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Background Antibody-mediated rejection after ABO-incompatible kidney transplantation (ABO-I KTx) is a major barrier to transplantation success. The advent of immunosuppressive therapy has markedly improved graft survival in ABO-I KTx. However, compared with normal KTx, clinical conditions during ABO-I KTx are difficult to control because of overimmunosuppression. To reduce the need for immunosuppression, we aimed to develop a novel blood group antigen-neutralizing therapy. Methods We screened for an ABO blood group antigen-targeting peptide (BATP) by screening of T7 phage-displayed peptide library. After screening, hemagglutination inhibition assays, enzyme-linked immunosorbent assay, and cytotoxicity assay were used to analyze the blood group antigen-blocking effect and toxicity of BATP. We also tested the inhibitory effects on anti-blood group antibody binding in normal human kidney tissues blocked with BATP and excised kidneys perfused ex vivo with BATP. Results We identified six peptide sequences that efficiently suppressed hemagglutination of red blood cells by anti-ABO blood group antibodies and binding of these antibodies to ABO histo-blood group antigens in kidney tissues. Surprisingly, ex vivo perfusion of BATP in kidneys excised from renal cell carcinoma patients caused significant suppression of anti-blood group antibody binding to antigen and IgG and IgM deposition in renal glomerular capillaries after ABO-I blood reperfusion. Conclusions These data indicate that A/B blood group antigens on red blood cells and in kidney tissues may be neutralized by BATP. This approach may enable the development of a novel blood group antigen-neutralizing therapy to overcome the challenges of ABO-I KTx.

Background: Chst10 transfers sulfate to glucuronic acid to form the HNK-1 antigen carried by glycoproteins and glycolipids in neurons and NK cells. Results: Chst10 transfers sulfate to glucuronidated steroid hormones, and Chst10-deficient mice exhibited subfertility. Conclusion: Subfertility in Chst10 null females is caused by a loss of steroid hormone dysregulation. Significance: This study identified a new regulatory mechanism mediated by sulfation of glucuronidated steroid., Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10−/− mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10−/− females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10−/− females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo.

In an effort to prime our mass spectrometry (MS)-based sulfoglycomic mapping platform technology for facile identification of sulfated lacdiNAc (GalNAcβ1-4GlcNAcβ1-), we have re-examined the N-glycans of bovine thyroid stimulating hormone. We showed that MALDI-MS mapping of permethylated glycans in negative ion mode can give an accurate representation of the sulfated glycans and, through MS/MS, diagnostic ions can be derived that we can collectively define the presence of a terminal sulfated lacdiNAc moiety at high sensitivity. Based on these ions, which can also be produced by nanoESI-MS(n), we demonstrated that the glycome of an ovarian carcinoma cell line, RMG-1, comprises a high abundance of sulfated lacdiNAc epitopes carried on multiantennary complex type N-glycans alongside fucosylated, sialylated and/or sulfated lacNAc antennae. This represents the first report of a natural glycomic occurrence of sulfated lacdiNAc on a cell line, as opposed to other better-characterized presence on secreted glycoproteins from a handful of sources. It is anticipated that with improved methods of detection such as that developed in this work, we are likely to identify a wider occurrence of sulfated lacdiNAc and be able to more accurately delineate the regulatory mechanism dictating the choice of a cell type in synthesizing sulfated, sialylated, fucosylated and/or non-substituted lacdiNAc.

信州大学博士（医学）・学位論文・平成24年3月31日授与（甲第916号）・唐澤文寿, Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal alpha 1,4-linked N-acetylglucosamine residues (alpha GlcNAc). Previously, we identified human alpha 1,4-N-acetylglucosaminyltransferase (alpha 4GnT), which is responsible for the O-glycan biosynthesis and characterized alpha GlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt(-/-) mice to better understand its role in vivo. A4gnt(-/-) mice showed complete lack of alpha GlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt(-/-) mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced alpha GlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of alpha GlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, alpha GlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation., Article, JOURNAL OF CLINICAL INVESTIGATION. 122(3):923-934 (2012)

Background: We produced a humanized monoclonal antibody, designated HMOCC-1, against cell surface carbohydrates presented by malignant ovarian cancer. Results: Co-transfection experiments predicted HMOCC-1 antigenic oligosaccharides structures, which were then chemically synthesized for testing antibody binding. Conclusion: HMOCC-1 antigen is the glycan structure composed of SO3→3Galβ1→4GlcNAcβ1→3(±SO3→6)Galβ1→4GlcNAcβ1→. Significance: The approach employed in this study will be useful in determining specificity of an undefined monoclonal anti-carbohydrate antibody., A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290–298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1 antigen formation. Because these results predicted that HMOCC-1 antigens are SO3→3Galβ1→4GlcNAcβ1→3(±SO3→6)Galβ1→4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer.

Annexin A1 is a multifunctional, calcium-dependent phospholipid binding protein involved in a host of processes including inflammation, regulation of neuroendocrine signaling, apoptosis, and membrane trafficking. Binding of annexin A1 to glycans has been implicated in cell attachment and modulation of annexin A1 function. A detailed characterization of the glycan binding preferences of annexin A1 using carbohydrate microarrays and surface plasmon resonance served as a starting point to understand the role of glycan binding in annexin A1 function. Glycan array analysis identified annexin A1 binding to a series of sulfated oligosaccharides and revealed for the first time that annexin A1 binds to sulfated non-glycosaminoglycan carbohydrates. Using heparin/heparan sulfate microarrays, highly sulfated heparan sulfate/heparin were identified as preferred ligands of annexin A1. Binding of annexin A1 to heparin/heparan sulfate is calcium- but not magnesium-dependent. An in-depth structure-activity relationship of annexin A1-heparan sulfate interactions was established using chemically defined sugars. For the first time, a calcium-dependent heparin binding protein was characterized with such an approach. N-Sulfation and 2-O-sulfation were identified as particularly important for binding. © 2011 American Chemical Society., This study was supported by the Max Planck Society, the ETH Zurich, the Swiss National Science Foundation, Novartis (fellowship to P.B.), NIH Grant CA71932 (to M.N.F.), and NIH Grant GM62116 to the Consortium for Functional Glycomics. We thank the FGCZ for technical assistance in microarray printing (J. Sobek) and SPR experiments (M. Scott and S. Schauer).

