SIALIC ACID O-ACETYLATION IN BLOOD CELL INTERACTIONS
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 1P01HL057345-01A10004 from National Heart, Lung, And Blood Institute
Abstract: Sialic acid (Sia) residues of N-linked sugar chains on blood and vascular proteins are usually attached in alpha2-6 or alpha2-3 linkages, and some molecules are selectively O-acetylated at the 9- position. This diversity can affect recognition of Sia by certain vascular lectins in vitro. We will elucidate the in vivo biological significance of these findings by manipulation of certain genes in the mouse. Sialic acid O-acetyltransferase(s) have been difficult to purify or clone. We have isolated cDNA and genomic clones of a mouse esterase (Lse) that selectively removes 9-O-acetyl groups from Sias. Expression of Lse is highly regulated e.g., in murine hematopoietic development. 9-O-acetylation also occurs preferentially on the sequence Siaalpha2-6Galbeta1-4GlcNAc, whose expression is determined by the alpha2-6 sialyltransferase gene (ST6Gal I). This gene is regulated by differential action of multiple promoters in cells such as hepatocytes, endothelial cells, erythrocytes and lymphocytes. Thus, alterations in activity of ST6Gal I and/or the competing alpha2-3 sialyltransferases (ST3Gal III and IV) may also alter 9-O-acetylation. To elucidate the in vivo regulation of 9-O-acetylation and to pursue specific hypotheses about its roles,we will do the following 1. Eliminate expression of the Lse gene systemically, and/or selectively in the cell types listed above. 2. Alter ratios of alpha2-6 and alpha2-3 Sia by disrupting expression of alpha2-6 and alpha2-3 sialyltransferases (Collaboration with Project 1) and by "knocking in" the cDNA for ST3Gal III into the endogenous ST6Gal I gene, disrupting the latter. In the latter mice, alpha2-6 and alpha2-3 Sia linkage ratios should be inverted on N-glycans. 3. Elucidate the changes in 9-O- acetylation occurring in these mice, and structural and functional consequences of these genetic manipulations. Changes will be detected in situ by immunohistology using specific probes for sialylation and O-acetylation, and directly demonstrated by chemical analyses. Particular attention will be directed towards hepatocytes, endothelial cells, erythrocytes and lymphocytes. Biological effects upon interactions involving the Sia-binding lectins H protein, CD22, CD33 and Sialoadhesin will be studied (some in collaboration with Projects 1 and 3). The sensitivity of red cells to complement and the turnover of red cells and plasma proteins will also be assessed. Selected aspects of blood coagulation, hematopoiesis, and the distribution and function of lymphocytes will also be examined. These studies will help to define the in vivo regulation and biological roles of Sia O-acetylation in the blood and vascular systems.
Keywords: acetylation, cell cell interaction, gene expression, lectin, sialate, sialyltransferase, chemical binding, complement, developmental genetics, erythrocyte, genetic regulatory element, hematopoiesis, lymphocyte, in situ hybridization, laboratory mouse, transgenic animal
Sponsored Links Excellgen http://Excellgen.com
GENETIC MODULATION OF BLOOD AND VASCULAR GLYCOSYLATION
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 1P01HL057345-01A1 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Oligosaccharides such as the N- and 0-linked chains of glycoproteins and the heparan sulfate chains of proteoglycans are major structural components of cells and tissues, with a complexity rivaling that of nucleic acids and proteins. This Program is focused upon exploring the biological roles in the blood and vasculature of two anionic modifications of these sugar chains sialic acid and N-sulfation. The structures of the N- and 0-linked sugar chains of many plasma glycoproteins and blood cells and are well known. Specific lectins such as the selectins (on leucocytes, platelets and endothelium), the I-type lectins (on B cells, macrophages or myeloid precursors), and the H protein of the complement pathway, can differentially recognize these chains in the context of their sialic acid residues. The N-sulfate esters of heparan sulfate glycosaminoglycan chains are recognized by various proteins involved in blood coagulation, cell growth, platelet interactions and leucocyte migration. Despite many clues, most physiologic roles of sialylation and N-sulfation are not well observed in cultured cells, and must be explored in the intact organism. However, these features of mammalian glycosylation are not well represented in model invertebrates, and few genetic defects affecting these pathways have been described in intact mammals. Our labs have been among the first to genetically dissect the in vivo roles of glycosylation in the mouse and to develop new technologies for efficient analysis of oligosaccharides from mouse tissues. Thus, the central theme of this proposal is the genetic manipulation of sialic acids and N-sulfate esters in the intact mouse. When required, we will use our recently developed method to selectively inactivate mouse genes in a cell type-specific manner. Replacement of wild type alleles with recombinant ones carrying loxP target sites at innocuous positions allows selective gene deletion, by mating with mice transgenic for Cre recombinase constructs driven by specific transcriptional control sequences. This allows specific focus on blood cells, endothelium and plasma proteins, and will be important in instances where systemic gene inactivation models are non-viable. We also have the necessary expertise to analyze the consequences of the genetic manipulations on the structure of hematopoietic, vascular and lymphoid tissues, the mechanisms of hemostasis, the structure of the oligosaccharides, and the functional consequences to hematopoiesis and lymphocyte biology. These studies are expected to reveal many important functions for oligosaccharides in health and disease.
Keywords: carbohydrate structure, glycosylation, hematology, immunogenetics
Project start date: 1997-09-30
Project end date: 2002-08-31
1P01HL057345-01A1 (1997): $1458161
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 1P01HL057345-01A19005 from National Heart, Lung, And Blood Institute
Abstract: All of the projects within this Program, require the analysis of embryonic and/or adult murine tissues. The plan is to use the Tissue Core facilitates of the UCSD Cancer Center for routine histology, immunohistochemistry or in situ hybridization to analyse the various tissues obtained from the different projects. The facility is already set up and most of the major equipment is in place. Some methodologies will be modified or optimized specially for this program.
Keywords: biomedical facility, histology, immunocytochemistry, in situ hybridization
Grants awarded to Ajit P Varki
MOLECULAR ETIOLOGY AND THERAPY OF BREAST CANCER
Ajit P Varki, Professor Of Cellular & Molecular Medici
University Of California San Diego
9500 Gilman Dr, Dept 0934
la Jolla, Ca 920930934
Grant 1P20CA066215-01 from National Cancer Institute IRG: SRC
Abstract: The UCSD Cancer Center plans to develop a research program with the theme of mo1ecular approaches to prevention and treatment of breast cancer. The plan incorporates the advantages of a matrix interfacing site-oriented clinical programs with the established basic research programs of the Cancer Center, and also takes advantage of infrastructure recently put into place to facilitate translational research. The plan derives five pilot projects from several of the established programs of the Cancer Center (1) from the Growth Control, Cancer Genetics, and Cancer prevention and Control programs, a project to develop and compare two new methods for surveying the entire tumor genome for abnormalities (representational difference analysis, and differential mRNA display). These comparisons will be on specimens from several studies, including a soon-to-be-initiated privately funded UCSD adjuvant dietary intervention project in women with localized breast cancer, the Long island case cluster, and material from the other projects in this proposal; (2) from the immunology program, a project to develop vaccines against neu peptides, initially from transgenic mammary tumors and later from human tumors; (3) from the Clinical investigation and Developmental Therapeutics (CIDT) program, an attempt to predict the eventual clinical response to chemotherapy of breast cancer, based upon the 24-hour post-treatment activation of tumor cell genes that respond to cellular injury e.g. gaddi53 and c-jun; (4) from the CIDT program and collaborations with investigators at Salk institute, a study to detect the presence of and results from elimination of amplified genes contained in double-minute chromosomes harbored within breast cancer cells from patients with chemotherapy-refractory disease; and (5) from the Cancer Genetics Program, a project using pseudotyped retroviral vectors carrying IL-2, GMCSF, and B7 genes to infect human breast cancers after completion of a pre-clinical feasibility study in nude mice. Funding support is requested for the pilot projects, including the recruitment of three new key investigators a molecular biologist, a molecular cytogeneticist, and a molecular epidemiologist, and for administration and planning. It is anticipated these projects will develop into peer-review funded studies which will coalesce into the nucleus of a highly interactive site- oriented program. The cohesion, focus, multidisciplinary interaction, and productivity of the program will depend upon successful recruitment of the new investigators, the scientific and administrative competence of the P.I., Dr. William Hryniuk, and his standing as the Director of the UCSD Cancer Center. Success will also be fostered by input from the senior individuals comprising a program advisory committee, and reinforced by the substantial resources already committed to the breast program by UCSD School of Medicine, UCSD Medical Center, and affiliated institutions. The excellent basic science milieu of UCSD should sustain a high level of performance of participants in the program
Keywords: breast neoplasm, cancer prevention, molecular oncology, neoplasm /cancer epidemiology, neoplasm /cancer genetics, neoplasm /cancer therapy, nonhuman therapy evaluation cell growth regulation, colony stimulating factor, drug resistance, gene therapy, genetic marker, immunomodulator, interleukin 2, metastasis, neoplasm /cancer chemotherapy, neoplasm /cancer immunotherapy, prognosis, tumor progression athymic mouse, enzyme linked immunosorbent assay, human subject, transgenic animal
Project start date: 1994-09-30
Project end date: 1995-09-29
1P20CA066215-01 (1994): $380456
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 3P30CA023100-15S1 from National Cancer Institute IRG: CCS
Abstract: The UCSD Cancer Center is an Organized Research Unit of UCSD. Comprised of 129 members in 15 University departments, it is located at UCSD Medical Center near downtown San Diego as well as at the campus of UCSD in La Jolla. Its mission is to reduce the incidence of and morbidity and mortality from cancer in San Diego County. UCSD Cancer Center is the research component of a University-wide Cancer Program integrating research, teaching and patient care. The Cancer Center has had continuous CCSG funding since April 1, 1979 and is oriented toward excellent basic research with an emphasis on translational research. Over 500 patients are placed on clinical protocols annually; more than half of these on institutional protocols. Dr. William Hryniuk is the Center Director and is also Associate Dean for Oncology Programs. He reports to the Dean of the School of Medicine. Oversight and counsel regarding the Center is provided to the Dean by the Dean s Advisory Group configured specifically for this purpose, and comprised of senior University executives. The Center Director is advised by a Cancer Center Executive Committee with three subcommittees (research education, protocol review, and senior clinical advisory), and by members of the UCSD Cancer foundation, a group of committed and influential citizens of the community. The Cancer Center s research is conducted through seven highly productive large programs carried out in 58,602 (ASF) of space for cancer research of which 30,650 ASF are under the direct control of the Center Director. Grants in support of cancer research total over $20 million (direct costs) which represents a five-fold increase in seven years. This increase is attributable to expansion of existing programs, and recruitment and support of additional senior investigators. The dramatic increase in the size, scope, and number of the research programs is a reflection of the strong and continued commitment of the parent institution. The programs are Clinical Investigation and Developmental Therapeutics, Cancer Genetics, Basic and Applied Immunology, Glycobiology, Growth Control, Molecular Virology, and Cancer Control and Prevention. There is also a broad and intensive research education program. A stem cell transplantation program is in development. To facilitate translational research, the clinical care activities have been organized into a matrix of clinical services and site-oriented programs. The matrix interlocks with the research programs. Efforts are being made to increase the patient base in order to further facilitate translational research and clinical trials. Funds are requested in this application for shared resources which include Biostatistics, Clinical Trials Office, Flow Cytometry, Pharmacology Lab, Tissue Bank, Molecular Biology Lab, and Lab Support Services. In addition, support is requested for new shared resources of Glycobiology and a Transgenic Mouse Facility to support the basic research strengths of the Center. An amount is requested in the first year of $612,528 for shared resources, and $309,277 for senior leadership and administration.
