Driving Biomedical Projects (DBP)

We have carefully selected two distinct sets of DBPs that drive most of the technical innovation outlined in this proposal.  The first set of DBPs are focused on the development of specific tools to impact significantly externally funding research projects.  The second set of DBPs are more focused on systems biology approaches to gain a better understanding of mammalian glycosylation and the role it plays in disease.  These DBPs require robust datasets from a large collection of cell lineages and genetic perturbations in glycosylation machinery. Of course, these two sets of DBPs are also augmented by our multiple biomedical collaborations as well as our industry and academic mass spectrometry facility partners.

For the first set of investigators (Contessa, Dahms, Hart, Hoffmeister, Lau, and Wang), we specifically have chosen leaders and pioneers in their respective fields whose research endeavors are inhibited due to the lack of appropriate, robust, and/or high-throughput analytical techniques to address their biological questions and thus would significantly benefit from the development of particular new and improved tools to investigate glycosylation.  We have brought in several investigators, including Hoffmeister, Lau, and Contessa, who will drive the development of our glycoprotein cell surface “capture and define” technology, including proteomics, site-mapping and direct glycopeptide analysis, and will benefit from our ability to make specific hES-derived cell lineages with specific glycosyltransferases knocked-out by CRISPR-Cas methodology.  Likewise, we have partnered with several investigators, including Hart, Hoffmeister, and Lau, whose needs will drive our maturation of the static IDAWG approach for glycans and the need for developing our dynamic IDAWG approach for glycans and glycopeptides.  We continue to partner with 2 existing DBPs (Dahms and Strauss) who will push our tool development with their needs for high-throughput, quantitative N- and O-glycan, as well as GSL, analyses. Wang as a DBP helps to drive the development of glycotranscriptomics and visualization tools.  Along with these DBPs, several of our collaborators also help to drive the development of the tools and resources outlined in the TR&Ds.

For the second set of investigators (Aoki-Kinoshita, Lewis) we have chosen leaders in the fields of glycan pathway modeling and informatics analysis. In these cases the investigators are developing state-of-the-art approaches for modeling complex glycan pathway data, including pathway flux analysis, but have struggled to obtain appropriate data sets that can be used to test their modeling systems. The reporter glycoproteins expressed in pluripotent and differentiated hESC lines and in hESC lines harboring targeted glycogene knockouts will be analyzed as end-point glycosylated products in TR&D1. The resulting glycomic and transcriptomic profiles are ideal data sets for the type of modeling done in both the Aoki-Kinoshita and Lewis DBPs that use distinct approaches for pathway-based flux analysis. These DBPs also bridge to the technologies developed in TR&D3 for integrated pathway visualization.  The Glycan Pathway Visualization tool will provide the ability to integrate pathway modeling output with comparative data from glycan and transcriptome profiling efforts. Interpretation of the integrated data in the context of a unified pathway diagram display framework will inform not only additional modeling efforts and optimization but also direct additional genetic perturbations of the reporter cell lines. Thus, an interactive and reciprocal set of driving interactions is anticipated with these DBPs. Additional challenges will be found in the DBP with Wang, where expansion of pathway visualization (TR&D 3) will be required for other glycan classes (proteoglycan biosynthesis) in order to integrate proteoglycan transcriptome profiling (TR&D1) with proteoglycan structural data generated in the Wang lab.

Our DBPs for this renewal application include:

BTRR Personnel Involved:  Lance Wells, Richard Steet, Will York

TR&D projects involved:  #1, #2, and #3

DBP Driver Statement: This DBP acts as a test bed for technology development through the generation of inhibitors that selectively block N-glycosylation via effects on specific OST catalytic subunits (e.g. STT3A and STT3B).  The recent identification of novel N-glycosylation inhibitors that target the catalytic subunits of the oligosaccharyltransferase (OST) complex has created challenges in defining which glycosites are impacted on a wide range of protein substrates.  This DBP will engage multiple TR&Ds to help drive technology development for the rapid detection of site-occupancy on glycoproteins.  This information is critical for the discovery of receptors that are sensitive to N-glycosylation inhibition and thus the identification of cell surface glycoproteins is also a pressing need.  These receptors represent targets for the study of radiosensitivity in cancer cells.