α-Dystroglycan (α-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelium development and tissue organization. The α-DG–mediated epithelial cell-BM interaction is often impaired in invasive carcinomas, yet roles and underlying mechanisms of such an impaired interaction in tumor progression remain unclear. We report here a suppressor function of laminin-binding glycans on α-DG in tumor progression. In aggressive prostate and breast carcinoma cell lines, laminin-binding glycans are dramatically decreased, although the amount of α-DG and β-dystroglycan is maintained. The decrease of laminin-binding glycans and consequent increased cell migration were associated with the decreased expression of β3- N -acetylglucosaminyltransferase-1 (β3GnT1). Forced expression of β3GnT1 in aggressive cancer cells restored the laminin-binding glycans and decreased tumor formation. β3GnT1 was found to be required for laminin-binding glycan synthesis through formation of a complex with LARGE, thus regulating the function of LARGE. Interaction of the laminin-binding glycans with laminin and other adhesive molecules in BM attenuates tumor cell migratory potential by antagonizing ERK/AKT phosphorylation induced by the components in the ECM. These results identify a previously undescribed role of carbohydrate-dependent cell-BM interaction in tumor suppression and its control by β3GnT1 and LARGE.

Cell surfaces of epithelial cancer are covered by complex carbohydrates, whose structures function in malignancy and metastasis. However, the mechanism underlying carbohydrate-dependent cancer metastasis has not been defined. Previously, we identified a carbohydrate-mimicry peptide designated I-peptide, which inhibits carbohydrate-dependent lung colonization of sialyl Lewis X-expressing B16-FTIII-M cells in E/P-selectin doubly-deficient mice. We hypothesized that lung endothelial cells express an unknown carbohydrate receptor, designated as I-peptide receptor (IPR), responsible for lung colonization of B16-FTIII-M cells. Here, we visualized IPR by in vivo biotinylation, which revealed that the major IPR is a group of 35-kDa proteins. IPR proteins isolated by I-peptide affinity chromatography were identified by proteomics as Ser/Arg-rich alternative pre-mRNA splicing factors or Sfrs1 , Sfrs2 , Sfrs5 , and Sfrs7 gene products. Bacterially expressed Sfrs1 protein bound to B16-FTIII-M cells but not to parental B16 cells. Recombinant Sfrs1 protein bound to a series of fucosylated oligosaccharides in glycan array and plate-binding assays. When anti-Sfrs antibodies were injected intravenously into mice, antibodies labeled a subset of lung capillaries. Anti-Sfrs antibodies inhibited homing of I-peptide-displaying phage to the lung colonization of B16-FTIII-M cells in vivo in the mouse. These results strongly suggest that Sfrs proteins are responsible for fucosylated carbohydrate-dependent lung metastasis of epithelial cancers.

Aggressive behaviors of trophoblasts during embryo implantation resemble to those of malignant tumor cells. As much as 20-40% of all epithelial cancers in humans express human chorionic gonadotrophin (hCG), a marker for trophoblast. Therefore it is not surprising if some mechanisms are shared by cancer and trophoblast. However, the molecular basis of human embryo implantation is not well understood due to difficulties in studying the process in humans. Mechanisms of human embryo implantation are unique, as are features of trophoblastic cancer. This review describes trophinin, a cell adhesion/signaling protein, and its associated proteins, bystin and tastin, the proteins potentially involved in human embryo implantation, and presents examples of trophinin-expressing cancers.

Human bystin was identified as a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. Although the trophinin gene is unique to mammals, the bystin gene (BYSL) is conserved across eukaryotes. Recent studies show that bystin plays a key role during the transition from silent trophectoderm to an active trophoblast upon trophinin-mediated cell adhesion. Bystin gene knockout and knockdown experiments demonstrate that bystin is essential for embryonic stem cell survival and trophectoderm development in the mouse. Furthermore, biochemical analysis of bystin in human cancer cells and mouse embryos indicates a function in ribosomal biogenesis, specifically in processing of 18S RNA in the 40S subunit. Strong evidence that BYSL is a target of c-MYC is consistent with a role for bystin in rapid protein synthesis, which is required for actively growing cells.

Ovarian cancer is the leading cause of death in women with gynecological malignancies, with prognosis of advanced stage tumors determined by chemotherapeutic response and the success of tumor resection. Since aberrant RAS pathway activation is frequent in ovarian cancer, study of in vitro RAS-induced transformation and accompanying genomic expression changes in ovarian surface epithelial cells is imperative for development of new therapeutic modalities and for understanding tumorigenesis. cDNA microarray analysis revealed TROPHONIN (TRO), a homophilic adhesion molecule involved in blastocyst implantation, was among the genes most downregulated by RAS induction. TRO expression is higher in cisplatin-sensitive cancer cell lines and positively correlates with prognoses in ovarian cancers. TRO knockdown by RNA interference conferred cisplatin resistance and led to increased invasiveness of cultured ovarian cancer cells. These findings underscore the importance of TRO in tumorigenesis, and suggest that TRO may be a useful biomarker for cisplatin sensitivity and invasive potential.