Keywords: biomedical facility
Project start date: 1978-04-01
Project end date: 1999-04-30
3P30CA023100-15S1 (1997): $1455100
5P30CA023100-13 (1994): $1318854
2P30CA023100-12 (1993): $1297476
5P30CA023100-15 (1996): $1702696
BIOLOGY OF SIALIC ACIDS AND THEIR SUBSTITUTIONS
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R37GM032373-17 from National Institute Of General Medical Sciences IRG: PBC
Abstract: The more specific biological functions of sialic acids appear to depend upon diversity generated by different linkages to the underlying sugar chain, and by modifications such as O-acetylation. Alpha-2-6-linked sialic acids are present in B and T-cells, endothelial cells and hepatocytes. This sequence generates ligands for CD22, a B cell-specific member of the new family of "I-type lectins of the immunoglobulin superfamily. These ligands can be masked by O-acetylation of the sialic acids. Alpha-2-8-linked sialicacids on gangliosides can also be selectively O-acetylated in melanoma cells, neuroectodermal tissues, and certain leucocytes. Biosynthesis of these sequences involves specific sialyltransferases and O-acetyltransferase(s) - while some of the former have been purified and cloned, the latter have not. Ablation of 9-O- acetylation by Influenza C hemagglutinin-esterase (CHE) can help to elucidate functions of this modification. A recombinant soluble form of CHE has been developed for this purpose, and can be modified to probe for 9-O-acetylation in cells and tissues. Human melanoma gangliosidesialic acids can also be de-N-acetylated, and the tyrosine kinase inhibitor genistein increases expression of this modification. We now propose to focus upon the following 1. Compare the tissue distribution of 9-O-acetylation, among four mammalian species.This is made possible by a CHE-based probe. O- acetylation should be conserved in tissues where it is of broad relevance. 2. Cell cycle dependence of 9-O-acetylation and de-N-acetylation of sialic acids. Preliminary studies indicate that these modifications my be regulated in a cell-cycle-dependent manner. 3. Attempts at molecular cloning of the sialic acid-specific O- acetyltransferase(s). All attempts to purify these enzymes have failed - expression cloning will be attempted using multiple approaches. 4.Study the mechanisms of de- and re-N-acetylation of sialic acids in melanoma cells. If time allows, we will pursue the enzymatic mechanisms by which de-N-acetylation occurs with genstein treatment. 5. Study the effect of B-cells activation on CD22, CD22 ligands and O- acetylation. Polyclonally activated B-cells will be studied for changes in the expression and connection of CD22 and its ligands. 6. Compare sialic acid recognition by human CD22beta with that by mouse CD22. This comparative study is critical for the planning of the aim #7. 7. Use sialyloligosaccharides or 9-de-O-acetylation to perturb T-B cell interactions in vitro, and in vivo. The outcome will be studied both in terms of tissue morphology, as well as the immune response. 8. Abrogate CD22 expression in the mo use by gene disruption and/or tissue-specific gene deletion. This will provide critical in formation regarding the role of CD22 lectin in the immune response. 9. Study the structure of CD22 and CHIC in complex with cognate oligosaccharides. Recombinant CD22 and CHE will be used to obtain a crystal structure, and to define the interaction-with sialic acids. 10.Ultrastructurally localize the previously described "cytosolic" CD22. This pool of CD22 persists almost throughout the life span of a B- lymphocyte. Its location has an important bearing on CD22 function.
Keywords: acetylation, carbohydrate biosynthesis, sialate, B lymphocyte, T lymphocyte, acyltransferase, cell cycle, chemical substitution, enzyme mechanism, ganglioside, lectin, melanoma, oligosaccharide, sialyltransferase, species difference, expression cloning, gene deletion mutation, human tissue, laboratory mouse, laboratory rat, molecular cloning, monoclonal antibody, neoplastic cell, recombinant protein, tissue /cell culture, transgenic animal
Project start date: 1983-08-01
Project end date: 2000-07-31
5R37GM032373-17 (1999): $301115
5R37GM032373-16 (1998): $289289
5R37GM032373-15 (1997): $307852
OLIGOSACCHARIES OF NORMAL AND MALIGNANT CELLS
Ajit P Varki, Professor Of Medicine And Cell. & Mole.
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 2R01CA038701-24 from National Cancer Institute
Abstract: The long-term theme of this grant has been to compare and contrast oligosaccharides (glycans) of normal and malignant cells, and to define the pathological and therapeutic significance of the differences. Sialic acids (Sias) are attached to underlying glycans in various linkages, and can be modified in several ways. Altered expression of specific types and linkages of Sias are prominent features of human and murine tumors. All work in our lab is now focused on the biology and evolution of Sias and Sia-binding proteins in health and disease. Thus, the recent focus of this grant has been on "tumor-specific" changes in Sia-terminated glycans, and on their interactions with Sia-binding proteins such as Selectins and Siglecs. During the last funding period we completed all of our previously stated aims on Selectins and on alpha2-6-linked Sias in cancer, and also generated novel data about a human-specific diet- and antibody-mediated mechanism for chronic inflammation in tumor progression. The latter involves human metabolic incorporation of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) from dietary sources (primarily red meats), in the face of human-specific circulating anti-Neu5Gc antibodies. We also now have preliminary evidence for a second mechanism of human-specific chronic inflammation, involving the reduced expression of inhibitory CD33-related Siglecs (CD33rSiglecs) on leukocytes. Given the known importance of chronic inflammation in carcinoma progression, these two human-specific mechanisms may help explain the unusual propensity of humans (as compared to other primates) to develop carcinomas, and have possible relevance for diagnosis, prognosis and therapy. Thus, the current proposal will focus only on the human-specific changes in Sia biology. The general hypothesis is that the human propensity to develop carcinomas is partly related to Neu5Gc incorporation into tumors in the face of anti-Neu5Gc antibody production, and also to relative leukocyte over-reactivity, arising due to human-specific reduction of CD33rSiglec expression. Our studies will focus on carcinomas of human or syngeneic mouse origin - growing in Cmah-/- mice with a human-like defect in Neu5Gc production and/or in mice with deletions or over-expression of myelomonocytic CD33rSiglecs. We will further explore the significance of Neu5Gc incorporation into primary and metastatic tumors in Cmah-/- mice, in the face of circulating anti-Neu5Gc antibodies, and use mouse models to explore the role of human myelomonocytic cell over-reactivity associated with reduced CD33rSiglec expression in chronic inflammation and progression of primary and metastatic human tumors. Finally, we will ask if the combination of Neu5Gc and anti-Neu5Gc antibodies interactions along with Siglec-deficiency-related myelomonocytic cell hyper-reactivity has even greater facilitating effect on progression of carcinomas. As time allows, we will also do pilot studies of human tumor samples for Neu5Gc expression and infiltrating leukocyte Siglec expression to see if either or both are predictive of prognosis and/or disease progression. We have found that a non-human molecule in certain foods of animal origin is becoming incorporated from our diet into the human cancers, even while we are making antibodies against it, and that human immune cells are relatively over-reactive to stimuli do to loss of certain inhibitory molecules. These findings have implications for the human propensity to develop carcinomas, and also provide one explanation for the association of red meat consumption with cancer risk. We propose to study all of these issues in detail, using carefully designed mouse models
Keywords: Animal Feed; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Binding Proteins; Biology; Blood leukocyte; COX inhibitor; COX-2; COX2; Cancers; Carcinoma; Cells; Chronic; Cyclooxygenase Inhibitors; D-glycero-D-galacto-2-Nonulosonic acid, 3, 5-dideoxy-5-((hydroxyacetyl)amino)-; Data; Defect; Diagnosis; Diet; Disease; Disease Progression; Disorder; Epidemiology; Epithelial Neoplasms, Malignant; Epithelial Tumors, Malignant; Evolution; Face; Forecast of outcome; Funding; GcNeu; Generalized Growth; Genetic; Glycans; Goals; Grant; Growth; Health; Human; Human, General; INFLM; Immune; Incidence; Infiltration; Inflammation; Inflammatory; Inhibitors, Cyclo-Oxygenase; Leukocytes; Ligand Binding Protein; Link; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Mammals, Primates; Man (Taxonomy); Man, Modern; Marrow leukocyte; Meat; Mediating; Metabolic; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Mice; Murine; Mus; N-Acetylneuraminic Acids; N-glycoloylneuraminic acid; N-glycolylneuraminic acid; NGNA; Neoplasm Metastasis; Oligosaccharides; PGG/HS; PGHS-2; PHS-2; PTGS2; PTGS2 gene; Pathway interactions; Pilot Projects; Polysaccharides; Primates; Production; Prognosis; Prostaglandin Endoperoxide Synthase Inhibitors; Prostaglandin Synthase Inhibitors; Prostaglandin Synthesis Antagonists; Regulation; Relative; Relative (related person); Reticuloendothelial System, Leukocytes; Role; Sampling; Secondary Neoplasm; Secondary Tumor; Selectins; Sialic Acids; Source; Stimulus; T-Cells; T-Lymphocyte; Testing; Therapeutic; Thymus-Dependent Lymphocytes; Time; Tissue Growth; Tumor Cell Migration; White Blood Cells; White Cell; Work; angiogenesis; animal food; antibody biosynthesis; cancer cell; cancer metastasis; cancer progression; cancer risk; design; designing; disease/disorder; epithelial carcinoma; facial; hCOX-2; immunoglobulin biosynthesis; inhibitor; inhibitor/antagonist; malignancy; mouse model; neoplasm progression; neoplasm/cancer; neoplastic progression; novel; ontogeny; outcome forecast; pathway; pilot study; public health relevance; red meat consumption; sialic acid binding Ig-like lectin; siglec; social role; thymus derived lymphocyte; tumor; tumor progression; white blood cell; white blood corpuscle
