Collaborative Projects

This page describes nine separate projects that we have designated "Collaborative Projects". Each Core has its own complement of collaborators, but the projects that follow are considered to be formal "test-beds" for Resource technology development. Since the Resource is an Integrated endeavor, multiple Cores are involved in almost every Collaborative Project; the main Resource collaborators are listed, however, for each Project. If a project that is submitted to the Resource is deemed not to constitute a test-bed, i.e., the investigator simply wants a sample(s) to be analyzed, then the project is assigned to Dr. Azadi, who directs the Analytical Service program. Depending on work-load and potential, however, a service project may be worked on by the Cores in anticipation that initial results may promote it to Collaborative Project status. Below is a listing and brief summary of the nine Collaborative Projects included in this application, followed by the more detailed descriptions of the projects.


Collaborative Project 1: IDENTIFICATION OF GLYCAN EPITOPES ON THE SURFACE OF HUMAN EMBRYONIC STEM CELLS (hESCs) AND DIFFERENTIATED CELL LINEAGES
PI: Lara Mahal, Ph.D.,
University of Texas, Austin
Funding: NSF
Resource Collaborators: Stephen Dalton, Ph.D., Core 1;
Gerardo Alvarez-Manilla, Ph.D., Core 2
Michael Pierce, Ph.D., Core 2

This collaborative project with Dr. Mahal is focused on applying her lectin microarray technology to differentiating stem cells to compare its accuracy and relative sensitivity for detecting minor glycan species. The Mahal technology is based on producing large micellular vesicles from the plasma membrane, labeling them with fluorescent Cy dyes, and measuring their binding to over 75 lectins that comprise microarrays on glass slides. The technique is rapid and would have applications for many laboratories for the profiling of lectin binding differences (i.e., glycan expression differences) between differentiating cell populations.


Collaborative Project 2: SIALOMUCINS AND MUCIN-LIKE PROTEINS IN STEM CELL DIFFERENTIATION
PI: Paul Simmons, Ph.D.,
University of Texas Health Sciences Center at Houston
Funding: Project Grant (#350236),
National Health and Medical Research Council, Australia
Resource Collaborators: Lance Wells, Ph.D., Core 2
Michael Tiemeyer, Ph.D., Core 2
Ron Orlando, Ph.D., Core 2
Michael Pierce, Ph.D., Core 2
Stephen Dalton, Ph.D., Core 2
Kelley Moremen, Ph.D. Core 3

Dr. Simmons studies the differentiation of bone marrow stem cells, as well as other cells in the vascular system and is particularly interested in the glycoproteins known as the sialomucins. Several lines of evidence suggest that these molecules show differences in O-linked glycosylation depending on the cell type that is expressing them and its state of differentiation. This project will focus on defining these differences structurally using glycan analysis technologies under development in Core 2. The material to be analyzed will range from whole cells to immunoprecipitated mucins such as CD34. The ability to analyze complicated O-linked glycans from small amounts of starting material will push the sensitivities of the technologies developed by Core 2. The results of this collaboration have significant implications for understanding mucin function in vascular physiology, such as L-selectin binding prior to extravasation, and during differentiation of hematopoetic and mesodermal stem cells.


Collaborative Project 3: IDENTIFICATION OF GLYCOPROTEINS EXPRESSING THE GLYCAN, O-MAN, IN NORMAL MOUSE BRAIN AND THE CHARACTERIZATION OF GLYCOSYLATION CHANGES IN MOUSE BRAINS LACKING POMGnT-1 OR Large
PI: Huaiyu Hu, Ph.D.,
SUNY Syracuse, Department of Neuroscience and Physiology
Funding: NIH RO1 HD044011
Resource Collaborators: Michael Tiemeyer, Ph.D., Core 2
Lance Wells, Ph.D., Core 2
Kelley Moremen, Ph.D. Core 3
Michael Pierce, Ph.D., Core 2

A relatively newly discovered glycoprotein glycosylation involves the mannosylation of ser/thr residues, followed by beta(1,2)GlcNAc transfer to the mannose by the enzyme POMGnT-I. Mutations in POMGnT-I have been shown to cause a defect in cell adhesion between nerve and muscle, causing a form of muscular dystrophy known as muscle-eye-brain disease. The main glycoprotein that is glycosylated by POMGnT-I is alpha-dystroglycan. This enzyme is also highly expressed in brain tissue, but its glycoprotein acceptors are not known. Dr. Huaiyu Hu has produced a POMGnT-I null mouse with a distinct brain cell migration defect. The first part of this collaborative project with Cores 2 and 3 is to identify glycan structural changes in the POMGnT-I null mice and mice with that lack a glycosyltransferase-like protein known as Large, which may also participate in the expression of O-Man-GlcNAc glycans in brain. Core 3 will determine if there are transcript changes in other brain glycosyltransferase expression in these mutant mice. Alpha-dystroglycan is a likely candidate for at least one of the prominent O-mannosylated proteins in brain; thus, its glycosylation will be studied in detail (see also Collaborative Project #5). In addition, a novel means to label proteins with exposed O-Man residues that result from the elimination of GnT-I expression in the null mice has been developed to identify the set of brain glycoproteins that are O-mannosylated.


