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Background Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood–brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. Methods (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 μmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 μmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. Results (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. Conclusions IF7C(RR)-SN38 crosses the blood–brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/ Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/ Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/ Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/ Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/ Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/ Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development

IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminal domain, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the Anxa1 N-terminus as target. Peptide sequences were identified, synthesized as D-amino acids, and designated as dTIT7, which was shown to bind the ANXA1 N-terminus. Whole body imaging of mouse brain tumors modeled with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered geldanamycin (GA)-conjugated dTIT7 suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that Anxa1-binding D-peptide could be developed as an orally-administrable, tumor vasculature-targeted therapeutic.Role of each author: MN designed and performed experiments, analyzed data, and wrote the manuscript; HMA and DLJ produced recombinant ANXA1 protein; KY conducted NMR analysis and data analysis; TOA designed, performed and analyzed LC-MS/MS data; MN, TS, IKT, YS, and TY analyzed peptide-binding assays and performed in silico structural analysis; CTU produced lentivirus for luciferase expression; CNH performed peptide binding assays, tissue culture and animal experiments; and MNF supervised the project and wrote the manuscript.

Objectives To evaluate the expression of annexin A1 protein in patients with renal cell carcinoma. Methods Annexin A1 expression was examined in renal cell carcinoma specimens from 27 patients, and their disease-free survival was analyzed using the log-rank test. Annexin A1 knockdown in the human renal cell carcinoma cell line Caki-1 was carried out, and its proliferation, invasion, motility and adhesion were compared with those of control cells. Results In 13 out of 27 patients, annexin A1 was highly expressed in the membrane of renal cell carcinoma tumor cells, whereas in the rest of the patients, annexin A1 expression was weak or negligible in the membrane of those cells. Patients with high annexin A1 expression had significantly poorer disease-free survival than those with weak or negligible annexin A1 expression (P = 0.031). In the renal cell carcinoma cell line, annexin A1 knockdown cells showed significantly decreased proliferation, invasion, motility and adhesion relative to control cells, and expressed lower relative levels of membrane-type 1 matrix metalloproteinase and hypoxia-inducible factor 1-alpha transcripts, showing a potential pathway regulated by annexin A1. Conclusion Annexin A1 is associated with renal cell carcinoma malignant potential and could serve as a marker of poor prognosis.

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.

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)

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.

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.

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.

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.

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.

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.

Substantial evidence suggests that cell surface carbohydrate antigens, particularly those containing fucose residues, are related to cancer malignancy. To investigate the mechanisms underlying cell surface carbohydrate-dependent cancer metastasis, we developed a 7-mer peptide, designated as I-peptide, which is capable of inhibiting FUT3-transfected B16 cell colonization of lung in the mouse. I-peptide inhibited lung colonization of FUT3-B16 cells in E-/P-selectin doubly deficient mutant mice, suggesting the existence of an as yet unidentified carbohydrate-binding endothelial receptor distinct from E-/P-selectins. Subsequently, we isolated the endothelial receptors by I-peptide affinity chromatography and identified them by proteomics. Thus, I-peptide receptors were identified to be pre-mRNA splicing factor (Sfrs) proteins. We also found a fragment of annexin A1 (Anxa1) bound to I-peptide. Our study demonstrated the usefulness of carbohydrate-ligand mimicry peptide for the identification of novel carbohydrate-binding proteins.

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.

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.

Publisher Summary This chapter discusses the roles of cell surface carbohydrates in development, while focusing on embryo implantation, spermatogenesis, and tissue maturation. The outer surface of mammalian cells is covered by glycoproteins and glycolipids. Substantial biochemical and immunochemical evidence suggests that cell surface carbohydrates play significant roles in development and health. Functional studies of cell surface carbohydrates still leave many questions unanswered. In the last decade, genetic approaches and sophisticated chemical analyses have enabled us to reveal the function of specific carbohydrate structures in vivo, and as a result the role of carbohydrates in development and disease is understood. In the field of reproductive biology and embryology, it has been assumed that cell surface carbohydrates play important roles. These hypotheses are difficult to test because embryonic development is dynamic and the material of interest is often too limited to allow chemical analysis. Analyzing early stage embryos requires well-trained hands and skills that many biochemists and molecular biologists have only recently developed. Nonetheless, many attractive hypotheses await testing by new technologies.