Relevance: 7. We have found that a non-human molecule in certain foods of animal origin is becoming incorporated from our diet into the human cancers, even while we are making antibodies against it, and that human immune cells are relatively over-reactive to stimuli do to loss of certain inhibitory molecules. These findings have implications for the human propensity to develop carcinomas, and also provides one explanation for the association of red meat consumption with cancer risk. We propose to study all of these issues in detail, using carefully designed mouse models
Project start date: 1985-08-01
Project end date: 2015-04-30
Budget start date: 11-JUN-2010
Budget end date: 30-APR-2011
PFA/PA: PA-07-070
2R01CA038701-24 (2010): $377221
SPECIALIZED CANCER CENTER CORE SUPPORT GRANT
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 3P30CA023100-11S1 from National Cancer Institute IRG: CCS
Abstract: The Cancer Center at the University of California San Diego School of Medicine is 3 years old. Building upon a firm laboratory research base, a strong and rapidly expanding clinical research program has developed, utilizing both local and national trials. This has led to appointment of an Assistant Director to corrdinate the clinical cancer research program, chair the Center s Protocol Review Committee, and supervise the planned data management system. The basic research program has also grown in a number of areas, including studies of human tumors in athymic mice, tumor cell culture in serum-free medium, and development of monoclonal antibodies to lymphocyte subpopulations. Construction of the new Theodore Gildred Cancer Facility, which was funded jointly by an NCI grant and a community drive, began in February 1981. When the facility is completed in early 1983, it will house many of the Center s research programs as well as a combined oncology clinic. In the past year the University has greatly increased its fiscal and philosophical commitment to the Cancer Center. This includes providing salary support for the Director, planning new faculty positions with joint appointments to the Center and an academic department, stimulating the formation of a foundation which will raise research funds for the Center, providing increased adminstrative funds, and allocating future additional laboratory research space for the Center s use. The Cancer Center Core Support Grant currently supports shared resource laboratories with expertise in pharmacology, serum-free culture medium, athymic mice, surface markers, cytogenetics, radiobiology, radioiodination, and biostatistics. A new immunology shared resource is planned and developmental funds for new research projects are needed. The Center anticipates continued development and expansion in its varied basic research, clinical research and education programs.
Keywords: biomedical facility
Project start date: 1978-04-01
Project end date: 1993-07-31
3P30CA023100-11S1 (1992): $522000
Sponsored Links Excellgen http://Excellgen.com
NEU5GC AND ANTI-NEU5GC ANTIBODIES FOR DETECTION OF CANCER AND CANCER RISK
Ajit P Varki, Professor Of Medicine And Cell. & Mole.
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5U01CA128442-04 from National Cancer Institute
Abstract: Studies from many years ago suggested that the cell surface sialic acid N-glycolylneuraminic Acid (NeuSGc) is an "oncofetal antigen" in humans, and that patients with cancer express antibodies against it. Our application revisits this matter using modern glycomic and high-throughput approaches, and in the light of a new paradigm that humans are genetically defective in synthesizing NeuSGc and can metabolically incorporate it into tumors from certain dietary sources (particularly red meat and milk). Furthermore, preliminary evidence indicates that the polyclonal human anti-Neu5Gc antibody response detects a wide and highly variable spectrum of NeuSGc-containing epitopes. We therefore propose to study total body burden of NeuSGc, NeuSGc-containing glycans on secreted glyconjugates and specific anti-NeuSGc-antibodies-as biomarkers for the early detection of carcinomas of the lung, pancreas and ovary. To achieve our goals, we have assembled an expert team of glycobiologists, chemists, oncologists and biostaticians, along with specialists on glycomics and on antibody-screening by microarrays. Using various established and newly developed approaches we propose to obtain baseline information on the nature and structural complexity of NeuSGc-glycan expression in primary human tumors, and in serum and urine samples from subjects with early and late-stage tumors. In parallel, we are developing a sensitive and specific method to determine the total body burden of NeuSGc. In order to define the antibody response, we will synthesize/obtain matched sets of glycans containing alpha-linked-NeuSGc or NeuSAc, and will validate and optimize a novel glycan array utilizing these targets. Glycan synthesis and conjugation will be optimized, array substrate and hybridization conditions determined, and inter-assay and inter-subject variability defined. The goal is to identify cancer-specific anti-NeuSGc antibody patterns by comparing cancer cases and controls. Initial approximations of sensitivity and specificity will be made at predefined interim analyses with decisions to expand or narrow testing of cancer types. The emerging data from this approach will be also used to define the need for additional iterations of the glycan array. Finally, we will use combinations of total body NeuSGc burden and/or specific NeuSGc-glycans and/or anti-NeuSGc antibody patterns, to identify cancer-specific differences between ill subjects with and without cancer, for use in early diagnosis and prognosis
Keywords: Acids; Animals; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Antigenic Determinants; Appearance; Asialogangliosides; Assay; Binding; Binding (Molecular Function); Binding Determinants; Bioassay; Biologic Assays; Biological Assay; Blood Plasma; Blood Serum; Blood erythrocyte; Blood normocyte; Blotting, Western; Body Burden; Body Tissues; C element; Cancer Detection; Cancer Induction; Cancer Patient; Cancer Staging; Cancers; Carbon; Carcinoma; Carcinoma of Ovary; Cell Coat; Cell surface; Cells; Chickens; Chromatography, High Performance Liquid; Chromatography, High Pressure Liquid; Chromatography, High Speed Liquid; Clinical; D-glycero-D-galacto-2-Nonulosonic acid, 3, 5-dideoxy-5-((hydroxyacetyl)amino)-; Dairy Products; Data; Detection; Diagnosis; Diagnostic; Diagnostic Neoplasm Staging; Diet; EDRN; ELISA; Early Detection Research Network; Early Diagnosis; Endothelium; Enzyme-Linked Immunosorbent Assay; Epithelial Neoplasms, Malignant; Epithelial Tumors, Malignant; Epitopes; Erythrocytes; Erythrocytic; Food; Forecast of outcome; Funding; Future; Gallus domesticus; Gallus gallus; Gallus gallus domesticus; Gastrointestinal Tract, Pancreas; GcNeu; Genital System, Female, Ovary; Glycans; Glycocalyx; Glycosphingolipids; Goals; HPLC; High Pressure Liquid Chromatography; Human; Human, General; INFLM; Iceberg; Immunoblotting; Inflammation; Investigators; Label; Laboratories; Light; Link; Lung; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Marrow erythrocyte; Mass Spectrum; Mass Spectrum Analysis; Measurement; Measures; Meat; Methods; Methods and Techniques; Methods, Other; Milk; Molecular Interaction; Monitor; Mono-S; MonoS; N-Acetylneuraminic Acid; N-Acetylneuraminic Acids; N-glycoloylneuraminic acid; N-glycolylneuraminic acid; NCRR; NGNA; NIGMS; National Center for Research Resources; National Detection Research Network; National Institute of General Medical Sciences; Nature; Neoplasm Staging; O element; O2 element; Oncologist; Ovarian; Ovarian Carcinoma; Ovary; Oxygen; Pancreas; Pancreatic; Pathway interactions; Patients; Pattern; Performance; Photometry/Spectrum Analysis, Mass; Photoradiation; Plasma; Polysaccharides; Population; Primary Neoplasm; Primary Tumor; Principal Investigator; Prognosis; Programs (PT); Programs [Publication Type]; Reaction; Recovery; Red Blood Cells; Red Cell; Red blood corpuscule; Red cell of marrow; Research Personnel; Research Resources; Research Specimen; Researchers; Resources; Respiratory System, Lung; Reticuloendothelial System, Erythrocytes; Reticuloendothelial System, Serum, Plasma; Role; Sampling; Screening procedure; Sensitivity and Specificity; Serum; Serum, Plasma; Sialic Acid; Sialic Acids; Source; Specialist; Specificity; Specimen; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Sphingoglycolipids; Stage at Diagnosis; Staining method; Stainings; Stains; Structure; T-Stage; Techniques; Technology; Testing; Time; Tissues; Tumor Cell; Tumor Staging; Tumor stage; Universities; Urinary System, Urine; Urine; Validation; Western Blotting; Western Blottings; Western Immunoblotting; Work; antibody biosynthesis; base; biomarker; blood corpuscles; cancer cell; cancer progression; cancer risk; cancer type; carcinogenesis; case control; cell type; early detection; epithelial carcinoma; immunoglobulin biosynthesis; in vivo; lung Carcinoma; malignancy; neoplasm progression; neoplasm/cancer; neoplastic cell; neoplastic progression; new approaches; novel; novel approaches; novel strategies; novel strategy; oncofetal antigen; oncofetal proteins; outcome forecast; pathway; prognostic; programs; protein blotting; pulmonary; screening; screenings; social role; translational approach; tumor; tumor progression; uptake; urinary; validation studies
Project start date: 2007-08-01
Project end date: 2012-06-30
Budget start date: 9-AUG-2010
Budget end date: 30-JUN-2011
PFA/PA: RFA-CA-07-020
5U01CA128442-04 (2010): $408279
5U01CA128442-03 (2009): $413250
5U01CA128442-02 (2008): $414190
1U01CA128442-01 (2007): $436949
SIGLEC MODULATION OF INFLAMMATION IN HUMANS AND MICE
Ajit P Varki, Professor Of Medicine And Cell. & Mole.
