Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

BACKGROUND: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. METHODS: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case-control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. RESULTS: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. CONCLUSION: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

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.

Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I).

The CMP-Neu5Ac:Galbeta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites.

Efficient intra- and extracellular production of human beta-1,4-galactosyltransferase in Saccharomyces cerevisiae is mediated by yeast secretion leaders.

We have compared the function of homologous and heterologous secretion leaders to mediate production of human beta-1,4-galactosyltransferase (Gal-Tf) in the yeast Saccharomyces cerevisiae. Although all genes encoding leader/Gal-Tf fusions were transcribed by strong yeast promoters, only low production levels were obtained for full-length Gal-Tf containing the human membrane-anchor region. In contrast, a gene fusion encoding the membrane-anchor region of the yeast alpha-1,2-mannosyltransferase (Mnt1) fused to soluble Gal-Tf yielded high mRNA and intracellular protein levels. Gal-Tf could also be produced extracellularly using a fusion of the pre-pro region of the yeast Mf alpha 1 precursor (MF alpha 1) to soluble Gal-Tf; a fusion containing only the pre-region of Mf alpha 1 was synthesized intracellularly, but did not lead to Gal-Tf activity in the culture medium. All yeast-produced Gal-Tf proteins were enzymatically active. These results demonstrate that yeast secretion leaders are advantageous to achieve efficient production of active Gal-Tf in S. cerevisiae.

Golgi localization in yeast is mediated by the membrane anchor region of rat liver sialyltransferase.

To investigate the function of the membrane anchor region of a mammalian glycosyltransferase in yeast we constructed a fusion gene that encodes the 34 amino-terminal residues of rat liver beta-galactoside alpha-2,6-sialyl-transferase (EC 2.4.99.1) (ST) fused to the mature form of yeast invertase. Transformants of Saccharomyces cerevisiae expressing the fusion gene produced an intracellular heterogeneously N-glycosylated fusion protein of intermediate molecular weight between the core and fully extended N-glycosylated form of invertase, suggesting a post-endoplasmic reticulum (ER) localization. In two types of cell fractionation using sucrose density gradients the ST-invertase fusion protein cofractionated with Golgi marker proteins, whereas a minor fraction (about 30%) comigrated with a vacuolar marker; ST-invertase was not detected in other cell fractions including the ER and the plasma membrane. Consistent with Golgi localization, about 70% of the total amount of the ST-invertase fusion was immunoprecipitated with an antibody directed against alpha-1,6-mannose linkages. The results demonstrate that the membrane anchor region of a mammalian type II glycosyltransferase is able to target a protein to the secretory pathway and to a Golgi compartment of the yeast S. cerevisiae, indicating conservation of targeting mechanisms between higher and lower eukaryotes. Since typical yeast Golgi localization signals are missing in the ST-membrane anchor region the results also suggest that yeast as mammalian cells utilize diverse mechanisms to direct proteins to the Golgi.

Golgi localization and in vivo activity of a mammalian glycosyltransferase (human beta1,4-galactosyltransferase) in yeast.

Gene fusions encoding the membrane anchor region of yeast alpha1, 2-mannosyltransferase (Mnt1p) fused to human beta1, 4-galactosyltransferase (Gal-Tf) were constructed and expressed in the yeast Saccharomyces cerevisiae. Fusion proteins containing 82 or only 36 N-terminal residues of Mnt1p were produced and quantitatively N-glycosylated; glycosyl chains were shown to contain alpha1,6-, but not alpha1,3-mannose determinants, a structure typical for an early Golgi compartment. A final Golgi localization of both fusions was confirmed by sucrose gradient fractionations, in which Gal-Tf activity cofractionated with Golgi Mnt1p activity, as well as by immunocytological localization experiments using a monoclonal anti-Gal-Tf antibody. In an in vitro Gal-Tf enzymatic assay the Mnt1/Gal-Tf fusion and soluble human Gal-Tf had comparable Km values for UDP-Gal (about 45 microM). To demonstrate in vivo activity of the Mnt1/Gal-Tf fusion the encoding plasmids were transformed in an alg1 mutant, which at the non-permissive temperature transfers short (GlcNAc)2 glycosyl chains to proteins. Using specific lectins the addition of galactose to several yeast proteins in transformants could be detected. These results demonstrate that Gal-Tf, a mammalian glycosyltransferase, is functional in the molecular environment of the yeast Golgi, indicating conservation between yeast and human cells. The in vivo function of human Gal-Tf indicates that the yeast Golgi is accessible for UDP-Gal and suggests strategies for the construction of yeast strains, in which desired glycoforms of heterologous proteins are produced.