Melanoma is one of the most lethal skin cancers worldwide, primarily because of its propensity to metastasize. Thus, the elucidation of mechanisms that govern metastatic propensity is urgently needed. We found that protein kinase Ce (PKCe)-mediated activation of activating transcription factor 2 (ATF2) controls the migratory and invasive behaviors of melanoma cells. PKCe-dependent phosphorylation of ATF2 promoted its transcriptional repression of the gene encoding fucokinase (FUK), which mediates the fucose salvage pathway and thus global cellular protein fucosylation. In primary melanocytes and cell lines representing early-stage melanoma, the abundance of PKCe-phosphorylated ATF2 was low, thereby enabling the expression of FUK and cellular protein fucosylation, which promoted cellular adhesion and reduced motility. In contrast, increased expression of the gene encoding PKCe and abundance of phosphorylated, transcriptionally active ATF2 were observed in advanced-stage melanomas and correlated with decreased FUK expression, decreased cellular protein fucosylation, attenuated cell adhesion, and increased cell motility. Restoring fucosylation in mice either by dietary fucose supplementation or by genetic manipulation of murine Fuk expression attenuated primary melanoma growth, increased the number of intratumoral natural killer cells, and decreased distal metastasis in murine isograft models. Tumor microarray analysis of human melanoma specimens confirmed reduced fucosylation in metastatic tumors and a better prognosis for primary melanomas that had high abundance of fucosylation. Thus, inhibiting PKCe or ATF2 or increasing protein fucosylation in tumor cells may improve clinical outcome in melanoma patients.

Human bystin is a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. The present study shows that bystin is expressed in luminal and glandular epithelia in the mouse uterus at peri-implantation stages. In fertilized embryos, bystin was not seen until blastocyst stage. Bystin expression started during hatching and increased in expanded blastocyst. However, bystin disappeared from the blastocyst during implantation. After implantation bystin re-appeared in the epiblast. Targeted disruption of the mouse bystin gene, Bysl, resulted in embryonic lethality shortly after implantation, indicating that bystin is essential for survival of mouse embryos.

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease.

In the adult brain, the subventricular zone (SVZ) is one of the regions where active neurogenesis occurs. Relatively few specific markers are available to distinguish different types of cells in the SVZ and rostral migratory stream (RMS) of adult brain. Here, we showed that trophinin and bystin, both of which are required for early embryo implantation during development, were expressed in the SVZ and RMS of the adult rat brain, but not in the brain of embryos and early postnatal animals. Trophinin-expressing cells were immunopositive for both Ki-67 and nestin in the SVZ. Some of the trophinin-positive cells did not express either the type A cell marker polysialylated weakly adhesive form of the neural cell adhesion molecule (PSA-NCAM) or the type B cell marker glial fibrillary acidic protein (GFAP). Double-label immunohistochemistry revealed that bystin-positive cells co-expressed GFAP, Ki-67 and nestin, but not PSA-NCAM, suggesting that they are likely type B cells. Intracerebroventricular infusion of cytosine-beta-d-arabiofuranoside (Ara-C) eliminated trophinin-positive cells in the SVZ. Following its depletion, however, the remaining bystin-positive cells continued to divide and generate actively dividing trophinin-positive cells that were negative for PSA-NCAM, leading to reconstruction of SVZ network. These characteristics indicate that this subset of trophinin-positive cells in the SVZ is type C cells. Conversely in the RMS, trophinin co-localized with nestin and PSA-NCAM, suggesting that it is expressed in neuroblasts. Cultured neural precursor cells derived from the adult SVZ also expressed both trophinin and bystin. These findings provide insight into the molecular basis of adult neurogenesis in the SVZ and RMS.

Trophinin, tastin, and bystin have been identified as molecules potentially involved in human embryo implantation. Both trophoblasts and endometrial epithelial cells express trophinin, which mediates apical cell adhesion through homophilic trophinin-trophinin binding. We hypothesized that trophinin's function in embryo implantation is unique to humans and investigated the expression of trophinin, tastin, and bystin in ectopic pregnancy, a condition unique to humans. In tubal pregnancies, high levels of all three were found in both trophoblasts and fallopian tubal epithelia. Trophinin expression in maternal cells was particularly high in the area adjacent to the trophoblasts, whereas trophinin was barely detectable in intact fallopian tubes from women with in utero pregnancies or without pregnancies. When explants of intact fallopian tube were incubated with the human chorionic gonadotrophin (hCG), trophinin expression was enhanced in epithelial cells. Since the trophectoderm of the human blastocyst secretes hCG before and after implantation, these results suggest that hCG from the human embryo induces trophinin expression by maternal cells. As both β-subunit of hCG and trophinin genes have diverged in mammals, the present study suggests a unique role of hCG and trophinin in human embryo implantation, including the pathogenesis of ectopic pregnancy.

The surfaces of mammalian cells are covered by a variety of carbohydrates linked to proteins and lipids. N-glycans are commonly found carbohydrates in plasma membrane proteins. The structure and biosynthetic pathway of N-glycans have been analyzed extensively. However, functional analysis of cell surface N-glycans is just under way with recent studies of targeted disruption of genes involved in N-glycan synthesis. This review briefly introduces the potential role of processing alpha-mannosidases in N-glycan biosynthesis and recent findings derived from the alpha-mannosidase IIx (MX) gene knockout mouse, which shows male infertility. Thus, the MX gene knockout experiment unveiled a novel function of specific N-glycan, which is N-acetylglucosamine-terminated and fucosylated triantennary structure, in the adhesion between germ cells and Sertoli cells. Analysis of the MX gene knockout mouse is a good example of a multidisciplinary approach leading to a novel discovery in the emerging field of glycobiology.