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Abstract: Sialic acids (Sias) presented at the distal ends of vertebrate glycan chains mediate many biological roles, including binding by intrinsic Sia-recognizing receptors called Siglecs (Sia-recognizing Ig-like lectins). We have hypothesized that the CD33-related subset of Siglecs (CD33rSiglecs) in primates and rodents recognize host Sias as "self, thereby dampening the reactivity of immune cells. We also hypothesize that Sia-expressing bacterial pathogens take advantage of this mechanism to down-regulate innate immune reactivity against them. We earlier discovered a human-specific evolutionary loss of the common mammalian Sia N-glycolylneuraminic acid (NeuSGc). This would have resulted in loss of optimal CD33rSiglec ligands. A variety of human-specific genetic changes and adjustments in these lectins apparently then ensued, leaving the human immune system in an altered state relative to that of our great ape evolutionary relatives. The biological and pathological consequences of these differences are being studied by comparing humans and great apes, but many practical, ethical and fiscal issues limit this approach. We therefore propose to use transgenic mice to model and compare human and chimpanzee sialic acid and CD33rSiglec biology, elucidating functional consequences resulting from genetic changes during human evolution. The overall hypothesis being tested is that the human propensity to develop inflammatory diseases involving innate and adaptive immune cells, as well as infections by Sia-expressing bacteria are related to human-specific evolutionary changes in certain CD33rSiglecs. These include humanspecific changes in Sia-binding properties of Siglec-9 on neutrophils and monocytes; in the binding properties, expression and function of Siglec-11 and -12 on macrophages; of Siglec-11 on human brain microglia, and the selective down-regulation of Siglec-5 on human T cells. We will use a variety of genetically modified mice to mimic the ancestral human condition of constitutive CD33rSiglec "unmasking" in myelomonocytic cells; the current functional states of human and chimpanzee Siglec-9; human-specific changes in Siglec-11 and -12 on macrophages; human-specific Siglec-11 expression in microglia; and, the ancestral great ape state of Siglec-5 expression on T lymphocytes. These studies will be done in wild-type mice, and in strains deficient in the relevant murine CD33rSiglecs. Appropriate studies of innate and adaptive immune responses as well as challenges with Sia-expressing bacterial pathogens will test the original hypotheses. These studies involve collaborations with other program members and utilize all Core services in the program. Importantly, even if some of the taken approaches fail to accurately mirror human evolution, the results will illuminate various general underlying principles regarding the biology of CD33rSiglecs on innate and adaptive immune blood cells
Keywords: No Project Terms available
Budget start date: 31-DEC-2009
Budget end date: 30-DEC-2010
5P01HL057345-13_0007 (2010): $259360
BIOLOGY OF SIALIC ACIDS AND THEIR SUBSTITUTIONS
Ajit P Varki, Professor Of Medicine And Cell. & Mole.
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 2R01GM032373-27A1 from National Institute Of General Medical Sciences
Abstract: Sialic Acids (Sias) are molecules attached to the outer ends of sugar chains (glycans) on cell surface and secreted molecules of vertebrates. The long-term goal of this grant has been to unravel mechanisms regulating various kinds of Sia attachments and structural modifications, and to elucidate their biological and pathological roles. All attention is now focused on the human-specific genetic loss of the common mammalian Sia N- glycolylneuraminic acid (Neu5Gc), which differs by a single oxygen atom from its precursor N-acetylneuraminic acid (Neu5Ac), which is enriched in humans. We have found that human cells can take up and metabolically incorporate exogenous free or bound Neu5Gc in significant amounts, and that this is relevant to human dietary intake of Neu5Gc, allowing incorporation into certain human cell types in vivo, as well Neu5Gc incorporation into biotherapeutic products in vitro. Our general hypothesis is that the loss of Neu5Gc in humans has broad implications, ranging from basic biochemical and cell biological issues involving Neu5Gc uptake and incorporation into some cell types, to the origins, diversity and significance of anti-Neu5Gc antibodies in humans, and to the need to develop methods to eliminate Neu5Gc from human cells and tissues. Besides biochemical, cell biological and epidemiological studies of human cells and humans, we will use mice with a human-like genetic defect in Neu5Gc production. We will study the metabolism and fate of Neu5Gc in human and animal cells and in animal models, asking if humans are uniquely different. Since the mice with a human- like defect do not easily accumulate dietary Neu5Gc, we hypothesize that mammalian cells have an as yet unknown mechanism to turnover Neu5Gc (and/or the N-glycolyl group), and that this mechanism may be defective in humans (as >2 million years have elapsed since we lost the ability to synthesize Neu5Gc). We will follow the fate of double-labeled Neu5Gc in human animal cells, and propose biochemical, bioinformatics and genetic approaches to discover the putative gene ("Gene X") involved in turnover of Neu5Gc in rodent cells. At the organismal level, we will follow the fate of bound and free Neu5Gc in the mouse GI tract, addressing host and microbial factors affecting its metabolic incorporation. Meanwhile, we will explore mechanisms by which adult humans develop a diverse and variable range of anti-Neu5Gc antibodies, testing multiple hypotheses regarding this issue, and also asking whether pre- or postnatal exposure to Neu5Gc impacts antibody production. We will also study pathological consequences of in vivo interactions between human anti-Neu5Gc antibodies and Neu5Gc-containing glycans, with a particular focus on the exacerbation of atherosclerosis. If time allows, we will ask whether the Neu5Gc found on biotherapeutic agents has any negative impact. Finally, assuming we confirm the importance of Neu5Gc and anti-Neu5Gc antibodies for human disease and/or biotherapeutic agents, we will pursue promising preliminary data, to develop simple and non-toxic metabolic manipulations that eliminate Neu5Gc from cells in culture, and potentially from the human body. We have found that a non-human molecule in certain foods of animal origin is becoming incorporated from our diet into the human body, even while we are making antibodies against. This has implications for several diseases in which consumption of animal foods may play a role. We are studying all aspects of this matter, ranging from the basic biochemistry, all the way to trying to eliminate the molecule from the human body
Keywords: 21+ years old; Abnormal Assessment of Metabolism; Acetylation; Address; Adult; Affect; Alimentary Canal; Animal Feed; Animal Model; Animal Models and Related Studies; Animals; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Attention; Binding; Binding (Molecular Function); Bio-Informatics; Biochemical; Biochemistry; Bioinformatics; Biologic Products; Biologic Therapy; Biological; Biological Agent; Biological Products; Biological Response Modifier Therapy; Biological Therapy; Biology; Body Tissues; C element; Carbon; Cell surface; Cells; Chemistry, Biological; Chronic; Complement; Complement Proteins; Consumption; D-glycero-D-galacto-2-Nonulosonic acid, 3, 5-dideoxy-5-((hydroxyacetyl)amino)-; Data; Defect; Diet; Dietary intake; Digestive Tract; Disease; Disorder; Epidemiologic Research; Epidemiologic Studies; Epidemiological Studies; Epidemiology Research; Exons; Exposure to; Funding; GI Tract; Gastrointestinal Tract; Gastrointestinal tract structure; GcNeu; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genomics; Glycans; Glycoconjugates; Goals; Grant; Human; Human Cloning; Human Figure; Human body; Human, Adult; Human, General; INFLM; Immune response; In Vitro; Individual; Inflammation; Label; Mammalian Cell; Mammals, Mice; Mammals, Rodents; Man (Taxonomy); Man, Modern; Metabolic; Metabolic Studies; Metabolism Studies; Methods; Mice; Modeling; Modification; Molecular; Molecular Interaction; Murine; Mus; Mutation; N-Acetylneuraminic Acid; N-Acetylneuraminic Acids; N-glycoloylneuraminic acid; N-glycolylneuraminic acid; NGNA; O element; O2 element; Oligosaccharides; Oxygen; Pilot Projects; Play; Polysaccharides; Process; Production; Research; Rodent; Rodentia; Rodentias; Role; Sampling; Severities; Sialic Acid; Sialic Acids; Source; Testing; Time; Tissues; Vertebrate Animals; Vertebrates; adult human (21+); alimentary tract; animal food; anti-IgM; antibody biosynthesis; atheromatosis; atherosclerotic vascular disease; base; biopharmaceutical; biotherapeutic agent; biotherapeutics; biotherapy; cell type; digestive canal; disease/disorder; experiment; experimental research; experimental study; feeding; genome mutation; host response; human disease; immunoglobulin biosynthesis; immunoresponse; in vivo; interest; measurement of metabolism; metabolic abnormality assessment; metabolomics; microbial host; model organism; pilot study; postnatal; prenatal; public health relevance; research study; response; social role; sugar; unborn; uptake; vertebrata
Relevance: 7. We have found that a non-human molecule in certain foods of animal origin is becoming incorporated from our diet into the human body, even while we are making antibodies against. This has implications for several diseases in which consumption of animal foods may play a role. We are studying all aspects of this matter, ranging from the basic biochemistry, all the way to trying to eliminate the molecule from the human body
Project start date: 1983-08-01
Project end date: 2014-04-30
Budget start date: 15-JUN-2010
Budget end date: 30-APR-2011
PFA/PA: PA-07-070
2R01GM032373-27A1 (2010): $386250
Sialic Acids And Siglecs In Myeloid Cells
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P01HL057345-100004 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Sialic acids (Sias) are found to the outermost ends of sugar chains of most cell surface molecules. Their two most common linkages to underlying sugar chains are alpha2-3 or 2-6 to a galactose unit. The most common Sia is N-acetylneuraminic acid (Neu5Ac), and two common modifications are 9- O-acetylation and an 5-Nglycolyl group instead of the usual 5-N-acetyl group. Siglecs (sialic acid-binding Ig superfamily lectins) are a family of cell surface proteins that recognize Sias via their first Ig V-set domains. Sia recognition by Siglecs can be affected by the linkage of the Sia to the underlying sugar chain as well as by the above-mentioned modifications. Siglec recognition of Sias is partially or completely dependent on a conserved "critical arginine" residue in the amino-terminal V-set domain. The Sia binding sites of most Siglecs are "masked" by interactions with Sia residues on the same cell surface, and can be unmasked during activation. The CD33-related Siglecs are selectively expressed on certain blood cell types, and have tyrosine-based signaling motifs in their cytosolic tails. The primary functions of most Siglecs is unknown. We hypothesize that linkage differences and/or modifications of Sias on mature cells of the myelomonocytic lineage (neutrophils, monocytes and macrophages) cells can regulate the levels and signaling of Siglecs in bone marrow development and/or in innate immunity. We propose that Siglecs regulate cellular responses to microorganisms that express sialidases and/or their own cell surface sialic acids by acting as "biological sensors of sialylation states" of both self and non-self. To pursue these hypotheses, we will (a) generate and characterize mice with single or double mutations in Siglec-3/CD33 and Siglec-F the two major Siglecs on cells of the myelomonocytic lineage; (b) define the unmasking and signaling state of myelomonocytic Siglecs in mice with combinations of deficiencies in the ST3Gal I sialyltransferases; (c) generate and characterize mice with altered expression of modified sialic acids in cells of the myelomonocytic lineage; and, (d) study the consequences of the above genetic alterations on the responses of neutrophils and monocytes to defined microbes expressing sialidases or sialic acids. These studies will test if these Siglecs on myelomonocytic cells act as biologic sensors of sialylation states, and if the genetic alterations in mice result in pathologically altered responses to the microbes.
