Glycosylation, a commonly occurring post-translational modification, is highly expressed in several tumors, specifically in those of the digestive system, and plays a role in various cellular pathophysiological mechanisms. Although the importance and detection methods of glycosylation in digestive system tumors have garnered increasing attention in recent years, bibliometric analysis of this field remains scarce. The present study aims to identify the developmental trends and research hotspots of glycosylation in digestive system tumors.

To find and identify the developmental trends and research hotspots of glycosylation in digestive system tumors.

We obtained relevant literature from the Web of Science Core Collection and employed VOSviewer 1.6.19 and CiteSpace (version 6.1.R6) to perform bibliometric analysis.

A total of 2042 documents spanning from 1978 to the present were analyzed, with the research process divided into three phases: the period of obscurity (1978-1990), continuous development period (1991-2006), and the rapid outbreak period (2007-2023). These documents were authored by researchers from 66 countries or regions, with the United States and China leading in terms of publication output. Reis Celso A had the highest number of publications, while Pinho SS was the most cited author. Co-occurrence analysis revealed the most popular keywords in this field are glycosylation, expression, cancer, colorectal cancer, and pancreatic cancer. Furthermore, the Journal of Proteome Research was the most prolific journal in terms of publications, while the Journal of Biological Chemistry had the most citations.

The bibliometric analysis shows current research focus is primarily on basic research in this field. However, future research should aim to utilize glycosylation as a target for treating tumor patients.

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers and is projected to soon be the second leading cause of cancer death. Median survival of PDA patients is 6-10 mo, with the majority of diagnoses occurring at later, metastatic stages that are refractory to treatment and accompanied by worsening prognoses. Glycosylation is one of the most common types of post-translational modifications. The complex landscape of glycosylation produces an extensive repertoire of glycan moieties, glycoproteins, and glycolipids, thus adding a dynamic and tunable level of intra- and intercellular signaling regulation. Aberrant glycosylation is a feature of cancer progression and influences a broad range of signaling pathways to promote disease onset and progression. However, despite being so common, the functional consequences of altered glycosylation and their potential as therapeutic targets remain poorly understood and vastly understudied in the context of PDA. In this review, the functionality of glycans as they contribute to hallmarks of PDA are highlighted as active regulators of disease onset, tumor progression, metastatic capability, therapeutic resistance, and remodeling of the tumor immune microenvironment. A deeper understanding of the functional consequences of altered glycosylation will facilitate future hypothesis-driven studies and identify novel therapeutic strategies in PDA.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths globally, and the incidence rate in the United States is increasing. Studies have identified inter- and intratumor heterogeneity as histologic and/or molecular subtypes/variants associated with response to certain molecular targeted therapies. Spatial HCC tissue profiling of N-linked glycosylation by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) may serve as a new method to evaluate the tumor heterogeneity. Previous work has identified significant changes in the N-linked glycosylation of HCC tumors but has not accounted for the heterogeneous genetic and molecular nature of HCC. To determine the correlation between HCC-specific N-glycosylation changes and genetic/molecular tumor features, we profiled HCC tissue samples with MALDI-IMS and correlated the spatial N-glycosylation with a widely used HCC molecular classification (Hoshida subtypes). MALDI-IMS data displayed trends that could approximately distinguish between subtypes, with subtype 1 demonstrating significantly dysregulated N-glycosylation versus adjacent nontumor tissue. Although there were no individual N-glycan structures that could identify specific subtypes, trends emerged regarding the correlation of branched glycan expression to HCC as a whole and fucosylated glycan expression to subtype 1 tumors specifically. IMPLICATIONS: Correlating N-glycosylation to specific subtypes offers the specific detection of subtypes of HCC, which could both enhance early HCC sensitivity and guide targeted clinical therapies.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Glycans are essential biomolecules in regulating human physiology and pathology ranging from signal transduction to microbial infections. Developing complex human diseases, such as cancer, diabetes, and cardiovascular diseases, are a combination of genetic and environmental factors. Genetics dominates embryonic development and the passing of genes to the next generation whereas the information in glycans reflects the impact of internal and external environmental factors, such as diseases, lifestyle, and social factors, on a person's health and disease. The reason behind this is that glycans are not directly encoded in a genetic template. Instead, they are assembled dynamically by hundreds of enzymes organized in more than 10 complex biosynthetic pathways. Any environmental changes affecting enzymatic activities or the availability of high-energy monosaccharide donors in a specific location will disturb the final structure of glycans. The glycan structure-dependent biological activities subsequently enable or disable gene expressions, which partially explain that it is difficult to pinpoint specific genetic defects to aging-associated diseases. Glycan-based biomarkers are currently used for diagnosis of diabetes, cancers, and other complex diseases. We will recapitulate the discovery of glucose, glycated proteins, glycan-, and glycoprotein-based biomarkers followed by summarizing clinically used glycan/glycoprotein-based biomarkers. The potential serum/plasma-derived N- and O-linked glycans as biomarkers will also be discussed.