Expected Innovations:  The BTRR investigators will develop new technology that allows site occupancy by N-linked type (highMan/hybrid versus complex) to be assessed on a global scale on the cell surface using the SEEL surface glycoprotein capture and assignment technology we are continuing to develop.  Such analyses will be key as new inhibitors with different specificities are generated by the Contessa laboratory and these approaches will have far-reaching potential to be utilized by others interested in surface glycoproteins and site-occupancy.

Start Date:  1/16

External Funding:  NIH/NCI R01CA172391 Targeted N-linked Glycosylation to Enhance Radiation Therapy

Publications to Date:  Lopez-Sambrooks, Shrimal S, Khodier C, Flaherty DP, Rinis N, Charest JC, Gao N, Zhao P, Wells L, Lewis TA, Lehrman MA, Gilmore R, Golden JE, and Contessa JN, Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells. Nat Chem Biol. 2016, 12:1023-30.

BTRR Personnel Involved:  Michael Tiemeyer, Kazuhiro Aoki

TR&D projects involved:  #1 and #2

DBP Driver Statement:  Dr. Dahms has generated a novel rat model for Fabry Disease, a glycosphingolipid degradation disorder, that phenocopies the human disease better than currently available mouse models, offering new possibilities for assessing therapeutic strategies.  Comprehensive glycomic characterization of the impact of Fabry disease on human tissues has not been possible and the mouse model is not an ideal choice for glycomic characterization based on its phenotypic presentation.  The rat model provided an opportunity to challenge our comprehensive glycomic, glycopshingolipidomic, and glycoproteomic analytic platform across multiple tissue/sample types (blood, urine, brain, kidney, dorsal root ganglia, eye, liver, muscle) and multiple genotypes.

Expected Innovations:  Novel opportunities for characterizing broad glycomic responses to altered glycosphingolipid catabolism and for focused characterization of specific, rare glycosphingolipid degradation/storage products.

Start Date:  1/16

External Funding: NIH/NINDS R21NS095627, Fabry Disease in the GLA Knockout Rat:  Development of Novel Protein Therapeutics

Publications to date:  Manuscript submitted — James J. Miller1, Kazuhiro Aoki1, Francie Moehring, Carly A. Murphy, Paula E. North, Iris S. Kassem, Michael Tiemeyer2, Cheryl L. Stucky2, Nancy M. Dahms2 (2017) Rats deficient in α-galactosidase A recapitulate Fabry disease phenotypes.  1equal contributors, 2co-corresponding authors

BTRR Personnel Involved:  Lance Wells, Will York

TR&D projects involved:  #1 and #2

DBP Driver Statement: Dr. Hart is the discover of and leader in the field of the O-GlcNAc modification that occurs on thousands of nuclear and cytosolic proteins.  This modification, that is involved in a variety of diseases, is thought to be both inducible and dynamic.  However, the dynamic nature of the modification has only been directly measured on a small handful of proteins due to the difficulty of the experimental design.  With the advent of dynamic IDAWG, we have the opportunity to shift this approach from straight glycan work to looking at glycopeptides.  Thus, our hope is to be able to measure the dynamics of O-GlcNAc at many sites on a multitude of proteins simultaneously and then examine the impact of various stimulations on those dynamics with Dr. Hart and his group.

Expected Innovations:  This DBP drives TR&D1 taking the IDAWG approach and applying to glycopeptides (O-GlcNAc peptides) to use it for both comparative glycoproteomics but also to look at dynamics using the dynamic IDAWG approach.  This approach also requires that TR&D2 adjust their software solutions for IDAWG to be applicable to both free glycans as well as glycopeptides.  It is also possible that this DBP will expand to include TR&D3 as the Hart laboratory has specific interests in human cardiovascular lineages that are a specific strength of the Dr. Dalton’s group.

