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PubMed1978

Human serum galactosyltransferase: distinction, separation and product identification of two galactosyltransferase activities.

Two different galactosyltransferase activities have been found in normal sera from A and O donors. Galactosyltransferase A incorporated galactose from UDP-Gal into sialic-acid-free ovine submaxillary mucin (asialo-mucin), whereas galactosyltransferase B transferred galactose from UDP-Gal to free N-acetylglucosamine or N-acetylglucosamine-glycoproteins. Specificity, kinetic and stability differences permitted the distinction of the activity of galactosyltransferase A from that of galactosyltransferase B; the only substrate found for galactosyltransferase A was asialo-mucin, whereas galactosyltransferase B showed only low activity towards asialo-mucin and free N-acetyl-galactosamine, but had a main specificity for either free N-acetylglucosamine or N-acetylglucosamine-protein. Galactosyltransferase B was more stable on heat inactivation than galactosyltransferase A; galactosyltransferase B could be separated from galactosyltransferase A by affinity chromatography on N-acetylglucosamine-derivatized agarose. The products of both enzyme activities have been analyzed. The galactosyltransferase A product was cleaved from asialo-mucin by alkaline-borohydride treatment. The acceptor used to identify the galactosyltransferase B product was free N-acetylglucosamine. Periodate oxidation studies performed on the reduced disaccharides indicated the linkage type of the products. The anomeric configuration of the respective galactosyltransferase products were determined with specific galactosidases. Using these methods, galactosyltransferase A was found to form a Galbeta (1 leads to 3)GalNAc-protein linkage and galactosyltransferase B was found to form a Galbeta(1 leads to 4)GlcNAc-linkage.

Galactosyltransferases↗
PubMed1994

Expressing murine beta 1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype.

Beta 1,4-Galactosyltransferase is traditionally viewed as a biosynthetic component of the Golgi complex, but a portion of galactosyltransferase is also expressed on the cell surface, where it has been suggested to function as a receptor for extracellular oligosaccharide ligands. Although results from a variety of studies are consistent with a cell adhesion function for galactosyltransferase, the most rigorous test of surface galactosyltransferase function is to produce a surface galactosyltransferase-dependent phenotype in cells that normally express negligible levels of surface galactosyltransferase. In agreement with previous reports, human HeLa cells were found to express low levels of galactosyltransferase on their surface and, therefore, were stably transfected with cDNAs encoding murine galactosyltransferase. Murine galactosyltransferase was expressed both within the presumed Golgi complex and on the cell surface, as assayed by enzyme activity and with antiserum raised against the bacterially expressed murine enzyme. HeLa cell transfectants adhered more strongly to their extracellular substrates than did control transfectants, as evidenced by a flatter morphology in culture and a more rapid spreading upon plating. In contrast, cell spreading was low and similar among all cell types when plated on extracellular substrates that did not contain binding sites for galactosyltransferase. Antibodies and Fab fragments against recombinant murine galactosyltransferase inhibited the increased cell spreading characteristic of galactosyltransferase transfectants, as did soluble recombinant galactosyltransferase and a variety of galactosyltransferase perturbants. Thus, expression of heterologous galactosyltransferase produces a surface galactosyltransferase-dependent phenotype, confirming its function as a cell adhesion molecule.

Animals↗
PubMed1990

Transfer and expression of a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase gene in transfected Chinese hamster ovary cells. Competition reactions between the alpha 1,3-galactosyltransferase and the endogenous alpha 2,3-sialyltransferase.