Normal human physiology is dependent on a tight control of the fasting blood glucose (FBG) levels. The islets of pancreas maintains FBG levels within a narrow range of 4-6mmol/L by secreting various hormones, especially insulin and glucagon. However, the hormone secretions by the islets of pancreas are governed by a collective effort among pancreas-islet axis, brain-islet axis, liver-islet axis, gut-islet axis, and adipocyte/myocyte-islet axis. Furthermore, the damage of pancreas, vascular system, brain, liver, intestine, adipose, muscle, and other organs and tissues might affect FBG levels through insulin resistance or impaired insulin signaling, which is the hallmark of type 2 diabetes. In this study, 320,572 clinical lab test results of FBG levels from healthy individuals and patients with 64 different types of diseases during the past 5 years in our hospital were retrieved and analyzed. Based on the mean (SD), median, and p (-Log₁₀p) values, we found 57/64 diseases including type 2 diabetes, pancreatitis, diabetic nephropathy, and pancreatic cancer had significantly (p<0.05, -Log₁₀p>1.30) increased whereas 6/64 diseases including preeclampsia, Wilms' tumor, and lupus erythematous had significantly decreased FBG levels compared to that of healthy controls. These data indicated that the increased FBG levels might be a general pathophysiological property of diseased tissues or organs and the increased FBG levels might be a consequence but not the cause for either prediabetes or type 2 diabetes.

Sialic acids are cytoprotectors, mainly localized on the surface of cell membranes with multiple and outstanding cell biological functions. The history of their structural analysis, occurrence, and functions is fascinating and described in this review. Reports from different researchers on apparently similar substances from a variety of biological materials led to the identification of a 9-carbon monosaccharide, which in 1957 was designated "sialic acid." The most frequently occurring member of the sialic acid family is N-acetylneuraminic acid, followed by N-glycolylneuraminic acid and O-acetylated derivatives, and up to now over about 80 neuraminic acid derivatives have been described. They appeared first in the animal kingdom, ranging from echinoderms up to higher animals, in many microorganisms, and are also expressed in insects, but are absent in higher plants. Sialic acids are masks and ligands and play as such dual roles in biology. Their involvement in immunology and tumor biology, as well as in hereditary diseases, cannot be underestimated. N-Glycolylneuraminic acid is very special, as this sugar cannot be expressed by humans, but is a xenoantigen with pathogenetic potential. Sialidases (neuraminidases), which liberate sialic acids from cellular compounds, had been known from very early on from studies with influenza viruses. Sialyltransferases, which are responsible for the sialylation of glycans and elongation of polysialic acids, are studied because of their significance in development and, for instance, in cancer. As more information about the functions in health and disease is acquired, the use of sialic acids in the treatment of diseases is also envisaged.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography-mass spectrometry and nano liquid chromatography-tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery. Graphical abstract ᅟ.

Gastric cancer has one of the highest cancer mortality rates worldwide, largely because of difficulties in early-stage detection. Aberrant glycosylation in serum proteins is associated with many human diseases including inflammation and various types of cancer. Serum-based global glycan profiling using mass spectrometry has been explored and has already led to several potential glycan markers for several disease states. However, localization of the aberrant glycosylation is desirable in order to improve the specificity and sensitivity for clinical use. Here, we combined protein-specific immunoaffinity purification, glycan release, and MS analysis to examine haptoglobin glycosylation of gastric cancer patients for glyco-markers. Age- and sex-matched 60 serum samples (30 cancer patients and 30 healthy controls) were used to profile and quantify haptoglobin N-glycans. A T-test based statistical analysis was performed to identify potential glyco-markers for gastric cancer. Interestingly, abundances of several tri- and tetra-antennary fucosylated N-glycans were increased in gastric cancer patients. Additionally, structural analysis via LC/MS/MS indicated that the fucosylated complex type N-glycans were primarily decorated with antenna fucose, which can be categorized as sialyl-Le^a or sialyl-Le^x type structures. This platform demonstrates quantitative, structure-specific profiling of haptoglobin glycosylation for the purposes of biomarker discovery for gastric cancer.