Start Date:  08/17

External Funding: NIH/NHLBI P01HL107153 Glycoconjugates and Cardiovascular Disease

BTRR Personnel Involved:  Lance Wells, Will York, Richard Steet, and Steve Dalton

TR&D projects involved:  #1, #2, and #3

DBP Driver Statement: Dr. Hoffmesiter has a long-standing interest in understanding the role of cell surface glycans in platelets and glycan remodeling in hematopoetic cell lineages.  One of the major challenges presenting to the TR&Ds is the ability to capture cell surface glycoproteins, look at cell surface glycans as opposed to whole cell glycans, and to interrogate remodeling in a variety of derived cell lineages.  Dr. Dalton’s group will derive appropriate lineages in TR&D3, Dr. Wells’ and Dr. Steet’s group will use SEEL and IDAWG approaches to determine cell surface glycoproteins, perform cell surface glycomics, and then interrogate remodeling over time using the dynamic IDAWG approach being developed in TR&D1.  The automation of generating results from the complex datasets will be done by Dr. Will York’s group in TR&D2.

Expected Innovations:  This DBP drives both the cell surface capture and identification of glycoproteins, cell surface glycomic analyses, and the dynamic IDAWG technologies being developed by TR&D1 and result generation platforms of TR&D2.  Further, given the focus of Dr. Hoffmeister’s work on sialyltransferases and hematopoetic lineages, the efforts of TR&D3 will come into play with regards to glycosyltransferase knock-out and derived lineages from human ES cells.

Start Date:  09/17

External Funding: NIH/NHLBI R01HL089224 Carbohydrate mediated platelet clearance

BTRR Personnel Involved:  Lance Wells, Will York, Richard Steet, and Steve Dalton

TR&D projects involved:  #1, #2, and #3

DBP Driver Statement: Dr. Lau is the leader in the field in extrinsic signaling and as such has significant interest in the role of soluble ST6GAL1.  Dr. Lau and Dr. Hoffmeister (a separate DBP) have significant overlap in their interests and collaborate with one another as well as with investigators of the BTRR.  In particular, Dr. Lau is interested in determining cell surface proteins and specific glycan structures in specialized lineages that are targets for exogenous ST6GAL1.  TR&D3 will assist with generating any hES-derived cell lines needed while TR&D2 will continue to develop SEEL technology for improved surface protein and glycan assignment with the data analyses help of TR&D2. Dr. Lau is also interested in sialic acid turnover on the cell surface of cells and thus will be a driver for the dynamic IDAWG technology we have under development.

Expected Innovations: This DBP drives both the cell surface capture and identification of glycoproteins, cell surface glycomic analyses, and the dynamic IDAWG technologies being developed by TR&D1 and result generation platforms of TR&D2.  Also, given Dr. Lau’s interest in the cells modified and the source of soluble glycosyltansferases, TR&D3’s expertise in deriving lines and generating glycosyltransferase knock-outs will be called on.

Start Date: 08/17

External Funding:  NIH/NAID RO1AI56082 ST6Gal Sialyltransferase in Hematopoiesis and Alliance Foundation Grant from Roswell Park Cancer Institute

BTRR Personnel Involved:  Michael Tiemeyer, Kazuhiro Aoki, Will York

TR&D projects involved:  #1 and #2

DBP Driver Statement:  The Clinic for Special Children (CSC) offers access to a unique cohort of human genetic disorders that present with phenotypes consistent with altered protein or lipid glycosylation.  The Clinic serves the Amish and Mennonite communities in Pennsylvania and beyond and also includes a world-class facility for molecular genetic studies.  TR&D1 has collaborated closely with CSC to expand our capacity to perform glycosphingolipidomics on patient samples, primarily serum and blood cells, through the analysis of patients deficient in ganglioside biosynthesis.  The pipeline of samples from the CSC has allowed us to achieve higher throughput in our glycosphingolipidomic work flow by optimizing sample handling and by driving the development of software and databases for automated or semi-automated annotation of MS spectra.  The analysis of additional genetic disorders requires us to continue to increase our sample throughput in order to assess inheritance across pedigrees and the impact of homozygosity/heterozygosity on biochemical and clinical phenotypes.  The CSC has a stockpile of 40-50 disorders awaiting glycomic analysis.

Expected Innovations:  Enhanced throughput for glycomic analysis arising from optimized sample handling and increased automation of MS data.