Previously we found that the trophinin-binding peptide GWRQ (glycine, tryptophane, arginine, glutamic acid) activated human trophoblastic cells. Although trophinin is expressed in human sperm, to our knowledge the function of this protein is not known. In this study we tested the effect of GWRQ on human sperm to evaluate whether the peptide enhances human sperm motility.Immunohistochemistry was performed using monoclonal antibodies specific to trophinin, bystin or tastin. GWRQ-MAPS (multivalent 8-branched GWRQ peptide) was chemically synthesized. Human sperm from 4 volunteers with a mean +/- SD age of 35.75 +/- 3.4 years was suspended in buffer with GWRQ or control peptides. In 23 volunteers with a mean age of 25.5 +/- 2.5 years the number of immotile sperm was counted and subtracted from the total number of sperm to determine the number of motile sperm. A Transwell assay was used to measure swim-down motility. Levels of adenosine triphosphate and intracellular calcium in sperm cells incubated with GWRQ or control peptide were measured using a luminescent cell viability assay and a fluo-4 calcium assay, respectively.The presence of trophinin and the trophinin associated proteins bystin and tastin in human sperm was confirmed by immunohistochemistry. Human sperm incubated with GWRQ-MAPS showed enhanced motility on sperm count assay and swim-down Transwell assay. Sperm cells incubated with GWRQ-MAPS showed decreased adenosine triphosphate and increased intracellular calcium.Results suggest that GWRQ-MAPS may facilitate optimized in vitro fertilization outcomes and help avert the need for intracytoplasmic sperm injection in cases of severely low sperm motility. Trophinin may have a role in regulating adenosine triphosphatase in human sperm.

During embryo implantation, trophinin mediates cell adhesion by homophilic binding at the apical surfaces of trophectoderm and endometrium. Trophinin is expressed on the human endometrial epithelia in rare occasions. We developed hCG-coated agarose beads that mimic the physical and physiological features of an implantation-stage human blastocyst. When hCG-coated beads were applied to human endometrial epithelial cells in the presence of IL-1β, endometrial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin-expressing human trophoblastic cells. These results provide a mechanism for trophinin-mediated adhesion of human blastocyst to endometrium by a spatially and temporally restricted paracrine effect of hCG derived from the blastocyst.

Reexamination of the Akp3−/−mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the Akp3−/−gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel murine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner ( gIAP) as opposed to duodenum-specific IAP ( dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant ( kcat) and a 30% drop in Km, leading to a 4-fold reduction kcat/ Kmcompared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of Akp3−/−mice. Developmentally, Akp3 expression is turned on during postnatal days 13–15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in Akp3−/−mice fed a high-fat diet.

Human magphinin proteins are translation products of differentially spliced transcripts from the 5' region of the human trophinin gene (TRO), whose 3' region encodes trophinin, a unique cell adhesion molecule involved in human embryo implantation. Magphinins belong to the MAGE (melanoma-associated antigen) family, and a previous study of mouse magphinins showed their expression in male and female germ cells, suggesting a role in germ cell development. Here, we characterized the structure and subcellular localization of human magphinins. Confocal microscopy analysis of ectopically expressed magphinins revealed that magphinin-alpha and -beta localize in the cytoplasm, whereas magphinin-gamma lacking the peptide encoded by exon-3 is nuclear. Following Triton X-100 extraction, DNA digestion, and high salt extraction magphinin-gamma remained nuclear, suggesting strong association with the nuclear matrix. A series of magphinin-gamma deletion mutants were generated and assayed for localization, which showed that the N-terminal region of the MAGE homology domain is necessary for nuclear localization. When magphinin-gamma was expressed in NIH3T3 cells, cells underwent G1 arrest. These results suggest that human magphinin-gamma inhibits cell cycle progression through nuclear activity.