Keywords: glycosylation, immunoregulation, lectin, myeloid stem cell, protein structure function, sialate, N acetylneuraminate, bactericidal immunity, carbohydrate structure, developmental genetics, exo alpha sialidase, gene mutation, immunogenetics, macrophage, neutrophil, genetically modified animal, laboratory mouse, tissue /cell culture
BIOLOGY OF SIALIC ACIDS AND THEIR SUBSTITUTIONS
Ajit P Varki, Professor Of Medicine And Cell. & Mole.
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5R01GM032373-26 from National Institute Of General Medical Sciences
Abstract: Sialic acids (Sias) are a family of acidic sugars found on vertebrate glycoconjugates, being attached to underlying oligosaccharides (glycans) in different linkages, and modified in different ways. These linkages and modifications are molecule-specific, tissue-specific and developmentally regulated. The overall goal of this grant has been to unravel the mechanisms responsible for regulating some of these linkages and modifications, and to elucidate their biological and pathological roles. Thus, we have focused on understanding the biosynthetic mechanisms responsible for their expression, the specific intrinsic lectins that recognize them, the consequences for cellular signaling, differentiation, activation and survival, and for cellular interactions with the extracellular matrix. We are also interested in their roles in mediating cell-cell interactions and extracellular signaling during tissue organization and development, as well their recognition by specific lectins of extrinsic origin. In the past, this grant has supported studies of several such Sia modifications and linkages and the recognition of such modifications and linkages by various lectins. As our most recent interesting findings have been on the human-specific loss of the common mammalian Sia N-Glycolylneuraminic acid (Neu5Gc), we propose to focus exclusively on this issue during the next grant period. We hypothesize that this genetically determined loss has many implications, ranging from basic biochemical and cell biological effects involving uptake and incorporation into some cell types, to the presence and significance of anti-Neu5Gc antibodies, to an impact on the functions of both intrinsic and extrinsic receptors that recognize Sias. In addition to biochemical and cell biological studies, and studies of certain human samples, we will use mice with a human-like defect in Neu5Gc expression, and cell lines derived from such animals. This genetic approach increases the likelihood of definitive mechanistic conclusions to bolster the observational correlations in humans. Specific Aims are #1, determining the fate of exogenously-introduced Neu5Gc in Neu5Gc-deficient Human cells and mice; #2, exploring the mechanism and consequences of incorporation of Neu5Gc from dietary sources, and the accompanying anti-Neu5Gc antibody response; #3, studying the consequences of human Neu5Gc loss on the evolution and function of intrinsic Sialic acid-recognizing receptors (particularly the CD33-related Siglecs); and, #4, exploring the consequences of human Neu5Gc loss on the evolution and function of extrinsic Sialic acid recognizing receptors. The extent to which some aspects of Aims #3 and #4 and their ramifications are pursued will depend on the results of #1 and #2, and on further preliminary studies. In the long run, these studies should help elucidate the molecular and cellular basis for certain diseases that appear to be uniquely or preferentially manifested in humans
Keywords: Abbreviations; Acetylation; Animals; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Apes; Autoimmune Diseases; Award; Binding; Binding (Molecular Function); Biochemical; Biological; Biology; Blood Vessels; Blood erythrocyte; Blood normocyte; Body Tissues; C element; CMP-N-acetylneuraminate monooxygenase; CMP-N-acetylneuraminic acid hydroxylase; CMP-Neu5Ac hydroxylase; CMP-NeuAc hydroxylase; CMP-sialic acid hydroxylase; CMPacetylneuraminate monooxygenase; Carbon; Cell Communication; Cell Communication and Signaling; Cell Interaction; Cell Line; Cell Lines, Strains; Cell Signaling; Cell-Extracellular Matrix; Cell-to-Cell Interaction; CellLine; Cells; Chickens; Chimp; Chimpanzee; Chimpanzee troglodytes; Class; Complement; Complement Factor H; Complement Proteins; Condition; Coronary Arteriosclerosis; Coronary Artery Disease; Coronary Artery Disorder; Coronary Atherosclerosis; D-glycero-D-galacto-2-Nonulosonic acid, 3, 5-dideoxy-5-((hydroxyacetyl)amino)-; Data; Defect; Development; Dietary intake; Disease; Disorder; ECM; Epidemiologic Research; Epidemiologic Studies; Epidemiological Studies; Epidemiology Research; Erythrocytes; Erythrocytic; Evolution; Extracellular Matrix; Factor H; Family; Fetal Tissues; Funding Agency; Funding Source; Gallus domesticus; Gallus gallus; Gallus gallus domesticus; Gangliosides; GcNeu; Genes; Genetic; Glycans; Glycoconjugates; Goals; Grant; Hominidae; Hominids; Human; Human, General; IgY; Immune response; Individual; Intracellular Communication and Signaling; Intravenous; Label; Lectin; Malaria; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow erythrocyte; Mediating; Metabolic; Mice; Moab, Clinical Treatment; Modification; Molecular; Molecular Interaction; Monkeys; Monoclonal Antibodies; Murine; Mus; N-Acetylneuraminic Acid; N-Acetylneuraminic Acids; N-glycoloylneuraminic acid; N-glycolylneuraminic acid; NGNA; Neuraminic Acids; Numbers; Oligosaccharides; Paludism; Pan; Pan Genus; Pan Species; Pan troglodytes; Parasites; Peritoneal; Plasmodium Infections; Polysaccharides; Pongidae; Pressure; Pressure- physical agent; Principal Investigator; Programs (PT); Programs [Publication Type]; Range; Receptor Protein; Recommendation; Red Blood Cells; Red Cell; Red blood corpuscule; Red cell of marrow; Relative; Relative (related person); Resistance; Reticuloendothelial System, Erythrocytes; Role; Rotavirus; Running; Sampling; Severities; Sialic Acid; Sialic Acids; Sialoglycosphingolipids; Signal Transduction; Signal Transduction Systems; Signaling; Source; Specificity; Staging; System; System, LOINC Axis 4; Temperature; Testing; Time; Tissues; Tissues, Fetal; antibody biosynthesis; antigen binding; autoimmune disorder; base; biological signal transduction; blood corpuscles; carbene; cell type; cultured cell line; disease/disorder; extracellular; fetus tissue; great ape; host response; human study; human tissue; immunoglobulin Y; immunoglobulin biosynthesis; immunoresponse; interest; methylene; methylene radical; pathogen; preference; pressure; programs; receptor; resistant; sialic acid binding Ig-like lectin; siglec; social role; sugar; sugar nucleotide; tumor; uptake; vascular
Project start date: 1983-08-01
Project end date: 2010-07-31
Budget start date: 1-AUG-2008
Budget end date: 31-JUL-2010
5R01GM032373-26 (2008): $0
3R01GM032373-26S1 (2008): $0
Sponsored Links Excellgen http://Excellgen.com
5R01GM032373-25 (2007): $366235
5R01GM032373-24 (2006): $377173
2R01GM032373-23 (2005): $385104
SIALIC ACID O-ACETYLATION IN BLOOD CELL INTERACTIONS
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P01HL057345-050004 from National Heart, Lung, And Blood Institute
Abstract: Sialic acid (Sia) residues of N-linked sugar chains on blood and vascular proteins are usually attached in alpha2-6 or alpha2-3 linkages, and some molecules are selectively O-acetylated at the 9- position. This diversity can affect recognition of Sia by certain vascular lectins in vitro. We will elucidate the in vivo biological significance of these findings by manipulation of certain genes in the mouse. Sialic acid O-acetyltransferase(s) have been difficult to purify or clone. We have isolated cDNA and genomic clones of a mouse esterase (Lse) that selectively removes 9-O-acetyl groups from Sias. Expression of Lse is highly regulated e.g., in murine hematopoietic development. 9-O-acetylation also occurs preferentially on the sequence Siaalpha2-6Galbeta1-4GlcNAc, whose expression is determined by the alpha2-6 sialyltransferase gene (ST6Gal I). This gene is regulated by differential action of multiple promoters in cells such as hepatocytes, endothelial cells, erythrocytes and lymphocytes. Thus, alterations in activity of ST6Gal I and/or the competing alpha2-3 sialyltransferases (ST3Gal III and IV) may also alter 9-O-acetylation. To elucidate the in vivo regulation of 9-O-acetylation and to pursue specific hypotheses about its roles,we will do the following 1. Eliminate expression of the Lse gene systemically, and/or selectively in the cell types listed above. 2. Alter ratios of alpha2-6 and alpha2-3 Sia by disrupting expression of alpha2-6 and alpha2-3 sialyltransferases (Collaboration with Project 1) and by "knocking in" the cDNA for ST3Gal III into the endogenous ST6Gal I gene, disrupting the latter. In the latter mice, alpha2-6 and alpha2-3 Sia linkage ratios should be inverted on N-glycans. 3. Elucidate the changes in 9-O- acetylation occurring in these mice, and structural and functional consequences of these genetic manipulations. Changes will be detected in situ by immunohistology using specific probes for sialylation and O-acetylation, and directly demonstrated by chemical analyses. Particular attention will be directed towards hepatocytes, endothelial cells, erythrocytes and lymphocytes. Biological effects upon interactions involving the Sia-binding lectins H protein, CD22, CD33 and Sialoadhesin will be studied (some in collaboration with Projects 1 and 3). The sensitivity of red cells to complement and the turnover of red cells and plasma proteins will also be assessed. Selected aspects of blood coagulation, hematopoiesis, and the distribution and function of lymphocytes will also be examined. These studies will help to define the in vivo regulation and biological roles of Sia O-acetylation in the blood and vascular systems.