Many proteins, in particular those in the plasma membranes, are glycosylated with carbohydrates, which are grouped into O-glycans and N-glycans. O-glycans are synthesized step by step by glycosyltransferases, whereas N-glycans are synthesized by en-bloc transfer of the so-called high-mannose-type oligosachharide from lipid-linked precursor to polypeptide. The high-mannose-type N-glycans are then modified by processing α-mannosidases. Alpha-mannosidase IIx (MX) was identified as the gene product of processing α-mannosidase II (MII)-related gene. MX apparently plays subsidiary role for MII in many cell types, as N-glycan patterns of MX null mouse tissues are not altered significantly. Surprisingly MX null male mice are infertile due to a failure of spermatogenesis. This review provides a brief overview of the in vivo role of N-glycans which are revealed by the gene knockout mouse approach, and introduce our studies on the MX gene knockout mouse. The MX gene knockout experiments unveiled a novel function of a specific N-glycan, which is N-acetylglucosamine-terminated and has a fucosylated triantennary structure, in the adhesion between germ cells and Sertoli cells. The study of MX is a good example of how the in vivo roles of an apparently redundant gene product are determined by the gene knockout approach.

Alpha-mannosidase IIx (MX) is an enzyme closely related to the Golgi N-glycan processing enzyme alpha-mannosidase II (MII). The enzymatic activity of MX in vitro is minimal. Therefore, the in vivo role of MX in N-glycan processing is as yet unclear. The targeted disruption of the gene encoding MX in the mouse resulted in an obvious phenotype, i.e., MX-deficient males were found to be infertile. Testes from homozygous mutant male mice are smaller than those from wild-type or heterozygous littermates. Histology of the MX null mouse testis showed significant reduction of spermatogenic cells in the seminiferous tubules. Electron microscopy showed that prominent intercellular spaces surround MX-deficient spermatogenic cells, suggesting a failure of germ cell adhesion to Sertoli cells. Quantitative structural analyses of N-glycans from wild-type and MX-deficient mouse testis showed that wild-type testes contain GlcNAc-terminated complex type N-glycans, while they are significantly reduced in MX-deficient mutant testis. An in vitro assay for adhesion of spermatogenic cells to Sertoli cells was carried out. By testing the effect of each purified N-glycan oligosaccharide, it was demonstrated that a GlcNAc-terminated tri-antennary, fucosylated N-glycan has an activity on the adhesion between germ cells and Sertoli cells. Thus, the targeted disruption of the gene encoding MX uncovered a novel carbohydrate recognition system in a biologically important process, spermatogenesis.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.

Review on BYSL, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

Kruppel-associated box-containing zinc finger proteins (KRAB-ZFPs) regulate a wide range of cellular processes. KRAB-ZFPs have a KRAB domain, which binds to transcriptional corepressors, and a zinc finger domain, which binds to DNA to activate or repress gene transcription. Here, we characterize ZNF777, a member of KRAB-ZFPs. We show that ZNF777 localizes to the nucleus and inducible overexpression of ZNF777 inhibits cell proliferation in a manner dependent on its zinc finger domain but independent of its KRAB domain. Intriguingly, ZNF777 overexpression drastically inhibits cell proliferation at low cell density but slightly inhibits cell proliferation at high cell density. Furthermore, ZNF777 overexpression decreases the mRNA level of FAM129A irrespective of cell density. Importantly, the protein level of FAM129A strongly decreases at low cell density, but at high cell density the protein level of FAM129A does not decrease to that observed at low cell density. ZNF777-mediated inhibition of cell proliferation is attenuated by overexpression of FAM129A at low cell density. Furthermore, ZNF777-mediated down-regulation of FAM129A induces moderate levels of the cyclin-dependent kinase inhibitor p21. These results suggest that ZNF777 overexpression inhibits cell proliferation at low cell density and that p21 induction by ZNF777-mediated down-regulation of FAM129A plays a role in inhibition of cell proliferation.

Trophinin mediates apical cell adhesion between two human cell lines, trophoblastic teratocarcinoma and endometrial adenocarcinoma. In humans, trophinin is specifically expressed in cells involved in implantation and early placentation. The present study was undertaken to establish trophinin expression by the mouse uterus. In the pregnant mouse uterus, trophinin transcripts are expressed during the time which coincides with the timing of blastocyst implantation. Trophinin is also expressed in the nonpregnant mouse uterus at estrus stage. Uteri from ovariectomized mice did not express trophinin, whereas strong expression was induced by estrogen but not by progesterone. Trophinin transcripts and protein were found in the pseudopregnant mouse uterus. No differences were detected in trophinin expression by the uteri in the pregnant, pseudopregnant, and pseudopregnant received blastocysts. In delayed implantation model, trophinin proteins were found in both luminal and glandular epithelium, whereas dormant blastocysts were negative for trophinin. Upon activation with estrogen, however, no significant changes were detected either in the blastocyst or in the uterus. These results indicate that ovarian hormones regulate trophinin expression by the mouse uterus, and that an implanting blastocyst has no effect on trophinin expression in the surrounding endometrial luminal epithelial cells.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Using an expression cloning strategy, the cDNA encoding the human HNK-1 sulfotransferase (HNK-1ST) has been cloned. During this cloning we found that HNK-1ST and other Golgi-associated sulfotransferases cloned before share homologous sequences including the RDP motif (Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 223, 5190-5195). Using this conserved sequence in HNK-1ST as a probe, we identified two expressed sequence tags in EST data base which have 31.6 and 30.7% identity with HNK-1ST at the amino acid levels. Expression of these two full-length cDNAs failed to form HNK-1 glycan nor to add sulfate to CD34 or NCAM. Surprisingly, proteins expressed by these cDNAs transferred sulfate to the C-4 position of N-acetylgalactosamine in chondroitin and desulfated dermatan sulfate, thus we named these two enzymes, chondroitin 4-O-sulfotransferase 1 and -2 (C4ST-1 and C4ST-2). Both C4ST-1 and C4ST-2, however, did not form 4, 6-di-O-sulfated N-acetylgalactosamine when chondroitin sulfate C was used as an acceptor. Moreover, analysis of (35)S-labeled dermatan sulfate formed by C4ST-1 indicate that sulfation preferentially took place in GlcA-->GalNAc unit than in IdoA-->GalNAc unit, suggesting that 4-O-sulfation at N-acetylgalactosamine may precede epimerization of glucuronic acid to iduronic acid during dermatan sulfate biosynthesis. Northern analysis demonstrated that the transcript for C4ST-1 is predominantly expressed in peripheral leukocytes and hematopoietic tissues while the C4ST-2 transcript is more widely expressed in various tissues. These results indicate C4ST-1 and C4ST-2 play complementary roles in chondroitin and dermatan sulfate synthesis in different tissues.