Collaborative Project 4: N-LINKED GLYCOSYLATION IN ADULT NEURAL TISSUE OF DROSOPHILA WITH AN EMPHASIS ON SIALYLATION
PI: Vladislav Panin, Ph.D.,
Texas A&M University, Department of Biochemistry
Funding: NIH RO1 GM069952-01
Resource Collaborators: Lance Wells, Ph.D., Core 2
Michael Tiemeyer, Ph.D., Core 2
Parastoo Azadi, Ph.D., Analytical Service

With all of the genetic tools available for the study of Drosophila, little was known about N-linked glycans until recently Core 2 investigators Tiemeyer and Wells published the N-linked structures found in embryos using their TIM method of analysis. In this collaborative project with Dr. Panin, these investigators have already performed experiments analyzing the amino acid sequence preferences surrounding N-linked glycosylation sites on 197 Drosophila proteins. As part of this proposal, the collaborators will perform glycan analysis (N- and O-linked) of tissues enriched in neural cells and identify many of the glycoproteins that express N-linked glycans using lectin affinity chromatography to enrich for them. Now that the sialyltransferase (dSiaT) has been shown to synthesize endogenous glycans, it is of considerable interest to use this enzyme as a "reporter" glycoprotein and identify its site-specific glycosylation when expressed in S2 cells. This project will be performed by Dr. Azadi, Director of the Analytical Service program of this Resource and provide another means to integrate the Service program into the Core research programs and provide Service with newly developed analytical technologies.


Collaborative Project 5: GLYCAN AND GLYCOPROTEOMIC ANALYSIS OF ALPHA-DYSTROGLYCAN
PI: James Ervasti, Ph.D.
Funding: NIH RO1 AR042423-11 (J.E) and
Muscular Dystrophy Association Award (L.W.)
Resource Collaborators: Lance Wells, Ph.D., Core 2
Michael Tiemeyer, Ph.D., Core 2

This collaborative project continues to focus on the site-specific glycosylation of a glycoprotein, alpha-dystroglycan (alpha-DG), expressed both in muscle and neural tissues, that is known to function in nerve-muscle adhesion (also please see Collaborative Project #3 above). Core 2 has been involved in mapping the sites of glycosylation, including N-linked, O-GalNAc, and O-Man, on rabbit skeletal muscle alpha-DG. Over 30 O-linked sites have been mapped thus far. The collaboration will continue by completing this analysis and extending it to alpha-DG from other tissues, such as brain. In addition, using glycosidase treatments and site-directed mutagenesis, more information on the glycan structures and locations that function in laminin-alpha-DG binding will be explored using a surface plasmon resonance assay system.


Collaborative Project 6: GLYCOMICS OF PROTEOGLYCAN BIOSYNTHESIS IN MURINE EMBRYONIC STEM CELL DIFFERENTIATION
PI: Robert J. Linhardt, Ph.D., RPI
Funding: NIH grants HL62244, HL52622, and GM38060
Resource Collaborators: Alison V. Nairn, Ph.D., Core 3
Stephen Dalton, Ph.D., Core 1
William York, Ph.D., Core 4
Michael Pierce, Ph.D., Core 2
Kelley Moremen, Ph.D., Core 3
Robert J. Linhart, Ph.D., Core 2

A collaboration between Dr. Linhardt's laboratory and Core 3 has already brought the Resource squarely into glycosaminoglycan (proteoglycan) analysis and biosynthesis. This collaborative effort will continue as we progress in analyzing data and integrating the glycan structural results with those of the glycotranscriptome. These results will be used by Core 4 to populate databases that show proteoglycan biosynthesis and suggest how it is regulated during stem cell differentiation. These studies will then progress into human ESC differentiation and allow comparisons between mouse and human glycosaminoglycan biosynthesis regulation. The next step in understanding the regulation of glycan biosynthesis is likely to be the development of technologies such as the expression of siRNAs specific for particular enzymes in a biosynthetic pathway that show transcript changes during a particular differentiation step. We will use this collaborative project as a test-bed to determine if perturbing the expression of specific enzymes using RNAi knockdowns will indeed point to a means to relate transcript changes with observed glycan expression differences during differentiation.