Keywords: acetylation, cell cell interaction, gene expression, lectin, sialate, sialyltransferase, CD antigen, CD22 molecule, chemical binding, complement, developmental genetics, erythrocyte, genetic regulatory element, hematopoiesis, lymphocyte, in situ hybridization, laboratory mouse, transgenic animal
SULFATED OLIGOSACCHARIDES OF NORMAL AND MALIGNANT CELLS
Ajit P Varki, Professor Of Cellular & Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01CA038701-03 from National Cancer Institute IRG: PBC
Abstract: Previous studies have demonstrated the existence of a class of sulfated N-linked oligosaccharides in eukaryotic glycoproteins. However, little is currently known about their detailed structure, biosynthesis, regulation, or biological significance. In this proposal, we describe the establishment of an enzymatic method for quantitative and nondegradative release of intact sulfated N-linked oligosaccharides directly from whole proteins, and their complete separation from all other sulfated macromolecules. This method will be used to look for differences in the expression of sulfated N-linked oligosaccharides in total cell homogenates and on specific glycoproteins in the following continuous tissue culture systems (1)CHO and mouse lymphoma cells with specific defects in N-linked glycosylation and/or treated with inhibitors of processing glycosidases; (2)NIH 3T3 cells before and after transformation by specific classes of oncogenes; (3)metastatic and nonmetastatic tumors cells; and (4)leukemia cells in various stages of induced differentiation. On the basis of the results of this screening, one or two of these systems will be chosen for further study. The individual oligosaccharides will be fractionated and the types of sulfated monosaccharides identified. This will be followed by a detailed structural analysis of the sulfated oligosaccharides, with regard to the location, linkages, and types of component sugars, using conventional and newly developed methods. This will define any differences in structure found within the systems chosen for study. The information obtained from the structural analysis will be used to predict the biosynthetic pathways for the generation of the sulfated oligosaccharides in these cells, and to predict possible levels of modulation that could explain any differences found. Finally, we will design assays that will identify the enzyme and substrates involved in the sulfation reactions in question. The specific aim of this work will be to see if differences in the expression of the enzymes involved could explain differences in sulfation of N-linked oligosaccharides in these systems. In the long run, we hope to extend these studies to attempt to understand potential biological roles of the sulfation of N-linked oligosaccharides in processes such as tumorigenesis and metastasis formation. (B)
Keywords: GLYCOPROTEINS, NEOPLASTIC CELLS, OLIGOSACCHARIDES, PATHOBIOCHEMISTRY STUDY SECTION, SULFATES, CARBOHYDRASES, CARBOHYDRATES BIOSYNTHESIS, CARBOHYDRATES STRUCTURE, CARBOHYDRATES, MONOSACCHARIDES, CHEMICAL REACTIONS, GLYCOSYLATION, ENZYME INHIBITORS, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENES, ONCOGENES, NEOPLASMS CHARACTERISTICS, PROTEINS OF NEOPLASMS, NEOPLASMS OF BLOOD AND RE SYSTEM, LEUKEMIA, NEOPLASMS OF BLOOD AND RE SYSTEM, LYMPHOMA, NEOPLASTIC GROWTH, NEOPLASMS METASTASIS, neoplastic transformation, sulfation, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, RODENTS, MYOMORPHA, HAMSTERS, PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PRESSURE LIQUID, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, RADIOISOTOPES, SULFUR, RADIOTRACERS, TISSUE (CELL) CULTURE
Project start date: 1985-08-01
Project end date: 1988-11-30
Sponsored Links Excellgen http://Excellgen.com
ADMINISTRATIVE AND MOUSE MANAGEMENT CORE
Ajit P Varki, Professor Of Medicine And Cell. & Mole.
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Abstract: Overview. As before, this Core consists of two components A General Administrative Component, and a Mouse Management Component. The General Administrative Component works to maintain the cohesiveness of the whole program and its functions by organizing group meetings, retreats and journal clubs, and by dealing with any and all inter- Departmental or Institutional issues that arise. It also organizes internal and external reviews of the program, as well as making sure that renewal applications are submitted in a timely fashion. It regularly tracks the financial status of the program on a monthly basis, and assists in hiring and other personnel issues. In addition, the Core identifies newly published literature relevant to the program, and distributes to the appropriate investigators within the program. The goal of the administrative component is to make certain that the entire program functions as efficiently and optimally as possible. The Mouse Management Component supports 1 full time and one part-time animal technician. These individuals who are fully trained and engaged in the maintenance of gene-targeted and transgenic mouse models proposed in the individual projects. This component has the necessary equipment and space in a pathogen-free mouse-specific vivarium, as detailed below. The actual production of new mouse models will be done through the currently established UCSD Transgenic Mouse and Embryonic Stem Cell Gene Targeting Core, please see http//cancer.ucsd.edu/Research/Shared/tgm/default2.asp. This state-of-the-art facility was originally started with major support and advice from J. Marth (Project 1 leader) and has since developed an excellent track record in the production of genetically altered mice. Thus, rather than doing gene targeting ourselves, it now makes sense for this program to pay via fee-for-service for the generation of mice through this established UCSD Core. While the total costs are about the same, we avoid duplicative functions at the institution. Since our past experience with this approach has been good, we have decided to continue it in the current proposal. Budgetary Approach Based on our experience to date, we also feel it is best to keep the daily management and breeding of mice, the animal technicians involved, the costs of mouse cage space, and the use of approaches such a Speed Congenics (see below) as a Core function - rather than allocating resources to each project for these activities. This approach also allows the maximum flexibility in accommodating the needs of all the projects, which will vary from year to year. While this makes for a rather large budget for an "Administrative Core", most of the budget is actually for mouse-related costs. The administrative component budget remains very much the same as in the previous version of the grant
Keywords: No Project Terms available
Budget start date: 31-DEC-2009
Budget end date: 30-DEC-2010
5P01HL057345-13_9006 (2010): $884943
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P01HL057345-109001 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Overview This program project grant consists of a group of investigators from three different departments (Medicine, Pathology and Cellular and Molecular Medicine) and two institutions (The UCSD School of Medicine and nearby VA Medical Center). This Core consists of two components a General Administrative component and a Mouse Management Component (MMC). The General Administrative Component will work to maintain the cohesiveness of the whole group and its functions by organizing group meetings, retreats and journal clubs, and by dealing with any and all inter-Departmental or inter-institutional issues that may arise. It will also organize internal and external reviews of the program, as well as make sure that renewal applications are submitted in a timely fashion. It will also track the financial status of the program on a monthly basis, and assist in hiring and other personnel issues. In addition, the Core will identify newly published literature relevant to the program, and distribute it to the appropriate investigators within the program. The goal of the administrative component is to make certain that the entire program functions as efficiently and optimally as possible. The Mouse Management Component (MMC) of this Core will include two full time animal technicians who are fully trained and engaged in the maintenance of gene-targeted and transgenic mouse models proposed in the individual projects. The MMC has the necessary equipment and space in a pathogen-free mouse-specific vivarium, as detailed below. The Core will play a key role in the breeding, husbandry, and dissemination of the mouse model systems described in this Program Project.
Keywords: animal colony, biomedical facility, laboratory mouse, gene targeting, genetically modified animal
GENETIC MODULATION OF BLOOD AND VASCULAR GLYCOSYLATION
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P01HL057345-05 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Oligosaccharides such as the N- and 0-linked chains of glycoproteins and the heparan sulfate chains of proteoglycans are major structural components of cells and tissues, with a complexity rivaling that of nucleic acids and proteins. This Program is focused upon exploring the biological roles in the blood and vasculature of two anionic modifications of these sugar chains sialic acid and N-sulfation. The structures of the N- and 0-linked sugar chains of many plasma glycoproteins and blood cells and are well known. Specific lectins such as the selectins (on leucocytes, platelets and endothelium), the I-type lectins (on B cells, macrophages or myeloid precursors), and the H protein of the complement pathway, can differentially recognize these chains in the context of their sialic acid residues. The N-sulfate esters of heparan sulfate glycosaminoglycan chains are recognized by various proteins involved in blood coagulation, cell growth, platelet interactions and leucocyte migration. Despite many clues, most physiologic roles of sialylation and N-sulfation are not well observed in cultured cells, and must be explored in the intact organism. However, these features of mammalian glycosylation are not well represented in model invertebrates, and few genetic defects affecting these pathways have been described in intact mammals. Our labs have been among the first to genetically dissect the in vivo roles of glycosylation in the mouse and to develop new technologies for efficient analysis of oligosaccharides from mouse tissues. Thus, the central theme of this proposal is the genetic manipulation of sialic acids and N-sulfate esters in the intact mouse. When required, we will use our recently developed method to selectively inactivate mouse genes in a cell type-specific manner. Replacement of wild type alleles with recombinant ones carrying loxP target sites at innocuous positions allows selective gene deletion, by mating with mice transgenic for Cre recombinase constructs driven by specific transcriptional control sequences. This allows specific focus on blood cells, endothelium and plasma proteins, and will be important in instances where systemic gene inactivation models are non-viable. We also have the necessary expertise to analyze the consequences of the genetic manipulations on the structure of hematopoietic, vascular and lymphoid tissues, the mechanisms of hemostasis, the structure of the oligosaccharides, and the functional consequences to hematopoiesis and lymphocyte biology. These studies are expected to reveal many important functions for oligosaccharides in health and disease.