The glycocalyx is a coating of protein and sugar on the surface of all living cells. Dramatic perturbations to the composition and structure of the glycocalyx are frequently observed in aggressive cancers. However, tools to experimentally mimic and model the cancer-specific glycocalyx remain limited. Here, we develop a genetically encoded toolkit to engineer the chemical and physical structure of the cellular glycocalyx. By manipulating the glycocalyx structure, we are able to switch the adhesive state of cells from strongly adherent to fully detached. Surprisingly, we find that a thick and dense glycocalyx with high O-glycan content promotes cell survival even in a suspended state, characteristic of circulating tumor cells during metastatic dissemination. Our data suggest that glycocalyx-mediated survival is largely independent of receptor tyrosine kinase and mitogen activated kinase signaling. While anchorage is still required for proliferation, we find that cells with a thick glycocalyx can dynamically attach to a matrix scaffold, undergo cellular division, and quickly disassociate again into a suspended state. Together, our technology provides a needed toolkit for engineering the glycocalyx in glycobiology and cancer research.

Simple and complex carbohydrates (glycans) have long been known to play major metabolic, structural and physical roles in biological systems. Targeted microbial binding to host glycans has also been studied for decades. But such biological roles can only explain some of the remarkable complexity and organismal diversity of glycans in nature. Reviewing the subject about two decades ago, one could find very few clear-cut instances of glycan-recognition-specific biological roles of glycans that were of intrinsic value to the organism expressing them. In striking contrast there is now a profusion of examples, such that this updated review cannot be comprehensive. Instead, a historical overview is presented, broad principles outlined and a few examples cited, representing diverse types of roles, mediated by various glycan classes, in different evolutionary lineages. What remains unchanged is the fact that while all theories regarding biological roles of glycans are supported by compelling evidence, exceptions to each can be found. In retrospect, this is not surprising. Complex and diverse glycans appear to be ubiquitous to all cells in nature, and essential to all life forms. Thus, >3 billion years of evolution consistently generated organisms that use these molecules for many key biological roles, even while sometimes coopting them for minor functions. In this respect, glycans are no different from other major macromolecular building blocks of life (nucleic acids, proteins and lipids), simply more rapidly evolving and complex. It is time for the diverse functional roles of glycans to be fully incorporated into the mainstream of biological sciences.

Neoplastic transformation results in a wide variety of cellular alterations that impact the growth, survival, and general behavior of affected tissue. Although genetic alterations underpin the development of neoplastic disease, epigenetic changes can exert an equally significant effect on neoplastic transformation. Among neoplasia-associated epigenetic alterations, changes in cellular glycosylation have recently received attention as a key component of neoplastic progression. Alterations in glycosylation appear to not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Many of these changes may support neoplastic progression, and unique alterations in tumor-associated glycosylation may also serve as a distinct feature of cancer cells and therefore provide novel diagnostic and even therapeutic targets.

Mucin-type O-glycans are a class of glycans initiated with N-acetylgalactosamine (GalNAc) α-linked primarily to Ser/Thr residues within glycoproteins and often extended or branched by sugars or saccharides. Most secretory and membrane-bound proteins receive this modification, which is important in regulating many biological processes. Alterations in mucin-type O-glycans have been described across tumor types and include expression of relatively small-sized, truncated O-glycans and altered terminal structures, both of which are associated with patient prognosis. New discoveries in the identity and expression of tumor-associated O-glycans are providing new avenues for tumor detection and treatment. This chapter describes mucin-type O-glycan biosynthesis, altered mucin-type O-glycans in primary tumors, including mechanisms for structural changes and contributions to the tumor phenotype, and clinical approaches to detect and target altered O-glycans for cancer treatment and management.

Microbial communities coexisting with humans are collectively known as microbiome. It influences almost every aspect of an individual's body function. Microbiome is idiosyncratic for body condition and its alteration is indicative for several abnormalities. This article discusses about recent ideas for developing microbiology based cancer indicators using alterations in microbiome. It is noteworthy that large exploratory studies are required to identify cancer indicator microorganisms from complex and diverse microbiome constituents. This complexity also warrants that these markers should be used in conjunction with other routine cancer indicators. The present article concludes that such studies can spur development of novel microbiome based cancer diagnostics.

