray pin printing, an appropriate method when glycoT activity was high; (ii) microwells and microcentrifuge tubes, a suitable method for studies with cell lysates when enzyme activity was moderate; and (iii) C(18) pipette tips and solvent extraction, a method that enriched reaction product when the extent of reaction was low. In all cases, reverse-phase thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified the reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrated an increase in overall ß(1,3)galactosyltransferase and α(2,3)sialyltransferase activity and a decrease in α(1,3)fucosyltransferases when these cells differentiate toward cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Lewis(x)-type structures in mESCs to sialylated Galß1,3GalNAc-type glycans on differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated toward cardiomyocytes. Overall, simple, rapid, quantitative, and scalable glycoT activity analysis methods are presented. These use a range of natural and synthetic acceptors for the analysis of complex biological specimens that have limited availability.
Assuntos
Diferenciação Celular , Cromatografia em Camada Fina , Glicosiltransferases/metabolismo , Células-Tronco/citologia , Animais , Fucosiltransferases/metabolismo , Galactosiltransferases/metabolismo , Lectinas/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Ligação Proteica , Células-Tronco/enzimologiaRESUMO
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.
Assuntos
Mucinas/química , Neoplasias/química , Neoplasias/metabolismo , Polissacarídeos/química , Sialiltransferases/metabolismo , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Feminino , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Humanos , Masculino , Mucinas/sangue , Mucinas/metabolismo , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/análise , Polissacarídeos/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
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.
Assuntos
Domínio Catalítico , Glicoconjugados/química , Mucinas/química , Ácidos Siálicos/química , Sialiltransferases/química , Animais , Antígenos Glicosídicos Associados a Tumores/química , Bovinos , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/química , Gangliosídeos/química , Glicosilação , Humanos , Masculino , Mucinas/metabolismo , Ratos , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Suínos , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
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.
Assuntos
Acetilglucosamina/análogos & derivados , Adesão Celular , Leucócitos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/química , Acetilação , Acetilglucosamina/farmacologia , Animais , Western Blotting , Células da Medula Óssea/metabolismo , Movimento Celular , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Citometria de Fluxo , Glicosilação , Células HL-60 , Humanos , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Peritonite/imunologia , Peritonite/metabolismo , Peritonite/patologia , Ligação ProteicaRESUMO
Cumulative evidence indicates that the sialyltransferase ST6Gal-1 and the sialyl-glycans, which it constructs, are functionally pleiotropic. Expression of the ST6Gal-1 gene is mediated by six distinct promoter/regulatory regions, and we hypothesized that these promoters may be used differentially to produce ST6Gal-1 for different biologic purposes. To examine this hypothesis, we compared a mouse with a complete deficiency in ST6Gal-1 (Siat1 null) with another mouse that we have created previously with a disruption only in the P1 promoter (Siat1DeltaP1). We noted previously greater neutrophilic inflammation associated with ST6Gal-1 deficiency. Here, we report that ST6Gal-1-deficient mice also have significantly elevated eosinophilic responses. Upon i.p. thioglycollate elicitation, eosinophils accounted for over 20% of the total peritoneal inflammatory cell pool in ST6Gal-1-deficient animals, which was threefold greater than in corresponding wild-type animals. A principal feature of allergic respiratory inflammation is pulmonary eosinophilia, we evaluated the role of ST6Gal-1 in allergic lung inflammation. Using OVA and ABPA experimental models of allergic airways, we showed that ST6Gal-1 deficiency led to greater airway inflammation characterized by excessive airway eosinophilia. The severity of airway inflammation was similar between Siat1DeltaP1 and Siat1 null mice, indicating a role for P1-generated ST6Gal-1 in regulating eosinophilic inflammation. Colony-forming assays suggested greater IL-5-dependent eosinophil progenitor numbers in the marrow of ST6Gal-1-deficient animals. Moreover, allergen provocation of wild-type mice led to a significant reduction in P1-mediated ST6Gal-1 mRNA and accompanied decline in circulatory ST6Gal-1 levels. Taken together, the data implicate ST6Gal-1 as a participant in regulating not only Th1 but also Th2 responses, and ST6Gal-1 deficiency can lead to the development of more severe allergic inflammation with excessive eosinophil production.