Determining the molecular mechanism of human embryo implantation is an extremely challenging task due to the limitation of materials and significant differences in this process among mammalian species. Trophinin has been identified as an apical cell adhesion molecule with potential involvement in human embryo implantation. We found that trophinin-mediated cell adhesion triggers signal transduction in human trophoblastic cells for proliferation and invasion, implicating in trophectoderm cell activation for placental formation. Prior to cell adhesion trophinin arrests ErbB4 by binding through bystin, which prevents ErbB4 from activation. Trophinin-mediated cell adhesion causes dissociation of bystin from trophinin, freeing ErbB4 from arrest and enabling tyrosine phosphorylation. Therefore trophinin functions as an adhesion molecule on the cell surface and as a molecular switch for trophoblast activation in the cytoplasm.

Lymphocyte homing is mediated by binding of L-selectin on lymphocytes with L-selectin ligands present on high-endothelial venules (HEV) of peripheral and mesenteric lymph nodes. L-selectin ligands are specific O-linked carbohydrates, 6-sulfo sialyl Lewis X, composed of sialylated, fucosylated, and sulfated glycans. Abrogation of fucosyltransferase-VII (FucT-VII) results in almost complete loss of lymphocyte homing, but structural analysis of carbohydrates has not been carried out on FucT-VII null mice. To determine whether functional losses seen in FucT-VII null mice are caused by structural changes in carbohydrates, we elucidated the carbohydrate structure of GlyCAM-1, a major L-selectin counter-receptor. Our results show that most alpha1,3-fucosylated structures in 6-sulfo sialyl Lewis X are absent and 6-sulfo N-acetyllactosamine is increased in the mutant mice. Surprisingly, the amount of 6'-sulfated galactose (Gal) that bound to Sumbucus nigra agglutinin column was also increased. We found that structures of those oligosaccharides containing 6'-sulfated Gal are almost identical to those synthesized by keratan sulfate sulfotransferase (KSST). We then showed that overexpression of KSST suppresses the expression of sialyl Lewis X on Chinese hamster ovary (CHO) cells engineered to express sialyl Lewis X. Moreover, KSST expression in those cells suppressed lymphocyte rolling compared with mock-transfected CHO cells expressing 6-sulfo sialyl Lewis X. 6'-Sulfo sialyl Lewis X can neither be found in GlyCAM-1 from CHO cells expressing both KSST and FucT-VII nor be found in GlyCAM-1 from HEV of mice. These results combined together suggest that KSST competes with FucT-VII for the same acceptor substrate and downregulates the synthesis of L-selectin ligand by inhibiting alpha1,3-fucosylation.

Previous work has demonstrated the ability of the NG2 proteoglycan, a component of microvascular pericytes, to stimulate endothelial cell motility and morphogenesis. This function of NG2 depends on formation of a complex with galectin-3 and α3β1 integrin to stimulate integrin-mediated transmembrane signaling. In addition, the co-expression of galectin-3 and NG2 in A375 melanoma cells suggests that the malignant properties of these cells may be affected by interaction between the two molecules. Here, we extend the theme of co-expression and interaction of NG2 and galectin-3 to human glioma cells. We also establish a molecular basis for the NG2/galectin-3 interaction. The C-terminal carbohydrate recognition domain of galectin-3 is responsible for binding to the NG2 core protein. Within the NG2 extracellular domain, the membrane-proximal D3 segment of the proteoglycan contains the primary binding site for interaction with galectin-3. The interaction between galectin-3 and NG2 is a carbohydrate-dependent one mediated by N-linked rather than O-linked oligosaccharides within the D3 domain of the NG2 core protein. These studies establish a foundation for attempts to reduce the aggressive properties of tumor cells by disrupting the NG2/galectin-3 interaction. J. Cell. Biochem. 98: 115–127, 2006. © 2005 Wiley-Liss, Inc.