Keywords: carbohydrate structure, glycosylation, hematology, immunogenetics
Project start date: 1997-09-30
Project end date: 2003-01-31
5P01HL057345-05 (2001): $1633453
5P01HL057345-04 (2000): $1574056
5P01HL057345-03 (1999): $1530812
5P01HL057345-02 (1998): $1488822
SELECTIN-CARBOHYDRATE INTERACTIONS IN ACUTE LUNG INJURY
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P50HL023584-200011 from National Heart, Lung, And Blood Institute
Abstract: Neutrophil extravasation and fluid leak into the alveolar space is a hallmark of the acute lung injury that occurs in adult respiratory distress syndrome (ARDS) and other human disease conditions. The long- term objective of this work is to treat acute lung injury in humans by interrupting the initial adhesion of leukocytes to the blood vessel wall. E-, P-, and L-selectin are endothelial, platelet, and leukocyte glycoproteins that support adhesion through the recognition of carbohydrate ligands. Antibody-, peptide-, and carbohydrate-based approaches will be used to elucidate the molecular interactions of selectins and their ligands in vitro and in vivo. Four specific aims are proposed I. Determine the expression of selectins and their carbohydrate ligands in acute lung injury. Specific antibodies, selectin-Ig fusion proteins, and cDNA probes will be used to localize and quantitate antigen and mRNA expression in lung tissue of rodents given bacterial endotoxin and/or inflammatory cytokines by intratracheal and intravenous routes. Selectin and carbohydrate ligand expression will be determined in blood and bronchoalveolar lavage samples from patients undergoing surgical removal of pulmonary arterial thromboemboli. II. Develop specific selectin-blocking reagents based on antibodies and peptides. Monoclonal antibodies generated against recombinant murine selectins and peptides representing sequences found in their extracellular domains will be evaluated as blockers of adhesion and direct molecular binding in vitro. III. Identify oligosaccharide ligands of murine selectins. Cell adhesion assays and direct molecular binding studies, including a competitive ELISA, titration microcalorimetry and fluorescence spectroscopy, will be used to study the carbohydrate specificities of murine selectins. Comparisons will be made to ongoing investigations of human selectin-carbohydrate interactions. IV. Inhibit selectin-carbohydrate interactions in acute lung injury utilizing antibodies, peptides, and oligosaccharides. Potent blockers of selectin-carbohydrate interactions in vitro will be evaluated for efficacy in the treatment of acute lung injury in rodent models. Alternative routes of administration will be tested. We anticipate that our studies will provide new insights into the role of selectin-carbohydrate interactions in acute lung injury, and directly point to avenues for therapeutic intervention. In addition, the reagents generated in the course of these studies may prove useful in the study of selectin-carbohydrate interactions in other human disease conditions.
Keywords: adult respiratory distress syndrome, carbohydrate receptor, gene expression, glycoprotein, leukocyte adhesion molecule, lung injury, selectin, blocking antibody, ligand, membrane protein, oligosaccharide, peptide, pulmonary circulation obstruction, enzyme linked immunosorbent assay, fluorescence spectrometry, human tissue, laboratory mouse, lung lavage, microcalorimetry, nucleic acid probe
Project start date: 1997-12-01
Project end date: 1998-11-30
Sponsored Links Excellgen http://Excellgen.com
N-LINKED OLIGOSACCHARIDES OF NORMAL AND MALIGNANT CELLS
Ajit P Varki, Professor Of Cellular & Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01CA038701-10 from National Cancer Institute IRG: PBC
Abstract: Most mammalian cells and their secreted products are covered with a dense coating of sugar chains. In recent years, the technologies for studying these oligosaccharide chains on glycoconjugates have advanced rapidly, allowing an exploration of their biological roles. There is now increasing evidence that they play many important roles in complex organisms, ranging from simple structural integrity to the mediation of cell-cell interaction, intra- and inter-cellular trafficking, and cell growth and differentiation. Work in this laboratory is driven by the hypothesis that the more specific biological role of oligosaccharides are likely to be mediated by unusual terminal structures or by modifications such as O- acetylation, phosphorylation and sulfation. Our overall goal is to explore the biosynthesis, regulation and function of such oligosaccharide structures in health and disease. In work supported by this grant, we have identified and partially characterized novel families of sulfated N-linked oligosaccharides that are enriched in pulmonary artery endothelial cells, as well as in lung tissue. These include multi-antennary complex-type oligosaccharides carrying sialic acids and O-Sulfate esters (SSOs) and N-linked molecules carrying glycosaminoglycan-like chains attached to complex-type oligosaccharides (NLGs). We have also developed a novel technique for tagging free oligosaccharides via their reducing terminus with biotinylated amino- pyridine (BAP). In the current project period, we will continue the structural analysis of these molecules and use structurally defined BAP- SSO adducts to search for specific receptors for these oligosaccharides, and to raise monoclonal antibodies directed against them. In the course of these studies, we have identified a novel class of sulfated glycosaminoglycans that are calcium-dependent high affinity ligands for the leucocyte adhesion molecule, L-selectin. These appear to be distinct from the NLGs and SSOs mentioned above, and from the previously described mucin-like sulfated ligands for L-selectin on the high-endothelial venules (HBV) of lymph nodes. In the current project period, we will carry out a detailed structural comparison of these sulfated ligands, and identify the minimal oligosaccharide structural requirements for the biologically relevant interactions. We will also study the biosynthesis of these molecules in isolated intact Golgi-enriched preparations, and create novel fluorescent biotinylated primers for their biosynthesis in intact cells. Altered glycosylation is an universal feature of cancer cells, generating tumor antigens and diagnostic markers, and apparently mediating some of their abnormal behavior. It is likely that metastatic tumor cells subvert the mechanisms that normally mediate the migration and invasion of blood cells into tissues. Since endothelial cells must play crucial roles in the metastatic process, our study of their sulfated oligosaccharides could have an important bearing on the design of therapeutic interventions in cancer.
Keywords: mucopolysaccharide, neoplastic cell, oligosaccharide, sulfate, Golgi apparatus, adduct, aminopyridine, carbohydrate biosynthesis, carbohydrate receptor, carbohydrate structure, glycosylation, metastasis, oncoprotein, selectin, sialate, vascular endothelium, animal tissue, fluorescent dye /probe, high performance liquid chromatography, laboratory mouse, laboratory rabbit, laboratory rat, monoclonal antibody, tissue /cell culture
Project start date: 1985-08-01
Project end date: 1998-11-30
5R01CA038701-10 (1995): $344958
2R01CA038701-09 (1994): $352257
N LINKED OLIGOSACCHARIDES OF NORMAL AND MALIGNANT CELLS
Ajit P Varki, Professor Of Cellular & Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 3R01CA038701-13S1 from National Cancer Institute IRG: PBC
Abstract: Most mammalian cells and their secreted products are covered with a dense coating of sugar chains. In recent years, the technologies for studying these oligosaccharide chains on glycoconjugates have advanced rapidly, allowing an exploration of their biological roles. There is now increasing evidence that they play many important roles in complex organisms, ranging from simple structural integrity to the mediation of cell-cell interaction, intra- and inter-cellular trafficking, and cell growth and differentiation. Work in this laboratory is driven by the hypothesis that the more specific biological role of oligosaccharides are likely to be mediated by unusual terminal structures or by modifications such as O- acetylation, phosphorylation and sulfation. Our overall goal is to explore the biosynthesis, regulation and function of such oligosaccharide structures in health and disease. In work supported by this grant, we have identified and partially characterized novel families of sulfated N-linked oligosaccharides that are enriched in pulmonary artery endothelial cells, as well as in lung tissue. These include multi-antennary complex-type oligosaccharides carrying sialic acids and O-Sulfate esters (SSOs) and N-linked molecules carrying glycosaminoglycan-like chains attached to complex-type oligosaccharides (NLGs). We have also developed a novel technique for tagging free oligosaccharides via their reducing terminus with biotinylated amino- pyridine (BAP). In the current project period, we will continue the structural analysis of these molecules and use structurally defined BAP- SSO adducts to search for specific receptors for these oligosaccharides, and to raise monoclonal antibodies directed against them. In the course of these studies, we have identified a novel class of sulfated glycosaminoglycans that are calcium-dependent high affinity ligands for the leucocyte adhesion molecule, L-selectin. These appear to be distinct from the NLGs and SSOs mentioned above, and from the previously described mucin-like sulfated ligands for L-selectin on the high-endothelial venules (HBV) of lymph nodes. In the current project period, we will carry out a detailed structural comparison of these sulfated ligands, and identify the minimal oligosaccharide structural requirements for the biologically relevant interactions. We will also study the biosynthesis of these molecules in isolated intact Golgi-enriched preparations, and create novel fluorescent biotinylated primers for their biosynthesis in intact cells. Altered glycosylation is an universal feature of cancer cells, generating tumor antigens and diagnostic markers, and apparently mediating some of their abnormal behavior. It is likely that metastatic tumor cells subvert the mechanisms that normally mediate the migration and invasion of blood cells into tissues. Since endothelial cells must play crucial roles in the metastatic process, our study of their sulfated oligosaccharides could have an important bearing on the design of therapeutic interventions in cancer.