Congenital dyserythropoietic anemia type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a genetic anemia in humans caused by a glycosylation deficiency. Erythrocyte membrane glycoproteins, such as band 3 and band 4.5, which are normally glycosylated with polylactosamines lack these carbohydrates in HEMPAS. Polylactosamines accumulate as glycolipids in HEMPAS erythrocytes. Analysis of N-glycans from HEMPAS erythrocyte membranes revealed a series of incompletely processed N-glycan structures, indicating defective glycosylation at N-acetylglucosaminyltransferase II (GnT-II) and/or α-mannosidase II (MII) steps. Genetic analysis has identified two cases from England in which the MII gene is defective. Mutant mice in which the MII gene was inactivated by homologous recombination resulted in a HEMPAS-like phenotype. On the other hand, linkage analysis of HEMPAS cases from southern Italy excluded MII and GnT-II as the causative gene, but identified a gene on chromosome 20q11. HEMPAS is therefore genetically heterogeneous. Regardless of which gene is defective, HEMPAS is characterized by incomplete processing of N-glycans. The study of HEMPAS will identify hitherto unknown factors affecting N-glycan synthesis.

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching beta1, 6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta1, 6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta1, 4-galactosyltransferases (beta4Gal-Ts), beta4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta4Gal-TI and i-extension beta1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta1-->4GlcNAcbeta1-->3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta4Gal-TI adds a galactose poorly to beta1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta1, 3-N-acetylglucosaminyltransferase and beta4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.

Trophinin, tastin, and bystin comprise a complex mediating a unique homophilic cell adhesion between trophoblast and endometrial epithelial cells at their respective apical cell surfaces. In this study, we prepared mouse monoclonal antibodies specific to each of these molecules. The expression of these molecules in the human placenta was examined immunohistochemically using the antibodies. In placenta from the 6th week of pregnancy, trophinin and bystin were found in the cytoplasm of the syncytiotrophoblast in the chorionic villi, and in endometrial decidual cells at the utero placental interface. Tastin was exclusively present on the apical side of the syncytiotrophoblast. Tissue sections were also examined by in situ hybridization using RNA probes specific to each of these molecules. This analysis showed that trophoblast and endometrial epithelial cells at the utero placental interface express trophinin, tastin, and bystin. In wk 10 placenta, trophinin and bystin were found in the intravillous cytotrophoblast, while tastin was not found in the villi. After wk 10, levels of all three proteins decreased and then disappeared from placental villi.

Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage- and/or temporal-specific patterns. The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Lex determinant, are important participants in leukocyte adhesion and trafficking processes. Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans. However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian alpha-1,3-fucosyltransferase and alpha-1,2-fucosyltransferase gene families. To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-D-mannose-4,6-dehydratase (GMD). This enzyme generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to GDP-L-fucose. GMD is thus imperative for the synthesis of all fucosylated oligosaccharides. An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs. This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to an Arabidopsis thaliana GMD cDNA. This approach, together with 5'-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line. Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells. By contrast, the transcript encoding the FX protein, which forms GDP-L-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells. These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein. The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region.

Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin. Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases. Database searches revealed a 53% identity of the predicted peptide sequence with the Drosophila bys ( mrr ) gene. Direct protein–protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by in vitro protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule. Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H. It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation.

Alpha-mannosidase-II (alphaM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of alphaM-II should abolish complex N-glycan production as reportedly does inhibition of alphaM-II by swainsonine. We find that mice lacking a functional alphaM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct alpha-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for alphaM-II in the formation of erythroid complex N-glycans. alphaM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