The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT.

As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining.

Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific sialylated structures. These results provide leads for targeting aggressive and drug resistant forms of pancreatic cancer cells.

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.

Peanut agglutinin (PNA) recognizes tumor-associated carbohydrates. In this study, we aimed to identify the core protein harboring PNA-binding sugars in the human lung and to explore the relationship with the pathology of primary non-small cell lung cancers (NSCLC).

PNA lectin blotting was used to detect PNA-binding proteins in the microsomal fraction of lung tissue from 24 patients with NSCLC. The 55- to 65-kDa core peptide PNA-binding protein was characterized by enzymatic treatment and identified by immunoprecipitation and affinity chromatography. The expression level and increase in size of the 55- to 65-kDa PNA-binding protein/decay-accelerating factor (DAF) were compared between normal and tumor regions of the tumor tissue by Western blotting and quantitative PCR.

The 55- to 65-kDa PNA-binding protein was observed in human lung. This was a glycosylphosphatidylinositol-anchored membrane protein carrying O-linked carbohydrates. This core protein was identified as DAF, one of the complementary regulatory proteins. DAF was enlarged to 65 to 75 kDa in NSCLC tumor lesions due to sialylation in the sugar moiety. At the transcription level, DAF levels were significantly lower in tumor regions, suggesting its down-regulation in NSCLC cells.

DAF was identified as a new PNA-binding protein in the human lung. The down-regulation and heavy sialylation of DAF was associated with pathology in NSCLC, and these alterations make this protein a potential marker for NSCLC.

Ovarian cancer is the leading cause of death due to gynecological malignancies. The aim of our study was to investigate the status of circulating glycoprotein levels in ovarian cancer patients.

Thirty ovarian cancer patients and an equal number of age-matched, apparently healthy subjects as controls were involved in the study. Glycoprotein levels, as indicated by the concentration of plasma total sialic acid, protein-bound hexoses, hexosamine and fucose were estimated in circulation of both the ovarian cancer patients and controls.

Significantly elevated levels of plasma total sialic acid, protein-bound hexoses, hexosamine and fucose were observed in ovarian cancer patients as compared to the apparently healthy controls.

Plasma total sialic acid, protein-bound hexoses, hexosamine and fucose in the circulation of ovarian cancer patients are markedly elevated and the increase in these carbohydrate moieties of glycoproteins reflect the stage of cancer and may be an additional tool in the diagnosis and prognosis of ovarian carcinoma.

Beta-1,4-galactosyltransferase (beta-1,4-GalT) V is a constitutively expressed enzyme that can effectively galactosylate the GlcNAcbeta1-->6Man group of the highly branched N-glycans that are characteristic of tumor cells. Upon malignant transformation of cells, the expression of the beta-1,4-GalT V gene increases in accordance with the increase in the amounts of highly branched N-glycans. Lectin blot analysis showed that the galactosylation of highly branched N-glycans is inhibited significantly in SH-SY5Y human neuroblastoma cells by the transfection of the antisense beta-1,4-GalT V cDNA, indicating the biological importance of the beta-1,4-GalT V for the functions of highly branched N-glycans. We cloned the 2.3-kb 5'-flanking region of the human beta-1,4-GalT V gene, and we identified the region -116/-18 relative to the transcription start site as that having promoter activity. The region was found to contain several putative binding sites for transcription factors, including AP2, AP4, N-Myc, Sp1, and upstream stimulatory factor. Electrophoretic mobility shift assay showed that Sp1 binds to nucleotide positions -81/-69 of the promoter region. Mutations induced in the Sp1-binding site showed that the promoter activity of the beta-1,4-GalT V gene is impaired completely in cancer cells. In contrast, the promoter activity increased significantly by the transfection of the Sp1 cDNA into A549 human lung carcinoma cells. Mithramycin A, which inhibits the binding of Sp1 to its binding site, reduced the promoter activation and expression of the beta-1,4-GalT V gene in A549 cells. These results indicate that Sp1 plays an essential role in the transcriptional activity of the beta-1,4-GalT V gene in cancer cells.

