Carlito B. Lebrilla - US grants

This grant in the Analytical and Surface Chemistry Program is in the area of mass spectrometry of biomolecules. Professor Lebrilla and his students will use an external source Fourier transform mass spectrometer, FTMS, to measure gas-phase basicities of protonated biomolecules. Semi-empirical molecular orbital calculations will be used to determine the sites of protonation in oligosaccharides. In addition, hydrogen/deuterium exchange reactions with chiral alcohols will be used to determine the exact configuration of different exchange sites on the oligosaccharides. The goal of the proposed research is to increase our understanding of intramolecular interactions and proton migration pathways in gas-phase biomolecules. %%% Professor Lebrilla proposes to use Fourier transform mass spectrometry to elucidate the structure of gas-phase biomolecules. The research involves mass spectrometry, molecular orbital calculations and hydrogen/deuterium exchange reactions.

The broad long term goal of this research is to develop a new mass spectrometric method for the structural elucidation of oligosaccharides. Oligosaccharides (OS) play a central role in mammalian biology in many fundamental processes such as cell-cell recognition, adhesiveness, control of cell division, cellular differentiation and malignant transformation. Hence, in the study of cancer and other related diseases, a general, sensitive, and accurate method of analysis is clearly desired. Mass spectrometry has the potential to offer many of these features. Our immediate goal is to understand the production and unimolecular chemistry of complex gas-phase oligosaccharide ions. The newly developed external source Fourier transform mass spectrometry instrument will be utilized and further developed for the specific analysis of these compounds. This method is unique and allows ions to be produced and trapped for long periods of time. The addition of the external source has allowed the use of fast atom bombardment to generate gas-phase bio-organic ions. Preliminary experiments have been performed showing major differences between this technique and other mass spectrometric methods. We find that matrix interference in the spectra is minimal add substantial fragmentation is obtained. This is due to the relatively longer detection time scale of the instrument. During the detection period, slow metastable decay occurs and allows oligosaccharide ions to undergo fragmentation. Matrix cluster ions also undergo metastable decomposition. However, these ions fragment more quickly due to the loose nature of the bonding. The result is a spectrum of oligosaccharide ions in the near absence of matrix interference. Studies are proposed to gain a better understanding of the unimolecular decay. In addition, conditions will be obtained to optimize structural information from FAB/FTMS alone (i.e. without further tandem MS determinations). This would make powerful the coupling of liquid chromatography techniques such as flow FAB (and electrospray) to this mass spectral method to analyze complex oligosaccharide mixtures. Direct application to glycolipids and high-mannose compounds will be performed through a collaboration with a natural products chemist and an oligosaccharide biochemists.

This award is made to the University of California at Davis in support of a national career planning workshop for underrepresented minority undergraduate chemistry majors. The workshop to be held July 25-28, 1996, at the Davis campus is the third such workshop, the first having been held in 1992 at Purdue University and the second at UC Davis in 1994. The objective is to bring minority students together with high-achieving minority mentors from industry, academia, and government in order to introduce the students to the opportunities created by obtaining an advanced degree in chemistry. The workshop will be hosted by the University of California Davis Chemistry Department and will provide opportunities for the student participants to observe graduate research, advanced laboratory instrumentation, and to meet with graduate students to discuss areas of mutual interest. This award is viewed as a way of encouraging qualified minority undergraduates to continue their studies in chemistry by introducing them to successful minority role-models in a supportive setting. The longer term objective is to increase the participation of minorities in careers in chemistry that require advanced education.

This research project, supported in the Analytical and Surface Chemistry Program, uses electrospray and matrix assisted laser desorption ionization (MALDI) source Fourier Transform Mass Spectrometry (FTMS) to investigate the structure of gas phase macromolecular complexes. Hydrogen-deuterium exchange, coupled with deprotonation reactions, provides structural information about large peptide and protein complexes. Lysozyme-oligosaccharide complexes will be examined, and general methods for the structural elucidation of complex biological molecules using mass spectrometry will be developed. Systematic, quantitative studies of non- covalent bonding in these gas phase complexes will be carried out. The structural characterization of large biological molecules, including drug-protein complexes and macromolecular species of interest to biology, is essential for the understanding of life processes. Mass spectrometric approaches to the solution of these structural problems is the focus of this research project. The use of isotopic exchange and deprotonation reactions provides a non- destructive structural probe in this effort. The results of this research project will have immediate application in the structural analysis of many important biological systems.