Assuntos
Eosinófilos/imunologia , Hipersensibilidade/complicações , Pneumonia/complicações , Pneumonia/enzimologia , Regiões Promotoras Genéticas/genética , Sialiltransferases/deficiência , Sialiltransferases/genética , Doença Aguda , Animais , Apoptose , Aspergilose Broncopulmonar Alérgica/complicações , Aspergilose Broncopulmonar Alérgica/enzimologia , Aspergilose Broncopulmonar Alérgica/imunologia , Aspergilose Broncopulmonar Alérgica/patologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/sangue , Modelos Animais de Doenças , Eosinófilos/citologia , Eosinófilos/enzimologia , Hipersensibilidade/sangue , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Imunoglobulina G/sangue , Contagem de Leucócitos , Fígado/enzimologia , Fígado/patologia , Longevidade , Camundongos , Peritonite/induzido quimicamente , Peritonite/enzimologia , Peritonite/imunologia , Pneumonia/sangue , Pneumonia/imunologia , Sialiltransferases/metabolismo , Células-Tronco , Células Th2/imunologia , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
The application of systems biology methods in the emerging field of glycomics requires the collection and integration of glycosyltransferase data at the gene and enzyme level for the purpose of hypothesis generation. We systematically examined the relationship between gene expression, glycosyltransferase activity, glycan expression, and selectin-binding function in different systems, including human neutrophils, undifferentiated HL-60 (human promyelocytic cells), differentiated HL-60, and HL-60 synchronized in specific growth phases. Results demonstrate that 1) the sLe(X) (sialyl-Lewis-X) epitope is expressed in P-selectin glycoprotein ligand-1 (PSGL-1) from neutrophils at higher levels compared with HL-60. This variation may be due to differences in the relative activities of alpha1,3-fucosyltransferases and alpha2,3-sialyltransferases in these two cell types. 2) HL-60 cell differentiation along granulocyte lineage increased the activity of beta1,4GalT and beta1,3GlcNAcT by 1.6- to 3.2-fold. This may contribute to LacNAc chain extension as evidenced by the 1.7-fold increase in DSA-lectin (lectin recognizing LacNAc) binding to cells after differentiation. 3) The activity of enzymes contributing to sLe(X) formation in leukocytes likely varies as ST3[Galbeta1,4GlcNAc] < or = alpha1,3FT[sialyl-LacNAc] < beta1,3GlcNAcT. 4) O-glycan specific glycosyltransferase activity does not undergo periodic variation with cell cycle phases. Overall, gene expression and enzyme activity data combined with knowledge of biochemistry can predict the resulting glycan structures and yield viable experimentally testable hypothesis.
Assuntos
Glicosiltransferases/genética , Leucócitos/fisiologia , Polissacarídeos/biossíntese , Selectinas/metabolismo , Biologia de Sistemas/métodos , Ciclo Celular , Diferenciação Celular , Expressão Gênica , Glicômica/métodos , Células HL-60 , Humanos , Hidroxiureia/farmacologia , Glicoproteínas de Membrana/biossíntese , Neutrófilos/fisiologia , Selectina-P/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialiltransferases/genéticaRESUMO
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.
Assuntos
Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Monofosfato de Citidina/metabolismo , Glicolipídeos/metabolismo , Sialiltransferases/metabolismo , Animais , Galinhas , Cromatografia de Afinidade , Cromatografia Líquida , Monofosfato de Citidina/química , Ácido N-Acetilneuramínico do Monofosfato de Citidina/química , Glicolipídeos/química , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos/química , Polissacarídeos/metabolismo , Ratos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Sialiltransferases/química , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma/enzimologia , Fucosiltransferases/metabolismo , Polissacarídeos/metabolismo , Neoplasias Gástricas/enzimologia , Sulfotransferases/metabolismo , Glicosiltransferases/metabolismo , HumanosRESUMO
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.
Assuntos
Flúor/química , Glicosiltransferases/antagonistas & inibidores , Mucinas/química , Oligossacarídeos de Cadeias Ramificadas/síntese química , Oligossacarídeos de Cadeias Ramificadas/farmacologia , Sulfotransferases/antagonistas & inibidores , Configuração de Carboidratos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologiaRESUMO
Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.
Assuntos
Neoplasias da Mama/enzimologia , Carboidratos/química , Glicosiltransferases/metabolismo , Polissacarídeos/química , Sulfotransferases/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sequência de Carboidratos , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
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.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Carboidratos/química , Mucinas/metabolismo , Sialiltransferases/metabolismo , Antígenos Glicosídicos Associados a Tumores/química , Sequência de Carboidratos , Linhagem Celular Tumoral , Cromatografia , Clonagem Molecular , Glicosilação , Humanos , Lectinas , Ligantes , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Mucinas/química , Neoplasias da Próstata/patologia , Selectinas , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Especificidade por Substrato , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galbeta1,4GlcNAcbeta1,6(3-O-MeGalbeta1,3)GalNAcalpha-O-Bn (where Bn is benzyl) and the disaccharide Galbeta1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalbeta1,4GlcNAcbeta1,-6(Galbeta1,3)GalNAcalpha-O-Bn and Galbeta1,3GalNAcalpha-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galbeta1,3GalNAcbeta1,3Galalpha-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galbeta1,4GlcNAcbeta unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galbeta1,4GlcNAcbeta1,6(3-O-sulfoGalbeta1,3)GalNAcalpha-O-Bn but not 3-O-sulfoGalbeta1,-4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galbeta1,4(6-O-sulfo)-GlcNAcbeta-O-Al as acceptor, Gal3ST-2 acted efficiently on Galbeta1,3(6-O-sulfo)GlcNAcbeta-O-Al, and Gal3ST-4 acted efficiently on Galbeta1,3(6-O-sulfo)GalNAcalpha-O-Al. Mg(2+), Mn(2+), and Ca(2+) stimulated the activities of Gal3ST-2, whereas only Mg(2+) augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn(2+) concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.