The four essential building blocks of cells are proteins, nucleic acids, lipids, and glycans. Also referred to as carbohydrates, glycans are composed of saccharides that are typically linked to lipids and proteins in the secretory pathway. Glycans are highly abundant and diverse biopolymers, yet their functions have remained relatively obscure. This is changing with the advent of genetic reagents and techniques that in the past decade have uncovered many essential roles of specific glycan linkages in living organisms. Glycans appear to modulate biological processes in the development and function of multiple physiologic systems, in part by regulating protein-protein and cell-cell interactions. Moreover, dysregulation of glycan synthesis represents the etiology for a growing number of human genetic diseases. The study of glycans, known as glycobiology, has entered an era of renaissance that coincides with the acquisition of complete genome sequences for multiple organisms and an increased focus upon how posttranslational modifications to protein contribute to the complexity of events mediating normal and disease physiology. Glycan production and modification comprise an estimated 1% of genes in the mammalian genome. Many of these genes encode enzymes termed glycosyltransferases and glycosidases that reside in the Golgi apparatus where they play the major role in constructing the glycan repertoire that is found at the cell surface and among extracellular compartments. We present a review of the recently established functions of glycan structures in the context of mammalian genetic studies focused upon the mouse and human species. Nothing tends so much to the advancement of knowledge as the application of a new instrument. The native intellectual powers of men in different times are not so much the causes of the different success of their labours, as the peculiar nature of the means and artificial resources in their possession. T. Hager: Force of Nature (1)

It is generally believed that the lower the grade of differentiation of glycoprotein-producing cells, the more often modification by bisecting N-acetylglucosamine (GlcNAc) or fucose (Fuc) at the sugar chain of the glycoprotein or increase in branching of side chains occurs. We examined the characteristics of the alpha-fetoprotein (AFP) sugar chain stored in amniotic and exocoelomic fluid during 5-9 weeks of gestation and analyzed serum-derived AFP of patients with germ cell tumors.

Total AFP concentrations in embryonic fluid at 5-9 weeks of gestation (n = 11) and serum of patients with germ cell tumors (n = 7) were measured using a radioimmunoassay (RIA) method. The percentages of AFPs reactive with Lens culinaris agglutinin (LCA), concanavalin A (Con A), erythroagglutinating phytohemagglutinin-E4 (E-PHA) and Ricinus communis agglutinin-120 (RCA 120) were obtained by lectin-affinity electrophoresis coupled with antibody-affinity blotting.

It was revealed that at 5-9 weeks of gestation, AFP variants that had been modified by the Fuc residue, which bound to the GlcNAc residue at the reducing end of the sugar chain, and bisecting GlcNAc residues gradually decreased as pregnancy advanced; however, the presence of N-acetylneuraminic acid (Neu5Ac) at the nonreducing ends changed little.

It appears very likely that the changes in the relative amounts of AFP variants in the embryonic fluid during early pregnancy were due to differentiation of the yolk sac. The grade of differentiation of yolk sac tumors was very similar to that of the normal yolk sac at around 6 weeks of gestation.

An association between protein glycosylation and tumorigenesis has been recognized for over 10 years. Associations linking the importance of glycosylation events to tumor biology, especially the progression to metastatic disease, have been noted over many years, Recently, a mouse model in which beta1,6-N-acetylglucosaminyltransferase V (a rate-limiting enzyme in the N-glycan pathway) has been knocked out, was used to demonstrate the importance of glycosylation in tumor progression. By crossing mice lacking this enzyme with a transgenic mouse model of metastatic breast cancer, metastatic progression of the disease was dramatically reduced. These experiments provide in vivo evidence for the role of N-linked glycosylation in metastatic breast cancer and have significant implications for the development of new treatment strategies.

Finding of the deletion phenomenon of certain oligosaccharides in human milk and its correlation to the blood types of the donors opened a way to elucidate the biochemical basis of blood types in man. This success led to the idea of establishing reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins. N-Linked sugar chains were first released quantitatively as oligosaccharides by enzymatic and chemical means, and labelled by reduction with NaB3H4. After fractionation, structures of the radioactive oligosaccharides were determined by a series of methods developed for the studies of milk oligosaccharides. By using such techniques, structural rules hidden in the N-linked sugar chains, and organ- and species-specific N-glycosylation of glycoproteins, which afforded a firm basis to the development of glycobiology, were elucidated. Finding of galactose deficiency in the N-linked sugar chains of serum IgG from patients with rheumatoid arthritis, and malignant alteration of N-glycosylation in various tumors opened a new research world called glycopathology. However, recent studies revealed that several structural exceptions occur in the sugar chains of particular glycoproteins. Finding of the occurrence of the Galbeta1-4Fucalpha1- group linked at the C-6 position of the proximal N-acetylglucosamine residue of the hybrid type sugar chains of octopus rhodopsin is one of such examples. This finding indicated that the fucosyl residue of the fucosylated trimannosyl core should no more be considered as a stop signal as has long been believed. Furthermore, recent studies on dystroglycan revealed that the sugar chains, which do not fall into the current classification of N and O-linked sugar chains, are essential for the expression of the functional role of this glycoprotein. It was found that expression of many glycoproteins is altered by aging. Among the alterations of the glycoprotein patterns found in the brain nervous system, the most prominent evidence was found in P0. This protein is produced in non-glycosylated form in the spinal cord of young mammals. However, it starts to be N-glycosylated in the spinal cord of aged animals. These evidences indicate that various unusual sugar chains occur as minor components in mammals, and play important roles in particular tissues.