Helicobacter pylori infects the stomachs of nearly a half the human population, yet most infected individuals remain asymptomatic, which suggests that there is a host defense against this bacterium. Because H. pylori is rarely found in deeper portions of the gastric mucosa, where O -glycans are expressed that have terminal α1,4-linked N -acetylglucosamine, we tested whether these O -glycans might affect H. pylori growth. Here, we report that these O -glycans have antimicrobial activity against H. pylori , inhibiting its biosynthesis of cholesteryl-α-D-glucopyranoside, a major cell wall component. Thus, the unique O -glycans in gastric mucin appeared to function as a natural antibiotic, protecting the host from H. pylori infection.

Trophinin is a membrane protein that is potentially involved in human embryo implantation by mediating homophillic cell adhesion between trophoblastic cells and endometrial cells. Trophinin expression by maternal cells may be induced by the embryo that secretes human chorionic gonadotropin (hCG). Because the process of tumor metastasis resembles that of trophoblast invasion and proliferation during embryo implantation, we hypothesized that testicular cancers that synthesize hCG express trophinin thus becoming aggressive trophoblast-like cells. We screened paraffin-embedded orchiectomy specimens of 158 patients with testicular germ cell tumor by immunohistochemistry using antitrophinin antibody. This screening identified trophinin-positive specimens with the frequencies 39 of 91 (43%) in stage I, 14 of 24 (58%) in stage II, and 41 of 43 (95%) in stage III (P < 0.001). Thus, trophinin expression positively correlates with clinical stage. Remarkably, trophinin was found in all of the cases (33 of 33) with lung metastasis. The levels of serum hCG-β were significantly higher in the patients with trophinin-positive tumors than those with trophinin-negative tumors (P = 0.004). To determine whether trophinin promotes aggressiveness of the cell, trophinin-negative human seminona cell line JKT-1 was stably transfected with a mammalian expression vector containing trophinin cDNA. In vitro assays revealed that trophinin-expressing JKT-1-Tro cells are more invasive than JKT-1-mock cells, whereas there are no differences between JKT-1-Tro and JKT-1-mock in their proliferation activity. Upon orthotopic inoculation to athymic nude mice, JKT-1-Tro cells exhibited i.p. metastases in all of the mice (n = 5), whereas JKT-1-mock produced no metastases (n = 5). These results suggest strongly that trophinin enhances invasiveness of the cells and promotes metastasis of testicular germ cell tumor.

Elevated expression of sialyl Lewis X has been postulated to be a prognostic indicator of prostate cancer. However, direct evidence for the relationship between increased expression of sialyl Lewis X and malignancy of prostate cancer is still lacking. To determine whether increased levels of sialyl Lewis X leads to malignancy in prostate tumor, we transfected the human prostate cancer cell line PC-3 with α1,3-fucosyltransferase III (FTIII) to obtain stable transfectants, PC-3-FTIII lines, that highly express sialyl Lewis X. When inoculated in the prostate of nude mice, PC-3-FTIII cells produced large prostate tumors, while mock-transfected PC-3 cells, which are negative for sialyl Lewis X antigen, produced small prostate tumors. The aggressive tumor formation by PC-3-FTIII cells was inhibited by preincubation of the tumor cells with anti-sialyl Lewis X antibody, by the presence of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide but not by control peptide. PC-3-FTIII cells and mock-transfected PC-3 cells exhibited no significant difference in cell numbers when cultured in vitro. Remarkably, PC-3-FTIII adhered to prostatic stromal cells in vitro with higher affinity than mock-transfected PC-3. Such adhesion was inhibited by preincubation of PC-3-FTIII cells with antisialyl Lewis X antibody, by the addition of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide. However, anti-E-selectin, anti-P-selectin or anti-L-selectin antibodies did not inhibit the adhesion of PC-3-FTIII cells to the stromal cells. These results suggest that prostate cancer cells gain aggressiveness through adhesive interaction with prostatic stromal cells by a novel mechanism involving sialyl Lewis X. © 2003 Wiley-Liss, Inc.