Assuntos
Acetilglucosamina/química , Sulfotransferases/química , Sulfotransferases/genética , Animais , Antígenos Glicosídicos Associados a Tumores/química , Ligação Competitiva , Células CHO , Linhagem Celular Tumoral , Cromatografia em Camada Fina , Clonagem Molecular , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mucinas/química , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Sulfotransferases/metabolismo , Trissacarídeos/químicaRESUMO
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.
Assuntos
Neoplasias do Colo/enzimologia , Fucosiltransferases/metabolismo , Lectinas/metabolismo , Lectinas de Plantas/metabolismo , Animais , Bovinos , Cromatografia de Afinidade , Clonagem Molecular , Concanavalina A/metabolismo , Dissacarídeos/metabolismo , Fucosiltransferases/química , Fucosiltransferases/isolamento & purificação , Glicopeptídeos/metabolismo , Humanos , Imunoglobulina G/imunologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
The synthesis of the common and well-documented Siaalpha 2,6 to Galbeta 1,4GlcNAc structure (Sia6LacNAc) is principally mediated by the sialyltransferase ST6Gal I, which is particularly highly expressed in liver, lactating mammary gland, intestinal epithelia of newborn animals, and B cells. Multiple independent promoters govern the expression of Siat1, the ST6Gal I gene. In liver, elevation of hepatic and serum ST6Gal is part of the acute phase reaction, the hepatic response to systemic trauma, and is governed by the inducible, liver-specific promoter-regulatory region, P1. A constitutive and nontissue-specific promoter, P3, mediates low-level, basal hepatic Siat1 transcription. We generated a mouse specifically unable to use the transcriptional initiation site uniquely used in P1-mediated ST6Gal I expression. These animals, Siat1deltaP1, are viable and display reduced ST6Gal I mRNA in liver with concomitantly reduced sialyltransferase activities in liver and in serum. Siat1deltaP1 animals are unable to elevate hepatic Siat1 mRNA as part of the inflammatory response induced by turpentine. Surprisingly, serum glycoprotein components exhibit normal extent of sialylation, with no noticeable difference in binding to SNA, the alpha2,6-sialyl-specific lectin. Siat1deltaP1 animals also exhibit an outwardly normal B cell response. On intraperitoneal challenge with the pathogen Salmonella typhimurium, a significantly greater accumulation of neutrophils within the peritoneal space was observed in Siat1deltaP1 animals compared to wild-type mice. Siat1deltaP1 mice also exhibit a greater bacterial burden in liver and spleen, accompanied by more pronounced spleno-/hepatomegaly and greater leukocyte infiltration into affected organs than their wild-type counterparts.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Sialiltransferases/genética , Sialiltransferases/metabolismo , Reação de Fase Aguda/enzimologia , Reação de Fase Aguda/imunologia , Reação de Fase Aguda/microbiologia , Animais , Feminino , Fígado/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Especificidade de Órgãos , Recombinação Genética/genética , Infecções por Salmonella/enzimologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologia , Deleção de Sequência/genética , Sialiltransferases/imunologia , Baço/microbiologia , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
OBJECTIVE: Approximately 50% of patients with ovarian cancer who have normal CA 125 levels at the completion of therapy have persistent disease. In an effort to improve the ability to detect small volume disease, we have evaluated the usefulness of N-acetylglucosamine:beta1,4-galactosyltransferase as a potential biomarker for the detection of subclinical disease after the completion of primary therapy for ovarian cancer. STUDY DESIGN: The sera of 33 patients with stage IIIC epithelial ovarian cancer in complete clinical remission after chemotherapy (CA 125 <35 units/mL and negative computed tomography scan) who underwent second-look surgery were examined for N-acetylglucosamine:beta1,4-galactosyltransferase activity. The values were determined from sera that had been obtained before primary cytoreductive operation and before second-look surgery after the completion of platinum-based chemotherapy. Determinations of the levels of CA 125 were performed with the Bayer Immuno ITM CA-125 II assay. N-acetylglucosamine:beta1,4-galactosyltransferase activity was determined by measuring the transfer of galactose from uridine diphosphate- carbon 14-labeled galactose to the terminal N-acetylglucosamine residue of a very well-defined synthetic acceptor, N-acetylglucosamine:beta1,6GalNAc(alpha)-o-benzyl, which is a portion of the core structure of mucin glycoproteins. The cutoff value of N-acetylglucosamine:beta1,4-galactosyltransferase was determined to be 22,000 counts/min, based on the