Golgi alpha-mannosidase II is a key enzyme of N-glycan processing. Its genetic defect is associated with HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test). We previously cloned cDNAs of human alpha-mannosidase II (alpha-MII) and its isotype, alpha-mannosidase IIX [alpha-MIIX, Misago, M., Liao, Y. F., Eto, S., Mattei. M. G., Moremen. K. W.Fukuda, M. N. (1995) Proc. Natl Acad. Sci. USA 92, 11766-11770]. Constitutive expressions of alpha-MII and alpha-MIIX mRNA were shown in various human tissues. To investigate the transcriptional regulation of alpha-MIIX gene, we characterized the cosmid clone of 40-kb that includes the 5'-flanking sequence. This clone contains at least eight exons which encode 396 amino acid residues of a total of 1139 amino acid residues of alpha-MIIX. Primer-extension analysis revealed multiple transcription-initiation sites in the range from -70 to -58 relative to the translation-initiation site. No canonical TATA or CAAT boxes were observed, but a (G + C)-rich region was found in close proximity to the transcription-initiation site. To localize the transcriptional regulatory region of this gene, various regions of the 5' sequences were fused to the luciferase gene, and transient-expression assays were conducted in human melanoma G-361 cells. These studies indicated that sequence from -12 to + 11 relative to the most distal 5'-transcription-initiation site was involved in the promoter function. Within this region, the sequence GGGCGT similar to the consensus sequence of the Sp1 binding site, is present at positions -12 to -7. Enhancer activities were found in the region upstream of this site, notably from -4300 to -252. Thus, the alpha-MIIX promoter located in a CpG island is also regulated by upstream elements, indicating the complexity of alpha-MIIX gene expression.

Gangliosides of the C series such as GT3 are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of GT3 synthase that adds the second α-2,8-sialic acid to GD3, NeuNAcα2→8NeuNAcα2→3Galβ1→4Glc→Cer, thus forming GT3, NeuNAcα2→8NeuNAcα2→8NeuNAcα2→3Galβ1→ 4Glc→Cer. Unexpectedly, the cloned cDNA was found to be identical to the cDNA that encodes GD3 synthase. The newly identified enzyme was therefore named GD3/GT3 synthase (GD3/GT3ST). GD3/GT3ST synthesized GT3 most efficiently when GM3, NeuNAcα2→3Galβ1→4Glc→Cer, was incubated as an acceptor, indicating that GD3/GT3ST is a polysialyltransferase that can transfer more than one sialic acid residue via α-2,8 linkage to gangliosides. Moreover, a longer period of incubation of GD3 with GD3/GT3ST produced a significant amount of GT3 and higher polysialogangliosides. Among various cell lines expressing GD3/GT3ST, higher polysialogangliosides including GT3 were detected only in cell lines where the amount of GD3/GT3 mRNA is sufficiently high. The expression of GD3/GT3ST mRNA among human tissues is highly restricted to fetal and adult brains. The GD3/GT3ST gene was found to be located at chromosome 12, region p12. Taken together, these results indicate that C series polysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, GD3/GT3ST, that is specifically expressed in neural tissues.

Recent studies on proteins residing in the Golgi complex revealed that the membrane-spanning domain of these proteins are largely responsible for their retention in the Golgi complex. We show here that β-1,4-galactosyltransferase (GT) forms homodimers and large oligomers in vivo, and the formation of the homodimers is dependent on cysteine and histidine residues within the transmembrane domain. Double mutations of these residues, Cys29→ Ser and His32→ Leu, abolish homodimerization and simultaneously reduce the Golgi retention. Co-immunoprecipitation of GT and various GT chimeras with anti-GT and anti-reporter molecule antibodies revealed that large aggregates of GT are associated with α- and β-tubulins and also with other cellular proteins. This association between tubulins and GT suggests a supportive role of the cytoskeleton in the Golgi retention mechanism.

Although numerous carbohydrates play significant roles in mammalian cells, development of carbohydrate-based reagents and therapeutics are hampered by the technical difficulty of chemically synthesizing complex carbohydrate structures. Use of carbohydrate mimetic peptides circumvents this difficulty, as short peptide can be easily synthesized and modified. We as well as others identified carbohydrate mimetic peptides by screening peptide displaying phage library using anti-carbohydrate antibodies and lectins. This review introduces our experiences with I-peptide that was used for identification of new carbohydrate binding receptor expressed in the lung endothelial cells, and those with IF7 peptide that can be used as a therapeutic against malignant tumors.

The process of human embryo implantation is mediated not only by evolutionarily conserved mechanisms but by activities unique to humans. Among the latter, evidence suggests that the cell adhesion molecule trophinin plays a unique role in human embryo implantation. Here, we describe characteristics of trophinin protein and of the trophinin-associated proteins bystin and tastin. We then describe trophinin-mediated signal transduction in trophectoderm cells during human embryo implantation and events related to human sperm tail motility. We also report dual roles for trophinin in human cancers in terms of promoting malignancy in some tumor types and suppressing it in others.

Although numerous carbohydrates play significant roles in mammalian cells, carbohydrate-based drug discovery has not been explored due to the technical difficulty of chemically synthesizing complex carbohydrate structures. Previously, we identified a series of carbohydrate mimetic peptides and found that a 7-mer peptide, designated I-peptide, inhibits hematogenous carbohydrate-dependent cancer cell colonization. During analysis of the endothelial surface receptor for I-peptide, we found that I-peptide bound to annexin 1 (Anxa1). Because Anxa1 is a highly specific tumor vasculature surface marker, we hypothesized that an I-peptide-like peptide could target anticancer drugs to the tumor vasculature. This study identifies IFLLWQR peptide, designated IF7, as homing to tumors. When synthetic IF7 peptide was conjugated to fluorescent Alexa 488 (A488) and injected intravenously into tumor-bearing mice, IF7-A488 targeted tumors within minutes. IF7 conjugated to the potent anticancer drug SN-38 and injected intravenously into nude mice carrying human colon HCT116 tumors efficiently suppressed tumor growth at low dosages with no apparent side effects. These results suggest that IF7 serves as an efficient drug delivery vehicle by targeting Anxa1 expressed on the surface of tumor vasculature. Given its extremely specific tumor-targeting activity, IF7 may represent a clinically relevant vehicle for anticancer drugs.