The cDNA encoding a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase has recently been cloned and sequenced using a transient expression method (Larsen, R.D., Rajan, V.P., Ruff, M.M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8227-8231). This report describes the construction and analysis of a Chinese hamster ovary (CHO) cell line in which in vitro expression alpha 1,3-galactosyltransferase activity has been achieved via transfer and expression of the murine alpha 1,3-galactosyltransferase gene. A primary aim of this research was to explore the role of the alpha 1,3-galactosyltransferase in regulating glycoprotein and glycolipid biosynthesis. CHO cells were cotransfected with murine genomic DNA fragments from F9 cells and plasmid DNA containing a resistance gene to the antibiotic G418. Cells resistant to G418 were then selected for expression of surface glycoconjugates containing terminal alpha 1,3-galactosyl residues by isolating cells bound to immobilized Griffonia simplicifolia-I-B4, a lectin which binds to alpha 1,3-galactosyl residues. A positive, stable transfectant clone, designated Clone 3, was obtained and analyzed for expression of the murine of alpha 1,3-galactosyltransferase. Fluorescence-activated cell sorting demonstrated that Clone 3, but not parental, CHO cells bound significant amounts of fluorescein isothiocyanate-labeled G. simplicifolia-I-B4. Southern and Northern blot analyses using the murine alpha 1,3-galactosyltransferase cDNA demonstrated that clone 3, but not parental, CHO cells contain murine alpha 1,3-galactosyltransferase genomic DNA sequences, and express a homologous transcript that comigrates with the authentic 3.6 kilobase alpha 1,3-galactosyltransferase murine mRNA. Enzyme assays confirmed that clone 3, but not parental CHO cells, contained the alpha 1,3-galactosyltransferase activity and that the level of activity is comparable to that found in F9 cells. [3H]Galactose-labeled glycopeptides and glycolipids were obtained from metabolically radiolabeled parental and Clone 3 cells and were analyzed for the presence of terminal alpha 1,3-galactosyl residues. Complex-type, Asn-linked oligosaccharides from both parental and Clone 3 cells contain the repeating disaccharide [3Gal beta 1, 4GlcNAc beta 1]n or poly-N-acetyllactosamine sequences, but only the poly-N-acetyllactosamine chains from clone 3 cells contained the terminal sequence Gal alpha 1,3Gal beta 1,4GlcNAc beta 1-R.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Cloning and expression of a proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I. A seventh member of the human beta4-galactosyltransferase gene family.

A seventh member of the human beta4-galactosyltransferase family, beta4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of beta4Gal-T7 depicts a type II transmembrane protein with sequence similarity to beta4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the beta4Gal-T family. The genomic organization of beta4Gal-T7 was different from previous beta4Gal-Ts. Expression of beta4Gal-T7 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity with beta-xylosides, and the linkage formed was Galbeta1-4Xyl. Thus, beta4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein beta1, 4-galactosyltransferase; EC 2.4.1.133), which attaches the first galactose in the proteoglycan linkage region GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Sequence analysis of beta4Gal-T7 from a fibroblast cell line of a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, previously shown to exhibit reduced galactosyltransferase I activity (Quentin, E., Gladen, A., Rodén, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342-1346), revealed two inherited allelic variants, beta4Gal-T7(186D) and beta4Gal-T7(206P), each with a single missense substitution in the putative catalytic domain of the enzyme. beta4Gal-T7(186D) exhibited a 4-fold elevated K(m) for the donor substrate, whereas essentially no activity was demonstrated with beta4Gal-T7(206P). Molecular cloning of beta4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.

Amino Acid Sequence↗
PubMed1997

A family of human beta4-galactosyltransferases. Cloning and expression of two novel UDP-galactose:beta-n-acetylglucosamine beta1, 4-galactosyltransferases, beta4Gal-T2 and beta4Gal-T3.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of the human UDP-galactose:beta-N-acetylglucosamine beta1, 4-galactosyltransferase, designated beta4Gal-T1, revealed a large number of ESTs with identical as well as similar sequences. ESTs with sequences similar to that of beta4Gal-T1 could be grouped into at least two non-identical sequence sets. Analysis of the predicted amino acid sequence of the novel ESTs with beta4Gal-T1 revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of beta4Gal-T1. The likelihood that the identified ESTs represented novel galactosyltransferase genes was tested by cloning and sequencing of the full coding region of two distinct genes, followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represented genuine UDP-galactose:beta-N-acetylglucosamine beta1, 4-galactosyltransferases, thus designated beta4Gal-T2 and beta4Gal-T3. Genomic cloning of the genes revealed that they have identical genomic organizations compared with beta4Gal-T1. The two novel genes were located on 1p32-33 and 1q23. The results demonstrate the existence of a family of homologous galactosyltransferases with related functions. The existence of multiple beta4-galactosyltransferases with the same or overlapping functions may be relevant for interpretation of biological functions previously assigned to beta4Gal-T1.

Amino Acid Sequence↗
PubMed1998

A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose:beta-N-acetyl-glucosamine/beta-nacetyl-galactosamine beta-1,3-galactosyltransferase family.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:beta-N-acetyl-glucosamine beta-1, 3-galactosyltransferase, designated beta3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to beta3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:beta-N-acetyl-glucosamine beta-1, 3-galactosyltransferase (beta3Gal-T2) with similar kinetic properties as beta3Gal-T1. Another gene represented a UDP-galactose:beta-N-acetyl-galactosamine beta-1, 3-galactosyltransferase (beta3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. beta3Gal-T1 mRNA was expressed in brain, beta3Gal-T2 was expressed in brain and heart, and beta3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. beta3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous beta3-galactosyltransferase genes.