Recent evidence demonstrates that the changes in the size of N-linked oligosaccharides that correlate with cell transformation and tumorigenicity are due at least in part to the regulation of expression of a glycosyltransferase involved in the branching of N-linked structures, N-acetylglucosaminyltransferase V or GlcNAc-T V. Studies have shown that the increases in GlcNAc-T V expression after oncogenic transformation are most likely caused by direct effects on the GlcNAc-T V promoter by the Ets family of transcriptional activators, which are up-regulated by a cellular proliferation signaling pathway. This pathway begins with growth factor receptors that activate tyrosine kinases at the cell surface and proceeds through src, ras, and raf. Additional evidence for the association between cellular proliferation and GlcNAc-T V expression will be presented, as well as a discussion of the effects of beta(1,6) branching on several of the phenotypes of oncogenically transformed cells, including metastatic potential.

The aim of this study was to investigate plasma membrane fluidity in human keratinocytes using fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its cationic derivative 1-[4-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Keratinocytes from normal or psoriatic skin were isolated using trypsin-EDTA or dispase. In keratinocytes isolated from normal skin, TMA-DPH anisotropy values were higher than those observed using DPH; the difference must be related to the different localization of the two probes. In fact, DPH in whole cells localizes in plasma as well as intracellular membranes, yielding an average value of fluidity, while the cationic derivative TMA-DPH resides in the plasma membrane of the whole cells for a sufficient time for anisotropy measurements. Moreover, it has to be considered that plasma membrane is more ordered than intracellular membranes. The kinetics of incorporation of TMA-DPH was similar in keratinocytes isolated using trypsin-EDTA and those isolated using; dispase, however, the fluorescence anisotropy values were lower in keratinocytes isolated with dispase (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.029). This difference is probably related to modifications of lipid-protein interactions after trypsin treatment. Since no damage to plasma membrane after incubation with dispase seems to have been reported, we decided to use this separation procedure to study plasma membrane fluidity in psoriasis, a human pathological condition characterized by excessive cell proliferation and incomplete differentiation. Lower anisotropy values (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.001), indicating an increase in fluidity, were observed in keratinocytes isolated from skin of psoriatic patients than in epidermal cells isolated from normal human skin. We suggest that the measurement of fluorescence anisotropy in living cells is a convenient and useful tool to study membrane fluidity in human keratinocytes isolated from normal and diseased skin. Its application represents a technical advance because plasma membrane fluidity can be measured using very limited amounts of tissue, as obtained from biopsies.

AchatininH (ATNH) is a lectin, isolated from the hemolymph of Achatina fulica snail, which has been shown to have narrow specificity towards 9-O-acetyl sialic acid. Usually ATNH does not agglutinate normal human erythrocytes, however, it is capable of agglutinating erythrocytes of patients suffering from acute lymphocytic and acute myelogenous leukemia. Determination of binding constants, numbers of binding sites and lectin overlay experiments using patients' erythrocytes ghost, have suggested that some alterations in erythrocyte cell surface sialoglycoproteins or more precisely appearance of some O-acetylated sialoglycoprotein as a result of pathological transformations has caused this change in the binding of ATNH.

Serum sialic acid (N-acetylneuraminic acid) was evaluated as a tumour marker for prostate cancer and compared with serum prostate specific antigen (PSA). The records of 35 patients suffering from prostate cancer (9 with bone metastases) were analysed and compared with those of 21 healthy individuals. Total serum sialic acid was significantly elevated among the cancer patients. Levels in patients with distant metastases were significantly higher than in those with locally restricted disease and normal individuals, whereas no such difference was seen between the latter 2 groups. A direct association between serum sialic acid and tumour T category and grade could not be established. The difference between the cancer and the control groups was reflected more significantly by PSA. As sialic acid lacks tumour specificity, it is not helpful in screening for prostate cancer, yet might contribute towards the early detection of tumour progression and metastases during both therapy and follow-up.