Collaborative Project 7: GLYCAN EXPRESSION AND REGULATION IN MOUSE PANCREATIC ISLET CELLS FROM WILD-TYPE AND GnT-IVa DEFICIENT (Mgat4a null) MICE
PI: Jamey Marth, Ph.D.
Funding: RO1 DK48247 and
an Investigator Award from the Howard Hughes Medical Institute
Resource Collaborators: Michael Pierce, Ph.D., Core 2
Ron Orlando, Ph.D., Core 2
Gerardo Alvarez-Manilla, Ph.D., Core 2
Michael Tiemeyer, Ph.D., Core 2
Lance Wells, Ph.D., Core 2
Steve Dalton, Ph.D., Core 1
Kelley Moremen, Ph.D., Core 3

Dr. Marth's laboratory has recently demonstrated that GnT-IVa null mice show symptoms of Type 2 diabetes and that this disease is likely caused by changes in the N-linked glycosylation of the Glut-2 transporter in pancreatic islet cells. These changes in glycosylation attenuate the cell surface residence time of the transporter, and thus result in decreased glucose uptake in the presence of insulin. Wild-type animals fed a chronic high fat diet also show down-regulation of GnT-IVa transcription and mimic the phenotype seen in the GnT-IVa null mice. These results strongly suggest that diet can regulate a specific glycosylation event that results in the onset of Type 2 diabetes. This collaboration with Dr. Marth will focus on determining the glycan structural changes that result when the GnT-IVa is eliminated or down-regulated due to a high fat diet. Core 2 will analyze pancreases and islets, as well as immunoprecipitates of the Glut-2 transporter from these preparations to determine the precise structural changes that are involved. Lectin binding data show that the lectin L-PHA does not bind to the N-linked structures on Glut-2 from GnT-IVa null animals, which is not predicted, since this lectin does not normally bind to the branch synthesized by GnT-IVa. These and other data suggest the glycan changes on Glut-2 from the null mice may be unusual. Core 2 will examine in detail the glycans on Glut-2 from wild-type and null mice, as well as those fed a high fat diet to establish the structural basis for the changes in Glut-2 resident time on the cell surface. Another explanation of the data is that down-regulating or eliminating GnT-IVa transcripts affects the expression of other glycosyltransferases in a coordinated manner. Core 3 will examine transcripts from pancreases and islet cells from the various animals to determine if this is the case. This collaboration is likely to have a significant impact on our understanding of the coordination of N-linked glycosylation of receptors and how changes can affect receptor endocytosis.


Collaborative Project 8: GLYCOMIC ANALYSIS OF HUMAN EMBRYONIC STEM CELL (hESC)-DERIVED NEURAL PRECURSOR CELLS
PI: Hudson Freeze, Ph.D.,
Burnham Institute, San Diego
Funding: NIH
Resource Collaborators: Stephen Dalton, Ph.D., Core 1
Gerardo Alvarez-Manilla, Ph.D., Core 2
Lance Wells, Ph.D., Core 2
Michael Tiemeyer, Ph.D., Core 2
Kelley Moremen, Ph.D., Core 3
Michael Pierce, Ph.D., Core 2

One of the most interesting differentiation lineages for hESC is that toward neural precursor cells. The hNPC are the precursors for further differentiation into neurons, oligodendrocytes, and astrocytes. This collaboration is with a group at the Burnham Institute to define glycomic changes during differentiation of hESC to hNPC. Core 3 will define transcriptional changes in genes associated with regulation of the glycome. Core 2 will analyze changes in glycan expression during this differentiation, and this information will be overlayed with the transcript changes to obtain a picture of how the glycome changes, with input from the Bioinformatics Core 4. Significant changes in glycan expression will be exploited to attempt to separate specific subpopulations of differentiated neural cell types. We also anticipate utilizing the microarray described in Collaborative Project #1 to screen for differences in lectin binding that can be used for cell separations. In addition, we will use siRNA expression, as outlined in Collaborative Project 8, to test hypotheses that will allow us to implicate specific regulatory points for changes in specific glycan biosynthesis pathways during differentiation of hESC to hNPC.


Collaborative Project 9: DEVELOPMENT OF COMPREHENSIVE DATABASES FOR GLYCAN STRUCTURE, FUNCTION, AND ANALYTICAL DATA
PI: Claus W. von der Lieth, Dr. Dipl.,
German Cancer Research Center, Heidelberg
Funding: EU Sixth Framework Programme
Resource Collaborators: William York, Ph.D., Core 4
Krzysztof J. Kochut, Ph.D., Core 4
John A. Miller, Ph.D., Core 4

Interactions between Dr. von der Lieth and Core 4 Bioinformatics have been extensive during the first funding cycle of the Resource. The acceptance of GLYDE, the Resource's initial XML format for the unambiguous representation of glycan structures by Dr. von der Lieth, as well as those bioinformaticians in the Consortium for Functional Glycomics, is an example of the exchange of information and initial collaboration between the Resource and the European group (EUROCarbDB). In the next funding cycle, this collaborative project will drive GLYDE II to be expanded and improved in its ability to exchange structural data and display structural representations. A large set of glycan structural records will be generated by the EUROCarbDB Project, for example, searched for errors, and then used to ultimately generate an RDF representation of each structure to populate the GlycO ontology. There will be feedback to the European group that will then allow them to improve the accuracy of their database, which is a fundamental part of the world-wide effort to share data that involve glycan analysis and structural assignments.