Amino Acid Sequence↗
PubMed2006

The expression of beta-1,3 galactosyltransferase and beta-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation.

The regulation of beta-1,3 galactosyltransferase (3betaGalT) and beta-1,4 galactosyltransferase enzymatic (4betaGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two beta-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4betaGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3betaGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3betaGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17beta-oestradiol (E). The activity of 4betaGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3betaGalT and 4betaGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.

Animals↗
PubMed1988

Identification of the full-length coding sequence for human galactosyltransferase (beta-N-acetylglucosaminide: beta 1,4-galactosyltransferase).

A lambda gt11 human placenta cDNA library was screened using a cDNA probe encoding the COOH-terminal region of human beta 1,4-galactosyltransferase and with a synthetic oligonucleotide having a sequence corresponding to that of the 5' end of the cDNA probe. The newly isolated cDNA was found to code for the NH2-terminal and the 5'-untranslated region, primed at an (A)8 region in the coding sequence. A complete amino acid sequence has been deduced which shows only one membrane anchoring domain near the NH2-terminus. Comparison of the sequence to the soluble enzyme suggests proteolytic cleavage at Arg 77. Presently obtained information of human beta 1,4-galactosyltransferase makes it possible to study DNA mutations responsible for genetic defects such as the altered expression of galactosyltransferase found in a variant of congenital dyserythropoietic anemia type II (HEMPAS).

Amino Acid Sequence↗
PubMed1998

Molecular cloning of a human cDNA encoding beta-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc beta-1,4-galactosyltransferase.

A cDNA encoding a beta-1,4-galactosyltransferase named beta-1,4-GalT II was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3'-rapid amplification of cDNA ends by using two nested degenerate oligonucleotide primers based on a highly conserved amino acid sequence found in the catalytic domain of mammalian beta-1,4-galactosyltransferases and Lymnaea stagnalis beta-1,4-N-acetylglucosaminyltransferase (beta-1,4-GlcNAcT), both of which utilize the same sugar acceptor. This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human beta-1,4-galactosyltransferase (referred to as beta-1,4-GalT I) and of 28% with that of L. stagnalis beta-1,4-GlcNAcT. Study of the properties of the beta-1,4-GalT II fused to protein A expressed as a soluble form in COS-7 cells revealed that it is a genuine beta-1,4-GalT but has no lactose synthetase activity in the presence of alpha-lactalbumin. Northern blot analysis of 24 human tissues showed that they all express the beta-1,4-GalT II transcript, although the levels varied. These results indicate that human cells contain another beta-1,4-GalT.

Amino Acid Sequence↗
PubMed2001

Biosynthesis of the linkage region of glycosaminoglycans: cloning and activity of galactosyltransferase II, the sixth member of the beta 1,3-galactosyltransferase family (beta 3GalT6).

A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1, -2, -3, -4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.

Amino Acid Sequence↗
PubMed1983

Ganglioside biosynthesis in rat liver. Characterization of UDPgalactose--glucosylceramide galactosyltransferase and UDPgalactose-GM2 galactosyltransferase.

The conditions for the quantitative determination of UDP-Gal:glucosylceramide galactosyltransferase and of UDP-Gal:GM2 galactosyltransferase in Golgi-enriched preparations of rat liver were optimized. Triton X-100 was the detergent routinely used as octyl glucoside acted as a galactose acceptor forming octyl lactoside. Manganese ions were required for full activity, but Co2+ and Mg2+ could substitute to some extent. The nucleotide pyrophosphatase activity of the Golgi preparations which interfered with the GL2-synthase assay was inhibited by addition of 20 mM IMP; the latter is without appreciable effect on the rate of GL2 synthesis. Apparent Km values for UDP-Gal were 130 microM and 140 microM with Gl2-synthase and Gm1-synthase, respectively. That for glucosylceramide was 80 microM with GL2-synthase; for GM2 it was 10 microM with GM1-synthase. Competition experiments with variable concentrations of the lipid acceptors showed that the two synthase activities are independent catalytic entities. The specific activity of GM1-synthase exceeds that of GL2-synthase by a factor of ca. 25 under the optimized conditions used here.

Animals↗
PubMed2003

Galactosyltransferase associated with tumor in patients with ovarian cancer: factors involved in elevation of serum galactosyltransferase.