analysis of 25 healthy control subjects. Correlation between serum CA 125 and N-acetylglucosamine:beta1,4-galactosyltransferase levels was determined with the use of the Pearson correlation coefficient. The ability of galactosyltransferase to identify small volume disease correctly was also evaluated. RESULTS: There was a significant correlation between serum CA 125 and N -acetylglucosamine:beta1,4-galactosyltransferase levels before the operation (r = 0.57; P =.03) but not before second-look surgery (r = 0.10; P =.57). Thirteen patients (39.4%) had residual disease at second-look surgery. Elevated N-acetylglucosamine:beta-1,4galactosyltransferase activity >22,000 cpm correctly identified 10 of these patients (76.9%). The sensitivity, specificity, and positive and negative predictive values of N-acetylglucosamine:beta1,4-galactosyltransferase activity (>22,000 counts/min) for the prediction of residual disease at second-look surgery were 77%, 45%, 48%, and 77%, respectively. CONCLUSION: Our comparative study of serum CA 125 and N -acetylglucosamine:beta1,4-galactosyltransferase levels showed a significant correlation between the two tumor markers before the beginning of ovarian cancer therapy. This correlation disappeared before second-look surgery because 60% of patients with normal serum CA 125 and N-acetylglucosamine:beta1,4-galactosyltransferase levels. CA 125 antigen appears to be inferior to N -acetylglucosamine:beta1,4-galactosyltransferase in the detection of small-volume residual disease. N-acetylglucosamine:beta1,4-galactosyltransferase may be useful as a biomarker in the monitoring of patients with ovarian cancer when the serum CA 125 level is normal. These findings require confirmation in larger studies.
Assuntos
Biomarcadores Tumorais/sangue , N-Acetil-Lactosamina Sintase/sangue , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/terapia , Adulto , Idoso , Antígeno Ca-125/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasia Residual , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Ovarianas/sangueRESUMO
A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor 10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1-3 were then obtained by the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1-3 obtained, biological evaluations of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.
Assuntos
Oligossacarídeos/síntese química , Sequência de Carboidratos , Cristalografia por Raios X , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Metilação , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Ácidos Siálicos/química , Espectrometria de Massas por Ionização por Electrospray , Sulfatos/químicaRESUMO
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.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Epitopos/biossíntese , Fucosiltransferases/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/biossíntese , Sequência de Carboidratos , Cátions Bivalentes/farmacologia , Epitopos/química , Etilmaleimida/farmacologia , Fucosiltransferases/antagonistas & inibidores , Humanos , Cinética , Antígenos do Grupo Sanguíneo de Lewis/química , Masculino , Dados de Sequência Molecular , Oligossacarídeos/síntese química , Oligossacarídeos/química , Neoplasias da Próstata , Células Tumorais Cultivadas , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Affinity Gel-UDP was utilized to purify GlcNAc:beta1,4Gal/GalNAc transferases (Ts) from human lymphoma spleen, ovarian tumor, and ovarian cancer sera. Mn(2+) was found to be an absolute requirement for activity. Two molecular species containing both beta1,4Gal/GalNAc-T activities were discernible when the purified ovarian tumor microsomal enzyme was subjected to Sephacryl S-100 HR column chromatography as well as native polyacylamide gel-electrophoresis. Acceptor specificity studies of the affinity-purified lymphoma spleen and ovarian tumor microsomal enzymes and the conventionally purified, as well as the cloned, bovine milk GlcNAc:beta1,4Gal-Ts using a number of synthetic acceptors showed that the beta(1,6)-linked GlcNAc moiety to alpha-GalNAc was the most efficient acceptor. As compared to the purified milk enzyme, the recombinant form exhibited sixfold GlcNAc:beta1,4 GalNAc-T activity and up to eightfold GlcNAc6SO3beta-:beta1,4Gal-T activity. Further, the recombinant enzyme catalyzed the transfer of GalNAc to the terminal beta-linked GlcNAc6SO3 moiety. Alpha-lactalbumin (alpha-LA) inhibited up to 85%, the transfer of Gal to the GlcNAc moiety linked either to Man or GlcNAc. On the contrary, alpha-LA had no significant influence on the transfer of GalNAc to the above acceptors. alpha-LA had no appreciable effect on the recombinant enzyme, except for the transfer of Gal or GalNAc to Glc. Both alpha- and beta-glucosides, as well as alpha-N-acetylglucosaminide, did not serve as acceptors.