Cloning of a novel member of the UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glycosphingolipid biosynthesis.

A novel putative member of the human UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, designated beta4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of beta4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other beta1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of beta4Gal-T4 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal beta-GlcNAc; however, in contrast to beta4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalacto-transferrin, or ovalbumin. The catalytic activity of beta4Gal-T4 with monosaccharide acceptor substrates, N-acetylglucosamine as well as glucose, was markedly activated in the presence of alpha-lactalbumin. The genomic organization of the coding region of beta4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in beta4Gal-T1, -T2, and -T3. beta4Gal-T4 represents a new member of the beta4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.

Synthesis of poly-N-acetyllactosamine in core 2 branched O-glycans. The requirement of novel beta-1,4-galactosyltransferase IV and beta-1,3-n-acetylglucosaminyltransferase.

Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Lex. Poly-N-acetyllactosamines in mucin-type O-glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 beta-1,6-N-acetylglucosaminyltransferase. Using a beta-1, 4-galactosyltransferase (beta4Gal-TI) present in milk and the recently cloned beta-1,3-N-acetylglucosaminyltransferase, the formation of poly-N-acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAcbeta1-->6(Galbeta1-->3)GalNAcalpha-->R. Since the majority of synthesized oligosaccharides contained N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the beta4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant beta4Gal-Ts, beta4Gal-TIV was found to be most efficient in the addition of a single galactose residue to GlcNAcbeta1-->6(Galbeta1-->3)GalNAcalpha-->R. Moreover, beta4Gal-TIV, together with beta-1,3-N-acetylglucosaminyltransferase, was capable of synthesizing poly-N-acetyllactosamine in core 2 branched oligosaccharides. On the other hand, beta4Gal-TI was found to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans. In contrast to beta4Gal-TI, the efficiency of beta4Gal-TIV decreased dramatically as the acceptors contained more N-acetyllactosamine repeats, consistent with the fact that core 2 branched O-glycans contain fewer and shorter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that beta4Gal-TIV is responsible for poly-N-acetyllactosamine synthesis in core 2 branched O-glycans.

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.

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.

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1.

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.

Control of O-glycan branch formation. Molecular cloning and characterization of a novel thymus-associated core 2 beta1, 6-n-acetylglucosaminyltransferase.

Core 2 O-glycan branching catalyzed by UDP-N-acetyl-alpha-D-glucosamine: acceptor beta1, 6-N-acetylglucosaminyltransferases (beta6GlcNAc-Ts) is an important step in mucin-type biosynthesis. Core 2 complex-type O-glycans are involved in selectin-mediated adhesion events, and O-glycan branching appears to be highly regulated. Two homologous beta6GlcNAc-Ts functioning in O-glycan branching have previously been characterized, and here we report a third homologous beta6GlcNAc-T designated C2GnT3. C2GnT3 was identified by BLAST analysis of human genome survey sequences. The catalytic activity of C2GnT3 was evaluated by in vitro analysis of a secreted form of the protein expressed in insect cells. The results revealed exclusive core 2 beta6GlcNAc-T activity. The product formed with core 1-para-nitrophenyl was confirmed by (1)H NMR to be core 2-para-nitrophenyl. In vivo analysis of the function of C2GnT3 by coexpression of leukosialin (CD43) and a full coding construct of C2GnT3 in Chinese hamster ovary cells confirmed the core 2 activity and failed to reveal I activity. The C2GnT3 gene was located to 5q12, and the coding region was contained in a single exon. Northern analysis revealed selectively high levels of a 5.5-kilobase C2GnT3 transcript in thymus with only low levels in other organs. The unique expression pattern of C2GnT3 suggests that this enzyme serves a specific function different from other members of the beta6GlcNAc-T gene family.

Control of O-glycan branch formation. Molecular cloning of human cDNA encoding a novel beta1,6-N-acetylglucosaminyltransferase forming core 2 and core 4.

A novel human UDP-GlcNAc:Gal/GlcNAcbeta1-3GalNAcalpha beta1, 6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-alpha-D-glucosamine:acceptor beta1, 6-N-acetylglucosaminyltransferase (beta1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a beta1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in beta1,6GlcNAc-transferases capable of forming core 2 O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

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.