The project entitled "The Development and Analytical Application of Gas-phase Chiral Reactions" is directed by an established investigator with prior NSF support. Professor Carlito Lebrilla of the University of California is supported by the Analytical and Surface Chemistry program. The purpose of the research is: (1) to study the fundamental nature of the chiral specificity of gas phase ion/molecule exchange reactions (2) to optimize and quantify the exchange reaction for chiral amino acids; (3) to test the method with other classes of compounds including chiral molecules of pharmaceutical importance. The protonated amino acid-cyclodextrin complexes are stored in the homogeneous region of a 5.2 tesla superconducting magnet of a Fourier Transform Mass Spectrometer (FTMS). The positively charged complex will be allowed to undergo collisions with neutral gaseous alkylamines. The alkylamines undergo exchange reactions with the amino acids. The rate of exchange is dependent on the chirality with one enantiomer reacting at rates up to four times faster than the other enantiomer.

This study will directly impact separation chemistry and mass spectrometry. The methodology developed may prove useful to industry as a rapid means for the identification of new chiral stationary phases. It may also prove useful in the pharmaceutical industry, where accurate measurement and separation of enantiomers is vital, or in process control, where speed and specificity are needed, as in the analysis of chiral libraries.

This project involves the development and validation of a novel encoding technique for "one-bead one-compound" (OBOC) small molecule combinatorial libraries. Using a solid phase split-mix synthesis method, bead libraries can be obtained such that each bead expresses only one compound, with 1013 copies of that compound in one single bead. The bead-library can then be screened for biological, chemical and physical properties of interest, and positive beads are then isolated for structure determination. In terms of synthesis and screening, the OBOC technique is highly efficient. However, a major limitation in the small molecule OBOC combinatorial library method is the difficulty in elucidating the chemical structure of a small molecule compound on one single bead. A rapid, sensitive, and reliable encoding/decoding methodology is necessary for full exploitation of the OBOC combinatorial method. In this proposal, topologically segregated bi-functional beads with testing molecules in the outer layer of the bead and coding molecules in the interior of the beads will be developed. Triple or quadruple cleavable coding arms will be constructed in the interior of the bead. Each of these coding arms contains a functional group that is identical or related to the functional groups on the scaffold of the testing compound to be synthesized. In this encoding method, each building block will react with the testing arm and the encoding arms simultaneously, thus eliminating many synthetic steps and lengthy encoding times. Decoding is accomplished by cleaving all the coding tags at once and analyzing the released molecules by mass spectroscopy. This novel encoding method is highly versatile and efficient. Over 100 beads can easily be decoded in one day. There is room for automation in terms of sample preparation and data analysis. Twelve different model library compounds obtained from the literature will be used to optimize the procedure, and the encoding method will be validated by designing, synthesizing, and screening three encoded small molecule libraries.


In recent years, many organic chemists have turned their attention from the synthesis of single molecules to the preparation of large "libraries" of related compounds, permitting fast and efficient screening for desirable properties. These "combinatorial" synthesis approaches come with their own challenges, often relating to the ability to determine the structure of the specific molecule giving rise to the most promising properties. With the support of the Organic and Macromolecular Chemistry Program, Professors Kit S. Lam, of the Department of Hematology and Oncology, and Carlito B. Lebrilla, of the Department of Chemistry at the University of California - Davis, are developing new methods for the simple, rapid, and efficient identification of molecules in combinatorial libraries.

DESCRIPTION (provided by applicant): We will develop the methods and tools that will profile glycans in human fluids to enable the discovery of markers for diseases. Cancer is a disease known to accompany aberrant glycosylation. Glycoproteins shed by diseased cells will be harvested for their glycan content and profiled to obtain disease markers. This approach represents a new paradigm for disease marker discovery. It will focus primarily on the glycans as disease markers while neglecting, at first, the identity of the associated proteins. This approach will be multifaceted and primarily glycocentric;it contrasts and complements the proteome-centered approaches currently under extensive investigations. In this process, we will develop a set of mass spectrometry-based tools for clinical glycomics. These tools will be used to observe changes in glycosylation of diseases in two human fluids, sera and tear. The fluids are chosen by the needs of our collaborator in diseases that include ovarian cancer and ocular rosacea but will have direct implications in breast cancer and prostate cancer for sera and other dry eye diseases for tear fluids. While disease markers will be the future outcome of these studies, they will not be the only focus of this project. Instead we will also develop the tools that will be used by our collaborators to discover markers for these diseases and lay the foundation for the use of mass spectrometry in clinics for glycomic analyses.

