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Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galβ1–4(Fucα1–3)GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N -glycans between these cell types. O -glycans are highly differentiated, as RT4 cells synthesize core 2-based O -glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1–3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers. [ABSTRACT FROM AUTHOR]

Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs. [ABSTRACT FROM AUTHOR]

During malignant transformation, glycosylation is heavily altered compared with healthy tissue due to differential expression of glycosyltransferases, glycosidases and monosaccharide transporters within the cancer microenvironment. One key change of malignant tissue glycosylation is the alteration of sialic acid processing that leads to a general upregulation of sialylated glycans (hypersialylation) on cell surfaces and an increased introduction of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) instead of N-acetyl-neuraminic acid into cell surface glycans. These changes have been shown to be the result of altered sialyltransferase and sialidase expression. Functionally, cancer-associated hypersialylation appears to directly impact tumor cell interaction with the microenvironment, in particular the modulation of sialic acid-binding lectins on immune cells. Moreover, Neu5Gc expression in human tissues enhances inflammation due to an anti- Neu5Gc immune response, which can potentially influence inflammation-induced cancer and cancer- associated inflammation. In this review, we summarize the changes of sialic acid biology within the malignant microenvironment and the resulting effect on cancer immunity. [ABSTRACT FROM AUTHOR]

Mucin-type O-glycans are the primary constituents of mucins that are expressed on various mucosal sites of the body, especially the bacteria-laden intestinal tract. Mucins are the main components of mucus, which is secreted by goblet cells and forms a protective homeostatic barrier between the resident microbiota and the underlying immune cells in the colon. However, the specific role of mucin-type O-glycans in mucus barrier function has been uncertain. Recent studies utilizing mice deficient in key glycosyltransferases involved in O-glycan biosynthesis on intestinal mucins have underscored the importance of mucin-type O-glycosylation in mucus barrier function. This review will highlight recent advances in our understanding of mucin-type O-glycan function in the mucus barrier and how they promote mutualism with our resident microbiota. [ABSTRACT FROM AUTHOR]

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt−/−) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1−/−) mice. The Muc2 O-glycans were released by reductive β-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt−/− knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1−/− mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species. [ABSTRACT FROM AUTHOR]

The αGal (Galα1-3Gal) epitope is a xenoantigen that is responsible for hyperacute rejection in xenotransplantation. This epitope is expressed on the cell surface in the cells of all mammals except humans and Old World monkeys. It can be digested by the enzyme endo-β-galactosidase C (EndoGalC), which is derived from Clostridium perfringens. Previously, we produced EndoGalC transgenic mice to identify the phenotypes that would be induced following EndoGalC overexpression. The mice lacked the αGal epitope in all tissues and exhibited abnormal phenotypes such as postnatal death, growth retardation, skin lesion and abnormal behavior. Interestingly, skin lesions caused by increased proliferation of keratinocytes suggest the role of a glycan structure [in which the αGal epitope has been removed or the N-acetylglucosamine (GlcNAc) residue is newly exposed] as a regulator of signal transduction. To verify this hypothesis, we introduced an EndoGalC expression vector into cultured mouse NIH3T3 cells and obtained several EndoGalC-expressing transfectants. These cells lacked αGal epitope expression and exhibited 1.8-fold higher proliferation than untransfected parental cells. We then used several cytokine receptor inhibitors to assess the signal transduction cascades that were affected. Only SB431542 and LY364947, both of which are transforming growth factor β (TGFβ) receptor type-I (TβR-I) inhibitors, were found to successfully reverse the enhanced cell proliferation rate of EndoGalC transfectants, indicating that the glycan structure is a regulator of TβRs. Biochemical analysis demonstrated that the glycan altered association between TβR-I and TβR-II in the absence of ligands. [ABSTRACT FROM AUTHOR]

We have precisely analyzed the structures of glycosphingolipids of human cancer cells and normal epithelial cells using several methods, including enzymatic release of carbohydrate moieties, fluorescent labeling, and identification using 2D mapping, enzymatic digestion, and mass spectrometry. These analyses enabled the identification of novel tumor-associated carbohydrate antigens that can be used to elucidate the involvement of carbohydrates in cancer malignancy and could act as candidate tumor markers. In our previous study, we identified a novel glycosphingolipid that accumulates in colon cancer cells, NeuAcα2-6(Fucα1-2)Galβ1-4GlcNAcβ1-3Galβ1-4Glc (α2-6 sialylated type 2H, ST2H). Here, structural analyses of cancer cells and normal epithelial cells from 60 colorectal and five pancreatic cancer patients, including four and two Lewis-negative individuals, respectively, reveal the presence of an additional novel glycosphingolipid, NeuAcα2-6(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc (α2-6 sialylated type 1H, ST1H). ST2H was found in colorectal and pancreatic cancer cells from about half of the cases. Unlike ST2H, ST1H was found in cancer cells from three out of six Lewis-negative patients (i.e., two cases of colorectal and one case of pancreatic cancer). However, the moiety was not found in normal epithelial cells or cancer cells from 59 Lewis-positive patients. These findings suggest that the accumulation of this carbohydrate antigen occurs predominantly in cancer cells of Lewis-negative patients. When the ST1H epitope is also carried on mucins as well as glycosphingolipids, this epitope is a promising tumor marker candidate, especially for Lewis-negative individuals. [ABSTRACT FROM PUBLISHER]