The serum level of beta1,4-galactosyltransferase (beta1,4-GalT) is increased in both malignancy and benign diseases. Galactosyltransferase associated with tumor (GAT) is one of the soluble forms of beta1,4-GalT, and is a marker of ovarian cancer with a high specificity. GAT and normal soluble beta1,4-GalT are both derived from the same membrane-bound form of the enzyme. This study investigated the mechanism of GAT elevation in patients with ovarian cancer. The serum levels of GAT and normal beta1,4-GalT were measured using specific monoclonal antibodies. In addition, nude mice bearing human ovarian cancer were used to assess the kinetics of tumor-derived enzymes. GAT and normal beta1,4-GalT were both detected in ovarian cancer patients, but only GAT reflected the tumor status. In tumor-bearing nude mice, both soluble forms of beta1,4-GalT were released from tumor cells, but the half-life of GAT was far shorter than that of normal beta1,4-GalT. Addition of serum from healthy women to colostrum (which has a high GAT content) reduced the GAT level, while adding patient serum caused a significantly smaller reduction of GAT. Addition of the serum from mouse which includes no human beta1,4-GalT to colostrum also reduced the GAT level with no significant change of total soluble beta1,4-GalT. These findings indicate that human serum contains certain factors that decrease the GAT level, but these factors are inhibited in ovarian cancer patients so that a high GAT level persists. It seems that the decrease of GAT occurs as a result of conversion into normal beta1,4-GalT.

Animals↗
PubMed2005

Oligosaccharide preferences of beta1,4-galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.

beta-1,4-Galactosyltransferase-I (beta4Gal-T1) transfers galactose from UDP-galactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3-arm), and the other to the 6-hydroxyl-(1,6-arm) group of mannose, which is beta-1,4-linked to an N-linked chitobiose, attached to the aspargine residue of a protein. For a better understanding of the branch specificity of beta4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human beta4Gal-T1 (h-beta4Gal-T1) and the mutant Met340His-beta4Gal-T1 (h-M340H-beta4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcbeta1,2-Manalpha1,6 (GlcNAcbeta1,2-Manalpha1,3)Man; the 1,6-arm trisaccharide, GlcNAcbeta1,2-Manalpha1,6-Manbeta-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcbeta1,2-Manalpha1,3-Manbeta-OR (1,2-1,3-arm) and GlcNAcbeta1,4-Manalpha1,3-Manbeta-OR (1,4-1,3-arm); and the trisaccharide GlcNAcbeta1,4-GlcNAcbeta1,4-GlcNAc (chitotriose). With the wild-type h-beta4Gal-T1, the K(m) of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-beta4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0A resolution showed that beta4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest K(m) for the trisaccharide. Present studies suggest that beta4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1,3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan.

Carbohydrate Conformation↗
PubMed1999

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions.

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.

Animals↗
PubMed1981

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.

Animals↗
PubMed2002

Evaluation of beta1,4-galactosyltransferase as a potential biomarker for the detection of subclinical disease after the completion of primary therapy for ovarian cancer.

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.

Adult↗
PubMed2000

Implication of insulin and nutritional factors in the regulation of intestinal galactosyltransferase activity during postnatal development.

In the rat small intestine, galactosyltransferases are the enzymes implicated in the biosynthesis of glycoproteins of the brush-border membranes and mucins. During postnatal development, the circulating insulin level increased at weaning in parallel with the activities of intestinal galactosyltransferases on O-glycans and N-glycans. This study deals with the role of insulin in the regulation of galactosyltransferase activities during postnatal development. The treatment of immature suckling rats with insulin induced a precocious increase in the activities of the O-glycan and N-glycan galactosyltransferases, partly reproducing the increase in galactosyltransferase activity normally found at weaning, since the O-glycan galactosyltransferase activity increased more quickly than the N-glycan galactosyltransferase activity. The sensitivity of the two galactosyltransferase activities to insulin disappeared after weaning, a period when drastic diet changes occur. In 22-day-old rats submitted to prolonged nursing (high-fat diet), the activities of the O-glycan and N-glycan galactosyltransferases were lower than those found in age-matched normally weaned rats (high-carbohydrate diet), indicating a delay in the maturation of the intestine of prolonged-nursing rats. The circulating insulin level of these animals stayed lower than that of the age-matched weaned rats. When the prolonged-nursing animals were treated with insulin, the O-glycan and N-glycan galactosyltransferase activities reached levels similar to those of the weaned rats. These observations suggest that insulin is one of the maturation factors for intestinal glycoprotein galactosylation and may be partly responsible for the natural enhancement of intestinal galactosyltransferase activities observed during postnatal development in relation to the dietary changes at weaning.

Aging↗