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC) is a common and devastating disease of premature infants. Effective preventive agents and biomarkers predictive of high-risk infants are significant clinical needs. The most promising interventions shown to prevent NEC are breast milk feedings and probiotic microorganisms, although the mechanisms of action are unknown. Our proposal draws from the integrated, multi-disciplinary Milk Bioactives Consortium at the University of California Davis and includes exciting preliminary data: a clinical trial of two probiotic products in premature infants, evidence that human milk oligosaccharides selectively stimulate growth of specific bifidobacteria, and a novel potential biomarker of infant susceptibility. We hypothesize that a regimen of prebiotic oligosaccharides and/or probiotic microbes, which increases bifidobacteria colonization to mimic that of healthy term breast-fed infants, will improve infant growth and lead to an attractive regimen for larger trials of prevention of NEC. We further hypothesize that a low [unreadable]-defensin gene copy number polymorphism predisposes some premature infants to development of an intestinal microbiota low in bifidobacteria and the consequent deficit in normal microflora protection increases their susceptibility to NEC. Specific Aim 1 will conduct Phase 1 and Phase 2 clinical trials to identify and evaluate a preferred dietary supplement regimen to achieve a predominance of bifidobacteria in the fecal microbiota of preterm infants. Specific Aim 2 will conduct a series of in vitro experiments to (a) analyze biochemical and genetic properties of the bifidobacteria in the feces of infants receiving prebiotic oligosaccharides and/or probiotic microbes and (b) analyze the prebiotic properties of components of human milk. Specific Aim 3 will analyze the potential of a novel genetic biomarker for susceptibility to NEC: [unreadable]-defensin gene copy number. The proposed clinical trials and in vitro experiments are designed to answer important questions regarding the development of the intestinal microbiota, the effect of breast milk components on the developing intestinal microbiota, and the effect of changes in the intestinal microbiota on the health and growth of the premature infant. PUBLIC HEALTH RELEVANCE: Having more healthy bacteria in the intestines may improve growth and prevent infections in premature infants. By giving different doses and combinations of live healthy bacteria (probiotics) and fiber (prebiotics) to premature infants, we aim to find the best way to change the bacteria in the intestines to be more like those of healthy breast-fed term infants. The genes of premature infants who get intestinal infections may be slightly different from those who don't;we will test one group of particularly promising genes to see if that is true.

DESCRIPTION (provided by applicant): Complex oligosaccharides have been proposed to exert important biological properties for human health. However, despite advances in the commercialization of simple plant oligosaccharides and polysaccharides, these products of plant metabolism are unable to substantially alter the human gut microbial ecology nor its metabolism nor the physiology and disease risks of humans. Human milk, on the other hand, is an attractive source of oligosaccharides precisely because human milk produces a unique microbiota whose persistence yields scientifically established evidence of benefits to human infants. The free oligosaccharides in human milk have emerged through evolution in remarkable abundance and with significant structural diversity. Research in the collaborating laboratories has demonstrated that these oligosaccharides constitute a symbiotic system in human infants stimulating the competitive growth of commensal bifidobacteria. In this project we propose to build the analytic, genetic and biological tools to establish the structures and functions of the oligosaccharides in human milk as thematic principles to guide the development, research and industrialization of bioactive oligosaccharides for human health. To achieve this goal, we will: (A) elucidate in precise chemical detail the entire human milk glycome and categorize a diverse pool of human lactating subjects differing in the composition of expressed oligosaccharides, (B) develop analytical tools to rapidly determine human secretor versus non- secretor status based on the oligosaccharide profile and link maternal secretor status to milk oligosaccharide composition and microbiota diversity, and (C) establish the genetic basis of bifidobacterial selection by the specific structures in the ensemble of human milk oligosaccharides using whole genomic analysis of a range of bifidobacterial strains, relating specific oligosaccharide consumption by bacteria to functional analysis of genes and proteins in and on bacterial surfaces.