The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, and cancer-associated MUC1 glycoforms represent potential targets for immunodiagnostic and therapeutic measures. We have recently shown that MUC1 with GalNAcα1-O-Ser/Thr (Tn) and NeuAcα2-6GalNAcα1-O-Ser/Thr (STn) O-glycosylation is a cancer-specific glycoform, and that Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance in human MUC1 transgenic mice and induce humoral immunity with high specificity for MUC1 cancer-specific glycoforms (Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, et al. 2006. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 16:96–107). In order to further characterize the immune response to Tn/STn-MUC1 glycoforms, we generated monoclonal antibodies with specificity similar to the polyclonal antibody response found in transgenic mice. In the present study, we define the immunodominant epitope on Tn/STn-MUC1 glycopeptides to the region including the amino acids GSTA of the MUC1 20-amino acid tandem repeat (HGVTSAPDTRPAPGSTAPPA). Most other MUC1 antibodies are directed to the PDTR region, although patients with antibodies to the GSTA region have been identified. A panel of other MUC1 glycoform-specific monoclonal antibodies was included for comparison. The study demonstrates that the GSTA region of the MUC1 tandem repeat contains a highly immunodominant epitope when presented with immature short O-glycans. The cancer-specific expression of this glycopeptide epitope makes it a prime candidate for immunodiagnostic and therapeutic measures. [ABSTRACT FROM AUTHOR]

Sialyl Lewis A (SLea), Lewis A (Lea), and Lewis B (Leb) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves β1,3-galactosyltransferases (β3Gal-Ts) and a combined action of α2- and/or α4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of β3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLea, Lea, and Leb synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. β3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only β3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Lea and Leb synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct α2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Leb-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics. [ABSTRACT FROM AUTHOR]

Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, β3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAcα1-peptide, in which C6-OH of αGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcα1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides. [ABSTRACT FROM AUTHOR]

Cell surface carbohydrates expressed on epithelial cells are thought to play an important role in tumor progression. Previously, we have shown that expression of core 2-branched O-glycans is closely correlated with vessel invasion and depth of invasion in colon and lung carcinomas. In this study, we found that expression of core 2 β1,6-N-acetylglucosaminyltransferase-1, Core2GnT, is positively correlated with the progression of prostate cancer in human patients. Statistical analysis demonstrated that Core2GnT is an independent predictor for progressed pathological stage (pT3) and for prostate-specific antigen (PSA) relapse. To determine directly the roles of Core2GnT in prostate cancer progression, we set up an experimental tumor model using the LNCaP prostate cancer cell line. Because this line does not express Core2GnT, we established an LNCaP line stably expressing Core2GnT, LNCap-Core2GnT, by transfecting cDNA encoding Core2GnT. When mock-transfected LNCaP cells and LNCaP-Core2GnT were inoculated in the prostate of nude mice, LNCaP-Core2GnT cells produced three times heavier prostate tumors than mock-transfected LNCaP cells. Furthermore, we found that LNCaP-Core2GnT cells adhered more strongly to prostate stromal cells, type IV collagen and laminin than did LNCaP-mock cells, but LNCaP and LNCaP-Core2GnT cells grew almost at the same rate on plates coated with type IV collagen or laminin. These results indicate that Core2GnT is an extremely useful prognostic marker for prostate cancer progression. The results also suggest that acquiring Core2GnT in prostate carcinoma cells facilitates adhesion to type IV collagen and laminin, and this increased adhesion may be a cause for aggressive tumor formation by prostate cancer cells expressing Core2GnT. [ABSTRACT FROM AUTHOR]