Glycoproteins are widely distributed among species in soluble and membrane-bound forms, associated with many different functions. The heterogenous sugar moieties of glycoproteins are assembled in the endoplasmic reticulum and in the Golgi and are implicated in many roles that require further elucidation. Glycoprotein-bound oligosaccharides show significant changes in their structures and relative occurrences during growth, development, and differentiation. Diverse alterations of these carbohydrate chains occur in diseases such as cancer, metastasis, leukemia, inflammatory, and other diseases. Structural alterations may correlate with activities of glycosyltransferases that assemble glycans, but often the biochemical origin of these changes remains unclear. This suggests a multitude of biosynthetic control mechanisms that are functional in vivo but have not yet been unraveled by in vitro studies. The multitude of carbohydrate alterations observed in disease states may not be the primary cause but may reflect the growth and biochemical activity of the affected cell. However, knowledge of the control mechanisms in the biosynthesis of glycoprotein glycans may be helpful in understanding, diagnosing, and treating disease.

Altered glycosylation is widely observed in glycoproteins produced by tumors. One of the most consistently observed alterations is the increase of larger asparagine-linked sugar chains in the plasma membrane glycoproteins. This phenomenon is brought about by the increase of N-acetylglucosaminyltransferase V, which is responsible for the formation of the GlcNAc beta 1----6Man alpha-1----6 group. The enrichment of the complex-type sugar chains containing the -GlcNAc beta 1----6(-GlcNAc beta 1----2)Man alpha 1----6 group is correlated with tumorigenicity and metastasic potential of tumor cells. Comparative study of the sugar chains of human chorionic gonadotropin isolated from the urine of pregnant women and of patients with trophoblastic diseases including choriocarcinoma revealed that many new oligosaccharides are included in the tumor hCG. The altered glycosylation of hCG is brought about by the ectopic expression of N-acetylglucosaminyltransferase IV. With use of this altered glycosylation, a novel method useful for the diagnosis of choriocarcinoma was established.

One of the characteristics of malignant tumor cells is the production of glycocompounds with a high content of N-acetylneuraminic acid (sialic acid). Total sialic acid concentration was determined in the serum of 136 patients with an intracranial tumor. The concentration was determined enzymatically and using HPLC. Both methods had a relatively high specificity (90%), provided an inflammatory process due to an infection could be excluded. Sensitivity was 72.6%; the cut-off level of sialic acid concentration was determined to be 2.75 mumol/ml. There was a significant difference in the average sialic acid concentration of benign and malignant tumors. The test was least reliable in determining whether the growth of an astrocytoma is malignant.

Since the discovery of the "Clostridium tetani phenomenon", various apathogenic clostridia have been used for tumour lysis. Experiments have been conducted to achieve a tumour diagnosis using radiolabelled antibodies to clostridia. In addition, a method has been described that distinguishes, with variable success, between healthy and tumour-carrying animals by means of hemagglutination. The method outlined here uses the fact that malignant cells produce a multitude of sialic acid compounds which lie on the cell membrane and are also connected to the lipid layer of the tumour cell membrane. The apathogenic Clostridium oncolyticum M55 only germinates and multiplies in the malignant tumour tissue. Thus; bacterial hydrolases can enter the tumour tissue and lead to oncolysis. Subsequently the glycocompounds which can be detected by means of an enzymatic determination of the concentration of neuraminic acid (one of the sialic acids) in the serum are washed out into the peripheral blood. We observed these processes in mice in the Ehrlich ascites solid carcinoma and in the Lewis lung carcinoma. Using this method it was possible to detect tumour growth at an early stage with impressive accuracy. The Lewis lung carcinoma which secretes only small amounts of sialic acid glycocompounds cannot be distinguished from the control group by determination of sialic acid concentration. It was possible to detect a 52% increase in the amount of sialic acid after administration of spores of clostridia. This method makes it possible to increase the tumour marker sialic acid through manipulation of the tumour, using apathogenic clostridia, and to measure of sialic acid concentration as an indicator of the metabolic products of the tumour.