Keywords: mucopolysaccharide, neoplastic cell, oligosaccharide, sulfate, Golgi apparatus, adduct, aminopyridine, carbohydrate biosynthesis, carbohydrate receptor, carbohydrate structure, glycosylation, metastasis, oncoprotein, selectin, sialate, vascular endothelium, animal tissue, fluorescent dye /probe, high performance liquid chromatography, laboratory mouse, laboratory rabbit, laboratory rat, monoclonal antibody, tissue /cell culture
Project start date: 1985-08-01
Project end date: 1999-11-30
3R01CA038701-13S1 (1999): $12624
N-LINKED OLIGOSACCHARIDES OF NORMAL AND MALIGNANT CELLS
Ajit P Varki, Professor Of Cellular & Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01CA038701-13 from National Cancer Institute IRG: PBC
Abstract: Most mammalian cells and their secreted products are covered with a dense coating of sugar chains. In recent years, the technologies for studying these oligosaccharide chains on glycoconjugates have advanced rapidly, allowing an exploration of their biological roles. There is now increasing evidence that they play many important roles in complex organisms, ranging from simple structural integrity to the mediation of cell-cell interaction, intra- and inter-cellular trafficking, and cell growth and differentiation. Work in this laboratory is driven by the hypothesis that the more specific biological role of oligosaccharides are likely to be mediated by unusual terminal structures or by modifications such as O- acetylation, phosphorylation and sulfation. Our overall goal is to explore the biosynthesis, regulation and function of such oligosaccharide structures in health and disease. In work supported by this grant, we have identified and partially characterized novel families of sulfated N-linked oligosaccharides that are enriched in pulmonary artery endothelial cells, as well as in lung tissue. These include multi-antennary complex-type oligosaccharides carrying sialic acids and O-Sulfate esters (SSOs) and N-linked molecules carrying glycosaminoglycan-like chains attached to complex-type oligosaccharides (NLGs). We have also developed a novel technique for tagging free oligosaccharides via their reducing terminus with biotinylated amino- pyridine (BAP). In the current project period, we will continue the structural analysis of these molecules and use structurally defined BAP- SSO adducts to search for specific receptors for these oligosaccharides, and to raise monoclonal antibodies directed against them. In the course of these studies, we have identified a novel class of sulfated glycosaminoglycans that are calcium-dependent high affinity ligands for the leucocyte adhesion molecule, L-selectin. These appear to be distinct from the NLGs and SSOs mentioned above, and from the previously described mucin-like sulfated ligands for L-selectin on the high-endothelial venules (HBV) of lymph nodes. In the current project period, we will carry out a detailed structural comparison of these sulfated ligands, and identify the minimal oligosaccharide structural requirements for the biologically relevant interactions. We will also study the biosynthesis of these molecules in isolated intact Golgi-enriched preparations, and create novel fluorescent biotinylated primers for their biosynthesis in intact cells. Altered glycosylation is an universal feature of cancer cells, generating tumor antigens and diagnostic markers, and apparently mediating some of their abnormal behavior. It is likely that metastatic tumor cells subvert the mechanisms that normally mediate the migration and invasion of blood cells into tissues. Since endothelial cells must play crucial roles in the metastatic process, our study of their sulfated oligosaccharides could have an important bearing on the design of therapeutic interventions in cancer.
Keywords: mucopolysaccharide, neoplastic cell, oligosaccharide, sulfate, Golgi apparatus, adduct, aminopyridine, carbohydrate biosynthesis, carbohydrate receptor, carbohydrate structure, glycosylation, metastasis, oncoprotein, selectin, sialate, vascular endothelium, animal tissue, fluorescent dye /probe, high performance liquid chromatography, laboratory mouse, laboratory rabbit, laboratory rat, monoclonal antibody, tissue /cell culture
Project start date: 1985-08-01
Project end date: 1999-11-30
5R01CA038701-13 (1998): $346211
5R01CA038701-12 (1997): $334391
BIOLOGY OF SIALIC ACID SUBSTITUTIONS
Ajit P Varki, Professor Of Cellular & Molecular Medici
Moores Ucsd Cancer Centeruniversity Of California San Diego
9500 Gilman Dr, Dept 0934
la Jolla, Ca 920930934
Grant 5R01GM032373-12 from National Institute Of General Medical Sciences IRG: PC
Abstract: Many specific biological functions have been attributed to sialic acids. However, most studies of sialic acids do not take into account the diversity generated by modifications of the parent molecule, such as 0-acetylation of the side chain. During the prior funding period, we have developed new and sensitive methods for the analysis of modified sialic acids, identified some new and unexpected molecules, studied sialic acidO-acetyltransferases from E.Coli., rat liver and human melanoma, identified and characterized sialic acid-specific 0-acetyl-esterases, studied the biosynthesis and turnover of the N-glycolyl group of sialic acids, and discovered a novel de-N-acetylation/re-N-acetylation reaction in melanoma gangliosides. Most recently, we have developed a novel approach to abrogate O-acetylation in the early mouse embryo, and in selected tissues of transgenic mice. In the upcoming grant period, we will focus our attention upon the following 1. Purification, reconstitution and characterization of the rat liver Sialic Acid O-acetyltransferase(s). 2. Subcellular distribution of O-acetylated sialic acids, and the related enzymes in rat liver. 3. Purification, reconstitution and properties of human melanoma Sialic Acid O-acetyltransferase(s). 4. Mechanisms for de-N-acetylation and re-N-acetylation of sialic acids on gangliosides. 5. Molecular cloning of cDNAs encoding sialic acid specific O-acetyltransferase(s). 6. Abrogation of O-acetylation in the early mouse embryo. 7. Abrogation of O-acetylation in selected tissues of transgenic mice. In the long run, these studies will help us to understand the biological roles of sialic acids and their modifications in health and disease. Our findings to date implicate these molecules in a variety of important roles, including protection from microbial organisms on mucosal surfaces, the modulation of complement activation, the expression of oncofetal antigens in various tumors, and the regulation of cell-cell interaction during the development of certain organs
Keywords: acetylation, carbohydrate biosynthesis, sialate enzyme mechanism, enzyme reconstitution, ganglioside, hydrolase, liver metabolism carbon, complementary DNA, embryo /fetus, enzyme linked immunosorbent assay, laboratory mouse, laboratory rabbit, laboratory rat, molecular cloning, monoclonal antibody, neoplastic cell, protein purification, radionuclide, radiotracer, tissue /cell culture, transgenic animal, tritium, western blotting
Project start date: 1983-08-01
Project end date: 1995-07-31
5R01GM032373-12 (1994): $269390
5R01GM032373-11 (1993): $254213
COMPARTMENTATION OF OLIGOSACCHARIDE BIOSYNTHETIC MACHINE
Ajit P Varki, Professor Of Cellular And Molecular Medici
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P01CA058689-100010 from National Cancer Institute
Abstract: Many normal and pathological functions have been attributed to the oligosaccharide chains on glycoconjugates. With few exceptions, the biosynthesis of these chains and their modifications takes place during trafficking through the Endoplasmic Reticulum-Golgi-Plasmalemma pathway. These glycosylation changes are convenient signposts for the definition of distinctive compartments in these pathways. On the other hand, the pathways can be viewed as kinetic events in the step-wise biosynthesis of biologically important glycoconjugates, that are eventually expressed at the cell surface to serve many biological roles. Each step in the N-linked oligosaccharide (NLO) processing pathway occurs in compartments of the ER- Golgi-PM pathway. Most steps requires a specific nucleotide donor, the corresponding nucleotide transporter, the correct transferase enzyme, and an appropriate endogenous acceptor to all be present at the same time in the same compartment. These complex interactions are not easily studied by pulse-chase analyses, because of the rapid rate of inter-compartmental trafficking. On the other hand, approaches that artificially arrest transport may alter or distort the natural situation. We have demonstrated that all of these steps can be successfully reconstituted in vitro by adding labelled nucleotides to isolated intact membrane preparations from normal tissues and cells. In this setting, inter-compartmental transport is absent, incorporation of label requires intactness of the compartment, and the endogenous products re found to be in the correct topology. This approach also makes it possible to simultaneously study different compartment without fear of mixing them together. The labelled oligosaccharides can then be structurally characterized, giving valuable information regarding the organization and function of the compartments in question. This project focuses on two specific biosynthetic reactions involving N-linked chains the addition of GlcNAc-1-P units to mannose residues of lysosomal enzymes (an early step), and the addition of 9-0- acetyl esters to the side chains of sialic acid residues on complex-type plasma membrane glycoproteins (a very late step). Studies will be done both in rat liver membrane preparations in permeabilized CHO cells previously engineered to express specific lysosomal enzymes and/or glycosyltransferases. Inter-compartmental transport will be reconstituted by adding back cytosolic factors, and changes in the pre-labelled chains used to elucidate the requirements for trafficking. Finally, these reactions will be compared between the golgi apparatus of normal and malignant hepatocytes, exploring the hypothesis that altered glycosylation in tumor cells is partly explained by a loss of fidelity in Golgi organization. This project directly relates to the primary theme of this program, the study of membrane trafficking, and the role of cytosolic factors in controlling this process. In the long run, the goal is to understand the normal organization of N-linked glycosylation in the Golgi apparatus, its relationship to inter-compartmental trafficking, and the changes that occur in malignant disease.
Keywords: biological signal transduction, carbohydrate biosynthesis, intracellular transport, membrane activity, oligosaccharide, Golgi apparatus, cell membrane, endoplasmic reticulum, enzyme activity, enzyme mechanism, ester, glycoprotein, glycosylation, guanosinetriphosphatase, lysosome, sialate, laboratory mouse, laboratory rat, liver cell
Project start date: 2002-04-01
Project end date: 2003-03-31
Sponsored Links Excellgen http://Excellgen.com