Acute heart allograft rejection is characterized by leukocyte infiltration and myocyte damage, key elements in the histological grading of rejection. The induction of selectins and their ligands on the graft postcapillary venular endothelium increases leukocyte tethering to, rolling on, and extravasation through the endothelium into graft parenchyma. We have characterized peptide mimicking selectin ligands by screening phage peptide libraries using anti-Lewis A antibodies and E-selectin as target molecules. The effect of this selectin- binding peptide, IELLQAR, on the prevention of inflammation and tissue damage and on the prolongation of graft survival in inbred DA (RT1a) rat heart allografts transplanted to WF (RT1v) recipients was tested. Bovine serum albumin (0.1%, solvent), VTSIAQA (control peptide), or IELLQAR were either continuously infused into the peritoneum via osmotic mini pumps or injected twice daily IV. Treatment with bovine serum albumin and VTSIAQA did not alter the number of graft infiltrating leukocytes or the histological grade of acute rejection, all scored as grade 4. On the contrary, the selectin binding peptide, IELLQAR, dose-dependently reduced inflammation and at the highest dose (6.0 mg/kg per day) eliminated the majority of graft infiltrating leukocytes, reduced the histological grade from 4 to 1B, but had no effect on graft survival. These data indicate that the intensity of inflammation related to the allograft rejection does not correlate to the graft survival.

Spermatogenesis is a precisely regulated process in which the germ cells closely interact with Sertoli cells. The molecular basis of this cell-cell adhesion is unknown. Here, we demonstrate that targeted disruption of Man2a2 , a gene encoding α-mannosidase IIx (MX), an enzyme that forms intermediate asparagine-linked carbohydrates ( N -glycans), results in Man2a2 null males that are largely infertile. The Man2a2 null spermatogenic cells fail to adhere to Sertoli cells and are prematurely released from the testis to epididymis. We identified an N -glycan structure that plays a key role in germ cell–Sertoli cell adhesion and showed that a specific carbohydrate was required for spermatogenesis.

Elucidation of the implantation mechanism in humans at the molecular level has been difficult because of methodological restrictions. Instead of using human materials during the implantation period, two human tumor cell lines that respectively mimic the biological behaviors of a blastocyst and uterine luminal epithelial cells were utilized successfully to identify three novel adhesion molecules named trophinin, bystin, and tastin. Trophinin is a membrane protein strongly expressed both on the apical surface of the trophectoderm of a simian blastocyst and at a putative implantation site of the human endometrium. Bystin and tastin are cytoplasmic proteins that associate with trophinin by presumably forming an active adhesion machinery. The expression patterns of these molecules are suggestive of their involvement in the initial blastocyst attachment to the uterus as well as in the subsequent placental development. Future perspectives in molecular implantation research are also discussed in relation to breakthroughs in assisted reproduction.

Embryo implantation is a complex process consisting of multiple cross-talks between maternal and embryonic cells. Defining the mechanisms underlying implantation at molecular level is challenging task in reproductive biology. In order to identify molecules involved in cellular interactions between trophoblastic and endometrial epithelial cells, we have established two human cell lines, trophoblastic HT-H and endometrial epithelial SNGM. These two cell types exhibit cell adhesion at their respective apical cell membranes. Molecules involved in this unique cell adhesion were identified by expression complementary DNA cloning and were named trophinin, tastin, and bystin. Trophinin is a membrane protein thought to have self-binding activity and thus mediates homophilic cell adhesion. Tastin and bystin are cytoplasmic proteins required for trophinin to exhibit cell adhesion activity. Trophinin is strongly expressed in trophectoderm of monkey blastocysts. In human endometrium, trophinin is expressed for a limited period in the luminal epithelium at the time expected for implantation. In human placenta, trophinin, tastin, and bystin are strongly expressed in trophoblast and endometrium at the uteroplacental interface at an early stage in pregnancy. All these molecules disappear from the human placenta in the second trimester. The unique expression pattern and cell adhesion activity exhibited by trophinin, tastin, and bystin suggest strongly the involvement of these molecules in the initial attachment of blastocyst to uterus.