Galactosyltransferase (GT) belongs to the glycosyltransferases. In several tissues and cell lines, the enzyme is localized by immunocytochemistry to the two to three trans cisternae of the Golgi complex and may thus be considered a specific membrane component of this type of endomembrane. As a consequence, it is the most common Golgi "marker" enzyme in cell fractionation studies. Study of its biosynthesis, membrane orientation, and turnover in several tissues and cultured cell lines has broadened our knowledge about Golgi function itself. The enzyme is oriented towards the lumen of the cisternal space. In this orientation, it catalyzes the transfer of galactose to glycoprotein-bound acetylglucosamine and, in the presence of alpha-lactalbumin, to glucose, as shown in the Golgi complex of mammary gland epithelial cells. The enzymatic properties of GT are well known. The metabolism of GT has been extensively studied in HeLa and human hepatoma cells. The enzyme is synthesized in the rough endoplasmic reticulum (RER) and provided with one N-linked oligosaccharide and palmitate residues. In the Golgi complex, terminal sugars are attached to the N-linked oligosaccharide and extensive O-glycosylation takes place. The half-life of the enzyme is about 20 hr, after which a soluble form appears in the culture medium. Release of GT into the medium is observed in all cell lines studied. This phenomenon is in accordance with the presence of soluble GT in body fluids such as serum, ascites, milk, and saliva. In patients suffering from ovarian and breast cancer, increased levels of GT enzyme activity have been reported. Whether extracellular GT is of biological significance is still a point of discussion.

Carbohydrate moieties were released from human thyroglobulins prepared from normal and transformed, thyroid tissues by hydrazinolysis. The oligosaccharides thus prepared were fractionated by DEAE-cellulose, Bio-Gel P-4, concanavalin-A--Sepharose and Ricinus-communis-agglutinin--Sepharose columns and were characterized by exo-glycosidase and endo-glycosidase digestions, methylation analysis and 400-MHz 1H-NMR spectroscopy. These studies showed that the alterations accompanying malignancy occurred in complex-type carbohydrate chains and exhibited the following structural features: (a) a complex-type carbohydrate chain containing 1 phosphate in a diester linkage was present in all the malignant thyroglobulins. (b) Asialo-carbohydrate chains with biosynthetically incomplete structures were found in all of the malignant thyroglobulins. (c) Carbohydrate chains of higher molecular mass, which have repeating Gal-GlcNAc disaccharides and peripheral alpha-fucosyl residues were present in metastatic papillary carcinoma.

The species-specific and the interspecies cross-reactive melanoma antigenic determinants are defined by the monoclonal antibodies raised by syngeneic immunizations. The two types of monoclonal antibodies (M562 or M622 and M2590) were obtained by the fusion of P3U1 murine myeloma cell lines and spleen cells of C57BL/6 mice hyperimmunized with MMC-treated syngeneic B16 melanoma cells. The M2590 antibody recognizes the cross-species melanoma determinant commonly shared among at least mouse, hamster, and human, while the M562 or M622 antibody reacts with the mouse (B16) melanoma antigenic determinant. The immunochemical and physiochemical characteristics of the melanoma antigens on SDS-PAGE analyses show that these two characteristic determinants are present on the same molecule (molecular weight of 31,000) of a glycoprotein. Furthermore, the interspecies cross-reactive melanoma antigenic determinants are possibly composed of the sugar moiety, whereas the species-specific determinants seem to be proteinaceous in nature.

Adenylate cyclase activity was measured in plasma membranes isolated from Morris Hepatoma 7800 and from control and host livers. The only difference found in tumor enzyme activity was the lack of response to glucagon. The membrane-binding capacities for the pancreatic hormones insulin and glucagon were measured. Hepatoma membranes did not bind glucagon. Insulin-binding parameters could not be determined because of high non-specific binding. The plasma levels of insulin in the tumor-bearing animals were approximately half of those found in controls, whereas the glucagon levels in plasma were 50% higher in tumor-bearing animals. Thyroxine and triiodothyronine plasma levels were reduced in tumor-bearing rats, while the thyroid-stimulating hormone level was within normal limits. The amount of cAMP (275 pmol g-1) and cGMP (3.6 pmol g-1) in the tumor were lower than in the host and control livers, but the ratio of cGMP to cAMP in the tumor was increased by a factor of 2. These results are discussed with respect to control mechanisms of cell proliferation in comparison with other hepato-proliferative states.

With [3H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellular/cytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these returned glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.