Phage display technology is an emerging drug discovery tool. Using that approach, short peptides that mimic part of a carbohydrate's conformation are selected by screening a peptide-displaying phage library with anti-carbohydrate antibodies. Chemically synthesized peptides with an identified sequence have been used as an alternative ligand to carbohydrate-binding proteins. These peptides represent research tools useful to assay the activities of glycosyltransferases and/or sulfotransferases or to inhibit the carbohydrate-dependent binding of proteins in vitro and in vivo. Peptides can also serve as immunogens to raise anti-carbohydrate antibodies in vivo in animals. Phage display has also been used in single-chain antibody technology by inserting an immunoglobulin's variable region sequence into the phage. A single-chain antibody library can then be screened with a carbohydrate antigen as the target, resulting in a recombinant anti-carbohydrate antibody with high affinity to the antigen. This review provides examples of successful applications of peptide-displaying phage technology to glycobiology. Such an approach should benefit translational research by supplying carbohydrate-mimetic peptides and carbohydrate-binding polypeptides.

Summary Congenital dyserythropoietic anaemia type II (CDA II) is a rare genetic anaemia in humans, inherited in an autosomally recessive mode. CDA II is also called HEMPAS as this disease is characterized by hereditary erythroblastic multinuclearity with positive acidified serum lysis test. Analyses of CDA II erythrocyte membranes showed that the band 3 glycoprotein is underglycosylated. An aberrant glycosylation pattern is seen in the polylactosamine carbohydrates which are normally attached to the band 3 and band 4.5 glycoproteins. The polylactosamines are, however, accumulated in the form of glycolipids. Therefore a genetic factor in CDA II appears to block the glycosylation of protein acceptors and shift these carbohydrates to the lipid acceptors. Structural analysis of CDA II band 3 carbohydrates identified truncated hybrid-type oligosaccharides and suggests that the Golgi glycosylation enzyme(s), α-mannosidase II or N-acetylglycosaminyl-transferase II is defective in CDA II. By using a cDNA probe for α-mannosidase II, one CDA II case has been identified as being defective in the gene encoding α-mannosidase II. At present, it is not clear whether CDA II is a genetically heterogenous collection of glycosylation deficiencies, or genetically homogenous but apparently heterogenous in phenotype expression. Freeze-fracture electron microscopy and immunoelectron microscopy revealed that the band 3 glycoproteins are clustered in CDA II erythrocyte membranes. The abnormal distribution of band 3 might cause an unstable membrane organization. In CDA II erythroblasts, the membrane proteins might also be underglycosylated and abnormally distributed. When normal erythroblasts were cultured in vitro in the presence of swainsonine (α-mannosidase inhibitor) the erythroblasts became multinucleated. It is, therefore, quite possible that the enzymic defect of α-mannosidase II could cause various morphological anomalies including multinuclearity. Because the genes encoding glycosylation enzymes are housekeeping genes, the enzyme defect of CDA II is not restricted to erythroid cells and there is also an abnormal glycosylation of hepatocyte glycoproteins. On the other hand, there are many types of cells and tissues which appear not to be affected by the CDA II defect. A mechanism for the erythroid-specific manifestation of CDA II and its tissue specificity are also discussed.

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.

Alterations in glycosylation play an important role during intestinal cell differentiation. Here, we compared expression of mucin-type O-glycan synthases from proliferating and differentiated HT-29 and Caco-2 cells. Mucin-type O-glycan structures were analyzed at both stages by mass spectrometry. Core2 β1,6-N-acetylglucosaminyltransferase-2 (C2GnT-2) was markedly increased in differentiated HT-29 and Caco-2 cells, but the core3 structure was hardly detectable. To determine whether such differential expression of mucin-type O-glycan structures has physiological significance in intestinal cell differentiation, expression of sucrase isomaltase (SI) and dipeptidyl-peptidase IV (DPP-IV), two well known intestinal differentiation markers, was examined. Interestingly, the fully glycosylated mature form of SI was decreased in C2GnT-2 knock-out mice but not in core2 N-acetylglucosaminyltransferase-3 (C2GnT-3) nulls. In addition, expression of SI and DPP-IV was dramatically reduced in C2GnT-1–3 triple knock-out mice. These patterns were confirmed by RNAi analysis; C2GnT-2 knockdown significantly reduced cell surface expression of SI and DPP-IV in Caco-2 cells. Similarly, overexpression of the core3 structure in HT-29 cells attenuated cell surface expression of both enzymes. These findings indicate that core3 O-glycan structure regulates cell surface expression of SI and DPP-IV and that core2 O-glycan is presumably an essential mucin-type O-glycan structure found in both molecules in vivo. Finally, goblet cells in the upper part of the crypt showed impaired maturation in the core2 O-glycan-deficient mice. These studies are the first to clearly identify functional mucin-type O-glycan structures modulating cell surface expression of SI and DPP-IV during the intestinal cell differentiation.

Peptide-displaying phage technology has numerous applications. Using a specificity-defined monoclonal anticarbohydrate antibody, we can identify a series of short peptides that mimic the binding specificity of a specific carbohydrate. Identified peptides constitute alternatives to the use of carbohydrate ligands in affinity chromatography to isolate a carbohydrate-binding protein. In this chapter, we introduce methods for carbohydrate mimicry peptide affinity chromatographies and discuss their potential use in identifying yet undiscovered carbohydrate-binding proteins in normal and pathological conditions.