Start Date:  1/14

External Funding:  W.M. Keck Foundation, “The glycomics of human neurodegenerative, developmental and cognitive disorders.”

Publications to Date:  Manuscript submitted — Kazuhiro Aoki, Adam D. Heaps, Kevin A. Strauss1, Michael Tiemeyer1 (2017) Quantification of plasma glycosphingolipids by nanospray ionization mass spectrometry: application to human ST3GALGM3 ganglioside deficiency. , 1co-corresponding authors

Boccuto L1, Aoki K1, Flanagan-Steet H, Chen CF, Fan X, Bartel F, Petukh M, Pittman A, Saul R, Chaubey A, Alexov E, Tiemeyer M2, Steet R2, Schwartz CE2 (2014) A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation.Hum Mol Genet. 23, 418-33. PMID: 24026681.  1equal contributors, 2co-corresponding authors

BTRR Personnel Involved: Kelley Moremen, William York

TR&D projects involved: #1, #2, and #3

DBP Driver Statement: Dr. Wang has a continued interest in understanding the regulatory mechanisms for the biosynthesis and function of proteoglycans in animal systems. Their recent focus has been on the generation of cell lines and animal models harboring defects in heparan sulfate with a goal of determining the biological functions of proteoglcyans attached to various core proteins under various physiological and pathological conditions, such as angiogenesis, leukocyte trafficking/inflammation, cancer and stem cell biology. The studies in the Wang lab are complementary with the ESC studies proposed in TR&D3, where N-glycan, O-glycan and glycolipid biosynthetic genes are being targeted for gene knockouts with subsequent analysis of the resulting glycan chains. The gene targeting studies in the Wang lab focused on individual pathway steps in heparan sulfate and chondroitin sulfate biosynthesis and have begun to dissect the contributions of these genes on the resulting corresponding proteoglycan structures and subsequent biological function. The ability to profile transcriptome abundance in the respective cell lines has been critical in capturing the compensatory mechanisms for alterations in gene expression that occur in response to the gene knockouts. Surprisingly significant changes in gene expression have been observed for the proteoglycan biosynthetic machinery and the core proteins that harbor proteoglycan chains in the first of the heparan sulfate gene knockout cell lines. This analysis has led to new hypotheses regarding regulatory mechanisms that control proteoglycan structures in animal cells and drives the need for a broader transcript profiling approach by RNA-Seq to dissect the signaling pathways that lead to these feedback signals. In addition, the ability to map these transcript data on biosynthetic pathway maps with corresponding proteoglycan structure data will provide an intuitive view of the compensatory changes in gene expression in response to the gene knockouts and help in interpretation of the alterations in glycan structures that are observed in these cell samples. The overall goals are to provide analytical tools for the characterization of cell lines and visualization tools for interpretation of the compensatory changes that occur in response to pathway perturbation as a framework for understanding the cellular functions of proteoglycan structures in animal cells.

Expected Innovations:  This DBP drives the development of expanded capabilities for transcript profiling (TR&D1), mapping of genes to biosynthetic pathways (TR&D2), and visualization of glycan structures and transcriptome data in a unified pathway representation (TR&D3) for a different class of glycan structures beyond the scope of the analytical studies in TR&D1. The extension of the transcriptome analyses and visualization into new glycan pathways will be a demonstration of the extensibility of these tools to outside users in a flexible manner to address critical biological questions.

Start Date:  09/17

External Funding: NIH/NCI R21CA199878 Using CRISPR-Cas9 Technology to Develop a Mutant Cell Library for Heparan Sulfate Structure-Function Study, NIH/NIGMS P41GM103390 Resource for Integrated Glycotechnology

Publications to Date:  Zhang, B., Xiao, W., Qiu, H., Zhang, F., Moniz, H., Jaworski, A., Condac, E. Gutierrez-Sanchez, G., Heiss, C., Clugston, R., Azadi, P., Greer, J.J., Bergmann, C., Moremen, K.W. Li, D.Y., Linhardt, R., Esko, J.D., Wang, L., (2014) Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia. J. Clin. Invest.124, 209-221. [PMID:24355925] [PMC3871243], Kraushaar, D.C., Rai, S., Condac, E, Nairn, A., Zhang, S., Yamaguchi, Y., Moremen, K.W., Dalton. S., Wang, L. (2012) Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells. J. Biol. Chem. 287, 22691-700 [PMID 22556407] [PMC3391080]