The effects of the N-linked oligosaccharide inhibitors swainsonine and N-butyldeoxynojirimycin (NB-DNJ) on granulopoiesis was investigated using human bone marrow cells in in vitro liquid and agar cultures. The addition of the inhibitors into cultures containing granulocyte colony-stimulating factor (G-CSF) suppressed maturation from myelocytes into mature neutrophils. Swainsonine did not induce apoptosis, but NB-DNJ induced considerable apoptosis, especially in the presence of G-CSF. This result indicated that the decrease of mature neutrophils by swainsonine was not because of cell degeneration. In the case of NB-DNJ, it was thought to be because of both maturation suppression and apoptosis. In a colony-forming unit-granuloid (CFU-G) colony assay, the number of colonies was increased in the presence of the inhibitors, but the morphology of colonies was predominantly compact, or immature. The inhibitors also suppressed the expressions of mRNAs of CCAAT/enhancer binding protein epsilon (C/EBPepsilon) and G-CSF receptor as markers of terminal neutrophil maturation. These findings suggested that the incompleteness of N-linked oligosaccharide leads to the suppression of terminal neutrophil maturation.

We set out to determine the expression profiles of glycoproteins possessing N-acetyllactosamine, a precursor carbohydrate of sialyl Le(x), during colorectal cancer development. We immunohistochemically analyzed the distribution of N-acetyllactosamine as well as of beta4GalT-I, a member of the beta1, 4-galactosyltransferase family responsible for N-acetyllactosamine biosynthesis, in normal mucosa and in adenoma and carcinoma of the human colorectum. Using monoclonal antibody H11, N-acetyllactosamine was barely detectable in the normal mucosa. In low-grade adenoma, however, N-acetyllactosamine was weakly but definitely expressed on the cell surface, and its expression level was moderately increased in high-grade adenoma and markedly increased in carcinoma in situ as well as in advanced carcinoma. To detect beta4GalT-I, we used a newly developed polyclonal antibody (designated A18G), which is specific for the stem region of human beta4GalT-I. Faint expression of beta4GalT-I was detectable in normal mucosa, and the expression level was moderately increased in low-grade adenoma and in high-grade adenoma and markedly increased in carcinoma in situ and advanced carcinoma. The expression of N-acetyllactosamine was highly correlated with the expression of beta4GalT-I in these tumor cells. These results indicate that the expression level of beta4GalT-I is apparently enhanced during tumorigenesis in the colorectum and that beta4GalT-I mostly directs the carcinoma-associated expression of N-acetyllactosamine on the colorectal tumor cell surface. (J Histochem Cytochem 47:1593-1601, 1999)

Defining the cellular and molecular bases of embryo implantation is an important yet extremely difficult problem in developmental biology because of the lack of an appropriate in vitro model for implantation. In this regard two human cell lines, HT-H and SNG-M, are noteworthy, as these cells appear to mimic some, if not all, of the mechanisms involved in implantation. In particular, their unique manner of initial attachment at their respective apical cell surfaces and subsequent morphological changes leading to stronger adhesive interactions resemble the trophectoderm cells of blastocyst and endometrial epithelial cells at the implantation stage (1).

Implantation following placentation is a unique system for mammals to reproduce. The initial attachment of the embryo to the uterus occurs via the apical cell membranes of two epithelial cells, trophoblast of the blastocyst and surface epithelial cells of the endometrium. Analysis of the implantation at the molecular level has been a difficult problem in reproductive biology. Recently, a major break through was made in this area: A discovery of a novel cell adhesion molecule complex mediating the initial attachment of trophoblast to the endometrial epithelium. This review provides a brief overview of cell adhesion molecules involved in implantation and introduces identification and characterization of trophinin and tastin.

Two human epithelial cell lines, trophoblastic teratocarcinoma HT-H and endometrial adenocarcinoma SNG-M cells, adhere to each other at their respective apical cell surfaces in a divalent cation-independent manner. Two novel molecules responsible for the adhesion between these two cell types were identified by expression cDNA cloning. One, named trophinin, is an intrinsic membrane protein and mediates homophilic self-binding. Another, named tastin, is a cytoplasmic protein and is necessary for trophinin to function as a cell adhesion molecule. Trophinin and tastin appear to be associated with the cytoskeleton in HT-H and SNG-M cells. These molecules are normally not expressed in various types of human cells in tissues, with the exception of macrophages. Strong expression of these molecules was detected in the trophectoderm surface of monkey blastocyst. These molecules are also expressed in human endometrial surface epithelium on day 16/17 at the early secretory phase of human endometrium, the time consistent with that expected for the "implantation window."