Congenital dyserythropoietic anaemia type II, or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a genetic disease caused by membrane disorganization of erythroid cells. The primary defect of this disease lies in the gene encoding enzyme(s) which is responsible for the biosynthesis of Asn-linked oligosaccharides chains of glycoproteins (Fukuda et al, 1990). In order to know whether this gene defect affects the glycosylation in the cells other than the erythroid cells, the carbohydrate structures of the transferrin isolated from the sera of HEMPAS patients were analysed. Fast atom bombardment mass spectrometry analysis showed the presence of high mannose type and hybrid type oligosaccharides in the HEMPAS transferrin which is in contrast to the complex-type oligosaccharides found in the normal transferrin. The results strongly suggest that biosynthesis of Asn-linked oligosaccharide chains in HEMPAS hepatocytes is disturbed. As a result, the serum glycoproteins with incompletely processed carbohydrates are circulating in the plasma in HEMPAS patients, but they must have been absorbed by the cells in the liver and the reticuloendothelial cells. Upon intravenous infusion into rats, as much as 30% of the HEMPAS transferrin was cleared from the plasma circulation. The majority of the HEMPAS transferrins was taken up by the liver, and transferrin was distributed both in the hepatocytes and the Kupffer cells. The presence of enormous amounts of aberrantly glycosylated serum glycoproteins may lead to the liver cirrhosis and secondary tissue siderosis seen in HEMPAS patients.

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi.

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

The poly-N-acetyllactosamines on neutrophils and monocytes have been shown to serve as ligands for various selectins present on endothelial cells and platelets. We have previously shown that only a limited number of glycoproteins contain poly-N-acetyllactosamine and found that lysosomal membrane glycoproteins (lamps) are the major glycoproteins carrying poly-N-acetyllactosamine. In order to understand the reason why only certain glycoproteins can be modified by poly-N-acetyllactosamine, we have utilized 21 degrees C incubation conditions, which were previously shown to cause the accumulation of glycoproteins at the trans-Golgi. HL-60 cells were labeled with [3H]galactose at 21 or 37 degrees C for 6 or 24 h, and lamp-1 and lamp-2 were immunoprecipitated. Upon examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, each lamp from HL-60 cells incubated at 21 degrees C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37 degrees C. The number of N-glycans containing poly-N-acetyllactosamine, estimated by their binding to tomato lectin column, increased approximately 30-50% after incubation at 21 degrees C than incubation at 37 degrees C. The analysis of oligosaccharides released by endo-beta-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21 degrees C, and methylation analysis confirmed these results. The same analysis and the results obtained by ion-exchange chromatography also provided evidence that the N-glycans of lamps are sialylated at 21 degrees C as much as at 37 degrees C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21 degrees C than at 37 degrees C. These results therefore indicate that incubation at 21 degrees C causes the lamps to reside longer within the Golgi complex, and such longer residence allows lamps to acquire more polylactosaminoglycan. These results also suggest that the time necessary for moving through the Golgi complex is a critical factor for poly-N-acetyllactosamine formation.

Macular corneal dystrophy (MCD) is subdivided into three immunophenotypes, MCD types I, IA and II, based on the reactivity of serum and corneal tissue to an antibody that recognises sulfated keratan sulfate (KS).1 In MCD type I (MCD-I), antigenic KS is undetectable in both serum and cornea, while in MCD-II it is present at normal or subnormal levels in serum, and is evident immunohistochemically in the corneal stroma. In MCD-IA, antigenic KS is absent from serum and extracellular stromal matrix, but is detected in keratocytes. Mutations of the carbohydrate sulfotransferase gene, CHST6, have been identified as causative for MCD.2 This study investigated the distribution of differentially sulfated KS in cornea in the three main MCD immunophenotypes.

Determining molecular mechanisms of human embryo implantation is an extremely challenging task due to the limitation of materials and significant differences underlying this process among mammalian species. Recently, L-selectin and its ligand carbohydrate have been proposed as a system that mediates initial adhesion of human blastocysts to the uterine epithelia. We have also identified trophinin as a unique apical cell adhesion molecule potentially involved in the initial adhesion of trophectoderm of the human blastocyst to endometrial surface epithelia. In the mouse, the binding between ErbB4 on the blastocyst and heparin-binding epidermal growth factor-like growth factor on the endometrial surface enables the initial step of the blastocyst implantation. The evidence suggests that L-selectin and trophinin are included in human embryo implantation. This review summarizes findings relevant to the functions of L-selectin and trophinin in human embryo implantation, and proposes a model that reconciles these cell adhesion mechanisms.

Tastin was previously characterized as an accessory protein for cell adhesion that participates in early embryo implantation. Here, we report that tastin is also required for spindle assembly during mitosis. Tastin protein levels peaked in the G(2)/M phase and abruptly declined after cell division. Microscopy showed that tastin is primarily localized on the microtubules, centrosomes, and the mitotic spindle during the cell cycle. Tastin interacted with the dynein intermediate chain, p150(Glued), and gamma-tubulin in addition to Tctex-1 ( the light chain of dynein). Overexpression of tastin led to monopolar spindle formation, whereas loss of tastin expression caused profound mitotic block and preferentially induced multipolar spindles. These multipolar spindles were generated through a loss of cohesion in mitotic centrosomes; specifically, tastin depletion caused the fragmentation of pericentrosomal material and the splitting of the centrioles at the spindle poles. Tastin depletion induced centrosome abnormalities exclusively during mitosis and required both microtubule integrity and Eg5 activity. However, tastin depletion did not disrupt the organization of spindle poles, as revealed by localization of nuclear mitotic apparatus protein (NuMA) and the p150(Glued) component of dynactin. These data indicate that the major function of tastin during mitosis is to maintain the structural and dynamic features of centrosomes, thereby contributing to spindle bipolarity.