BTRR Personnel Involved:  Will York, Kelley Moremen, Lance Wells, Michael Tiemeyer, Steve Dalton, Richard Steet

TR&D projects involved:  #1, #2, and #3

DBP Driver Statement: Dr. Lewis has a continued interest in the computational modeling and regulation of N-glycan biosynthesis. Their group developed low parameter tools for modeling glycan biosynthetic pathway data for use in understanding regulatory nodes and pathway flux in mammalian cell factories for biopharmaceutical production and other cell systems. The glycan data sets on secreted reporter glycoproteins from wild type and gene disrupted ES cell lines are ideal data sets for developing computational models of glycan maturation in the mammalian secretory pathway. The ability to examine unique differentiated cell types and targeted pathway disruption will allow the testing of the modeled pathway flux in a controlled system with a single cellular genotype providing the ideal system to ensure the most accurate models and potential for new discoveries around the regulation and function of glycosylation pathways.  In addition, the visualization tools for glycan metabolic pathway display system will provide a unified and integrated visualization framework to represent changes in glycan structural abundance and correlate them with glycogene transcript abundance and modeled enzyme/reaction abundance values generated in the Lewis lab to streamline the identification of regulatory networks and biosynthetic bottlenecks that will aide in the interpretation of the modeling data.

Expected Innovations:  This DBP drives the development of appropriate glycoprotein reporter systems, targeted gene disruptions, and ESC differentiation pathways (TR&D3) that provide effective model systems for pathway analysis. It also drives the acquisition of comprehensive glycome data sets from glycoprotein reporters and total cellular glycome and transcriptome data sets as input for the modeling studies (TR&D1). It also drives the ability to process the raw data into appropriate input values for the modeling studies (TR&D2) and comprehensive pathway visualization (TR&D3).

Start Date:  09/17

External Funding: NIH/NIGMS R35GM119850 Unraveling the mammalian secretory pathway through systems biology and algorithm development

BTRR Personnel Involved: Kelley Moremen, Will York, Lance Wells, Michael Tiemeyer, Steve Dalton, Richard Steet

TR&D projects involved:  #1, #2, and #3

DBP Driver Statement: Dr. Aoki-Kinoshita’s laboratory is also developing computational N-glycan biosynthesis models using methodologies (e.g., rule based systems) (reference) that are distinct from those used by Dr. Lewis. Her ability to generate realistic and predictive models are currently hampered by the limited availability and inconsistent representation of suitable data sets that can be used to inform and test them. The comprehensive collection of data sets that will be provided by BTRR over the funding period will provide critical data for testing/validating Dr. Aoki-Kinoshita’s N-glycan biosynthesis models. Her simulation and data mining efforts have the potential to reveal new aspects of this complex process that are not otherwise apparent and help in formulating new hypothesis that will be tested using the methods developed in all three TR&Ds. For example, our plans to genetically modify stem cells (TR&D3) will be considerably influenced by her models and the hypotheses they generate; TR&D1 will analyze these cell lines to produce the analytical data used for model testing and validation; TR&D2 will develop methods required to assemble and exchange robustly annotated data sets. In addition, the curated metabolic pathways we develop (TR&D3 in collaboration with Prof. Kinoshita’s lab) will be shared with the Japanese GlyCosmos Database, which stores two integrated datasets (molecular structures of glycans and pathway information pertaining to glycogenes), making this knowledge more accessible to the scientific community and eliciting feedback that will enhance our ability to provide the most accurate models of glycan biosynthesis.

Expected Innovations: This collaboration will lead to the development of improved models for glycosylation pathway reactions and better methods to test the hypotheses that these models generate.  Taken together, our combined efforts will lead to an integrated modeling approach that includes algorithm development, model generation, identification of targets for genetic manipulation, and testing with explicit data obtained by analysis of the modified cell lines.

Start date: 10/17

External Funding: TBD