In culture the human teratocarcinoma cell line HT-H generates both adherent monolayer and free-floating aggregates. Some populations of aggregated cells develop further to form cystic bodies. A previous study showed the morphological resemblance of the cystic bodies to cells of blastocyst of preimplantation embryo. In this study, HT-H adherent cells were further separated into two subpopulations, fast adhering and slow adhering cells. Fast adhering cells produce fibronectin, spread well onto substratum, and do not proliferate. In contrast, slow adhering cells do not produce fibronectin. Trophoblastic markers were examined in each morphological stage of HT-H cells and the following results were obtained. Only fast adhering cells produce progesterone. Human chorionic gonadotropin was secreted preferentially by fast adhering cells, about six times less by slow adhering cells, and was not secreted by aggregates or cystic bodies. All stages of HT-H cells express c-fos but only fast adhering cells express c-fms oncogene. Cytokeratin 18 was expressed in all stages of HT-H cells. The level of cytokeratin 18 is modestly decreased from adherent to aggregates further into cystic bodies. These results indicate that HT-H cells share properties with cells in trophoblast, placenta, and extraembryonic endoderm. Spontaneous differentiation of HT-H cultures results in the appearance of fast adhering cells which exhibit biochemical properties expected for syncytiotrophoblast.

Congenital dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) is a genetic disease caused by membrane abnormality. Previously we have found that Band 3 and Band 4.5 are not glycosylated by lactosaminoglycans in HEMPAS erythrocytes, whereas normally these proteins have lactosaminoglycans (Fukuda, M. N., Papayannopoulou, T., Gordon-Smith, E. C., Rochant, H., and Testa, U. (1984) Br. J. Haematol. 56, 55-68). In order to find out where glycosylation of lactosaminoglycans stops, we have analyzed the carbohydrate structures of HEMPAS Band 3. By fast atom bombardment-mass spectrometry, methylation analysis, and hydrazinolysis followed by exoglycosidase treatments, the following structure was elucidated: (formula; see text) N-Linked glycopeptides synthesized in vitro by reticulocyte microsomes from HEMPAS were shown to be predominantly the above short oligosaccharide, whereas those from normal reticulocytes contain large molecular weight carbohydrates. The N-acetylglucosaminyltransferase II, which transfers N-acetylglucosamine to the C-2 position of the Man alpha 1----6Man beta 1----arm of the biantennary core structure, was therefore examined by using Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcol as an acceptor. N-Acetylglucosaminyltransferase II activity was demonstrated in the lymphocyte microsome fraction from normal individuals. However, this enzyme activity was found to be decreased in those from HEMPAS patients. These results suggest that the primary defect of HEMPAS lies in the lowered activity of N-acetylglucosaminyltransferase II.

The glycolipids of blood group type O adult, newborn, and fetal erythrocytes were compared. The total amount of glycolipids was indistinguishable between adult and newborn erythrocytes. However, glycolipids with long and neutral carbohydrates and the H determinant were greatly reduced in newborn cells. On the other hand, the amount of sialylated glycolipids (gangliosides) was significantly higher in newborn cells, suggesting that during erythropoiesis sialyltransferases are more active in fetuses than in adults. The amount of each core structure, lacto-N-tetraosyl, linear lacto-N-hexaosyl, and branched lacto-N-octaosyl, was compared between adult and newborn erythrocytes. It was found that branched lacto-series glycolipids were reduced in newborn cells compared with adult cells. Thus, development from fetal to adult human erythrocytes is associated with an increase of branching and a decrease of sialylation of N-acetyllactosaminyl carbohydrate chains. The study indicates that glycolipids are quantitatively different between adult and newborn or fetus.