A and B histo-blood group antigens are present on carcinoma cells at the early stages of cancerogenesis and tend to disappear at later stages, but it is not yet clear whether they take part to the process of tumor progression. To gain some insight into this issue, we used a rat colon carcinoma experimental model. To obtain expression of the A antigen, REG cells were cotransfected with the rat A enzyme cDNA and a rat α1,2fucosyltransferase cDNA, either FTA or FTB, whereas PRO cells that spontaneously have α1,2fucosyltransferase activity were only transfected with the A enzyme cDNA. All A antigen–expressing transfected cells derived from either REG FTA, REG FTB, or PRO parental cells were more resistant to apoptosis induced by either serum deprivation or heat shock than were their respective controls. When injected to syngeneic immunocompetent rats, A enzyme–transfected PRO cells formed tumors that grew faster than those formed by mock-transfected PRO cells. However, in immunodeficient SCID mice, no difference in growth could be observed between the two types of tumors, indicating that the faster tumor growth of the A antigen–positive cells in immunocompetent animals was due to their higher ability to escape immune control and that this was associated with their higher degree of resistance to apoptosis. These results might explain the slightly augmented incidence of carcinomas observed in A and B blood group individuals compared to O individuals. [ABSTRACT FROM PUBLISHER]

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 α1,2-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 × 109 LNCaP cells (~200-fold; 40% yield). The Km 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 α1,2-FT [Chandrasekaran et al. (1996) Biochemistry 35, 8914–8924]. N-Ethylmaleimide was a potent inhibitor (Ki 12.5 µM). 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 Galβ1,3GlcNAcβ-O-Al was the best acceptor among the acrylamide copolymers. Additionally, highly significant biological features of α1,2FT were identified in the present study. The synthesis of Globo H and Lewis b determinants became evident from the fact that Galβ1,3GalNAcβ1,3Galα-O-Me and Galβ1,3(Fucα1,4)Glc-NAcβ1,3Galβ-O-Me served as high-affinity acceptors for this enzyme. Further, D-Fucβ1,3Gal-NAcβ1,3Galα-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 α1,2-FT, namely, the expressions of blood group H, Lewis b from Lewis a, and Globo H. [ABSTRACT FROM PUBLISHER]

The biochemical and biophysical properties of mucinsare largely determined by extensive O-glycosylation of serine- andthreonine-rich tandem repeat (TR) domains. In a number of humandiseases aberrant O-glycosylation is associated with variationsin the properties of the cell surface–associated and secretedmucins. To evaluate in vivo the O-glycosylationof mucin TR domains, we generated recombinant chimeric mucins withTR sequences from MUC2, MUC4, MUC5AC, or MUC5B, which were substituted forthe native TRs of epitope-tagged MUC1 protein (MUC1F). These hybridmucins were extensively O-glycosylated and showed the expected associationwith the cell surface and release into culture media. The presenceof different TR domains within the chimeric mucins appears to have limitedinfluence on their posttranslational processing. Alterations inglycosylation were detailed by fast atom bombardment mass spectrometryand reactivity with antibodies against particular blood-group andtumor-associated carbohydrate antigens. Future applications of thesechimeras will include investigations of mucin posttranslationalmodification in the context of disease. [ABSTRACT FROM PUBLISHER]

Accumulation of histo-blood group antigens such as Lewis b, Lewis Y and H in colon cancer is indicative of poor prognosis. It is accompanied by increase in α1,2fucosyl­transferase activity, a key enzyme for synthesis of these antigens. Using a model of colon carcinoma, we previously showed that α1,2fucosylation increases tumorigenicity. We now show that tumorigenicity inversely correlates with the cells’ sensitivity to apoptosis. In addition, poorly tumorigenic REG cells independently transfected with three different α1,2fucosyltransferase cDNAs, the human FUT1, the rat FTA and FTB were more resistant than control cells to apoptosis induced in vitro by serum deprivation. Inversely, PRO cells, spontaneously tumorigenic in immunocompetent syngeneic animals and able to synthesize α1,2fucosylated glycans, became more sensitive to apoptosis after transfection with a fragment of the FTA cDNA in the antisense orientation. Expression of α1,2fucosyl­transferase in poorly tumorigenic REG cells dramatically enhanced their tumorigenicity in syngeneic rats. However, in immunodeficient animals, both control and α1,2fuco­syltransferase transfected REG cells were fully tumorigenic and metastatic, indicating that the presence of α1,2fucosylated antigens allowed REG tumor cells to escape immune control. Taken together, the results show that increased tumorigenicity mediated by α1,2fucosyl­ation is associated to increased resistance to apoptosis and to escape from immune control. [ABSTRACT FROM PUBLISHER]

The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N-acetylgalactosamine α2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O-glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situhybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s). [ABSTRACT FROM PUBLISHER]

Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. A series of small molecular weight analogs of the GalNAc-α-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2-deoxy-α-O-d-galactopyranoside have been synthesized and tested in the human colorectal cancer cell lines PC/AA/C1/SB10C and HCA7/C29. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-α-O-d-galactopyranoside, and the corresponding 2-azido- and C-glycoside analogs were screened in these colorectal cancer cell lines at 0.5 mM and showed induction of apoptosis and downregulation of proliferation. Treatment of both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The inhibition of glycosyltransferase activity in cell homogenates from human colorectal mucosal cells and cultured cell lines could be shown. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosyltransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells; however, there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and aryl-glycan formation. These events may play a part in growth arrest. [ABSTRACT FROM AUTHOR]

The αGal epitope (Galα1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-αGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-β-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the αGal epitope by cleaving the Galβ1-4GlcNAc linkage in the Galα1-3Galβ1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the αGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in β1,4-galactosyltransferase 1 (β4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation. [ABSTRACT FROM AUTHOR]

Transgenic (TG) mice that have systemically expressed Endo-β-galactosidase C (EndoGalC) have rough and flaky skin. This skin phenotype is detectable around 5 days postnatal and becomes obscure by 2 weeks after birth. Their epidermis is thickened but the dermis and hair follicles are normal in structure. EndoGalC, which removes the terminal Galα1-3Gal disaccharide (αGal epitope), was expressed in the epidermis of TG mice. GS-IB4 lectin staining showed that the αGal epitope did not exist in the epidermis in TG but existed in wild-type (WT) mice. In TG mice, N-acetylglucosamines were exposed by EndoGalC, which is detected using GS-II lectin. To understand the cause of the epidermal thickening and skin phenotype, we examined the proliferation and differentiation of kerationocytes. BrdU-pulse-labeling revealed that proliferating keratinocytes increased approximately three-fold in TG epidermis compared to WT one. In TG epidermis, the expression domain of cytokeratin 14 increased from 1–2 layers to 4–5 layers and co-expressed with cytokeratin 6 and 10 in the upper layers. The layers expressing involucrin and loricrin also increased but those expressing filaggrin and transglutaminase looked normal. The localization of E-cadherin was similar in both TG and WT mice. Although TG mice showed delayed development of the barrier function around 8 days postnatal, they acquired the function by 12 days after birth. These results suggest that the absence of the αGal epitope or the exposed N-acetylglucosamine terminal could play a critical role in the proliferation of basal keratinocytes and differentiation of them into the spinous cells in newborn mice. [ABSTRACT FROM AUTHOR]

Secreted mucins protect the underlying epithelium by serving as the major determinant of the rheological property of mucus secretion and the receptors for pathogens. These functions can be affected by the three branch structures, including core 2, core 4, and blood group I, which are synthesized by the mucus-type core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT-M). Decreased activity of this enzyme and expression of this gene have been found in colorectal cancer, which supports the important role of this enzyme in the protective functions of secreted mucins. We cloned full-length mouse (m) C2GnT-M cDNAs and showed that the deduced amino acid sequence was homologous to those of other C2GnT-Ms. The recombinant protein generated by mC2GnT-M cDNA exhibited core 2, core 4, and blood group I enzyme activities with a ratio of 1.00:0.46:1.05. We identified two different size transcripts by rapid amplification of cDNA ends and RT-PCR. Derived from the 6.6 kb mC2GnT-M gene composed of three exons and two introns, these two transcripts were intronless and differed by the length of the 3′ untranslated region. In addition, exon 2 was found to be heterogeneous in size. This gene was highly expressed in the gastrointestinal tract, including colon, stomach, and small intestine. Antibodies generated against mC2GnT-M identified this enzyme in the goblet cells and other mucus cells/glands. This report provides the basis for further characterization of the regulation of mC2GnT-M gene expression and the biological functions of this gene. [ABSTRACT FROM AUTHOR]

The carbohydrate determinants Sda and sialyl Lewis x (sLex) both result from substitution of an α2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) β1,4-linked to Gal and the second by an α1,3-linked fucose on N-acetylglucosamine. The Sda antigen is synthesized by Sda β1,4-N-acetylgalactosaminyltransferase II (β4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between β4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether β4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. β4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sda antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sda antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, β4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sda and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of α1,3-FucTs on 3′-sialyllactosamine parallels that of β4GalNAcT-II; and (iii) both β4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription–polymerase chain reaction analysis reveals that the transcripts of β4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3′-sialyllactosamine acceptors and that β4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues. [ABSTRACT FROM AUTHOR]