DESCRIPTION (provided by applicant): The proposed instrument will be the central analytical tool for the glycomics profiling in several NIH projects focused on infection and disease diagnosis. The instrument includes an integrated nanoflow liquid chromatography device with a quadrupole time-of-flight (Q-TOF) mass analyzer. The instrument is similar to an existing instrument that is on loan in the Principal Investigator's laboratory. The new instrument will include tandem MS capability for online structural analysis. The proposed instrument will be used to analyze oligosaccharides and determine their structures in highly complicated biological mixtures. Projects that the instrument will be used for include: (1) The determination of O-glycans in stomach mucin to determine the site of interaction with Helicobacter pylori;Serum glycan biomarkers for early detection of H. pylori-associated gastric cancer (Dr. Jay Solnick, School of Medicine), (2) The effect of prebiotics and probiotics in necrotizing enterocolitis (Dr. Mark Underwood, School of Medicine), (3) The profiling of oligosaccharides by nanoLC Chip/Q-TOF mass spectrometry (Dr. Carlito Lebrilla, Department of Chemistry and School of Medicine), (4) Mass spectrometry identification of molecular complexes in cell lysates and "hit compounds" (Dr. Xiangdong Zhu, Department of Physics), (5) The search of glycans markers for disease (Dr. Kit Lam, School of Medicine), (6) The analysis of products and intermediates in multiple-enzyme reactions (Dr. Xi Chen, Department of Chemistry), and (7) The design and synthesis of chemotherapeutics (Dr. Jacqueline Gervay-Hague, Department of Chemistry). PUBLIC HEALTH RELEVANCE: A nanoflow liquid chromatography time-of-flight mass spectrometry instrument with an integrated microchip separation device is requested for performing glycomics profiling of biological fluids. The instrument will be used for research related to human health in terms of gut health, cancer diagnosis, and infection.

With this award from the Chemistry Research Instrumentation and Facilities: Departmental Multi-User Instrumentation program (CRIF:MU), Professor Carlito Lebrilla and colleague R. David Britt from University of California at Davis will acquire an X-band EPR spectrometer. The proposal is aimed at enhancing research training and education at all levels, especially in areas of study such as (a) water splitting cobalt catalysts, (b) multielectron reduction of carbon dioxide, (c) studies of metalloproteins, (d) functionalized materials and catalysts in surfaces and zeolites, (e) silicon-doped nanoparticles, (f) polaron delocalization in conjugated polymer blends, and (g) transition metal complexes with uncommon coordination numbers and oxidation states.

An EPR spectrometer yields detailed information on the geometric and electronic structure of molecular and solid state materials. It may also be used to obtain information about the lifetimes of free radicals, short-lived, highly reactive species involved in valuable chemical transformations as well as the initiation of pathological tumor growth. These studies will impact a number of areas, from the synthesis of inorganic and organic molecules to the development of new solid state materials to compounds of biological interest. Employing examples inspired from ongoing research, this instrument will be an integral part of research and teaching at the undergraduate and graduate levels.

DESCRIPTION (provided by applicant): Probiotics and prebiotics are CAM interventions frequently employed by the American public to promote intestinal health and wellness. The presence of beneficial bacteria such as lactobacilli and bifidobacteria, whether delivered exogenously as probiotics or enriched via prebiotics, has been linked to positive health effects including reduction of gut inflammation, diarrhea and allergic reactions. At present however, our understanding of the mechanism of action underlying these biological effects is significantly lacking. Milk oligosaccharides are naturally-evolved prebiotic substrates that facilitate bifidobacterial enrichment and interaction within the infant host. Work from the UC Davis Milk Bioactives Program has shown that human milk oligosaccharides are utilized by select infant-borne bifidobacterial strains that demonstrated unique preferences in oligosaccharide consumption. In addition, genomic analysis of Bifidobacterium infantis and Bifidobacterium bifidum, two strains that grow well on milk oligosaccharides, has revealed unique gene clusters that are specifically induced during growth on these glycans. Further analysis has revealed that growth on milk glycans results in enhanced bifidobacterial interaction with the host epithelium. We hypothesize that the evolutionarily-driven relationship between milk oligosaccharides and cognate infant-borne bifidobacteria provides a model for enhanced probiotic persistence within, and interaction with, the human host. To address this hypothesis we will gain mechanistic insight into the specific catabolism of these unique milk glycans by bifidobacteria and comprehensively map their influence on bifidobacteria-host interaction via the following Specific Aims: 1. To comprehensively characterize human and bovine milk oligosaccharides and develop methods for their large scale fractionation and production for functional studies. 2. To completely characterize the bifidobacterial transporters and glycosyl hydrolases necessary to deconstruct these complex milk oligosaccharides. 3. To examine the influence of milk oligosaccharides on the interaction between bifidobacteria and the host. Successful completion of these Specific Aims will reveal specific mechanisms by which human milk oligosaccharides facilitate a protective enrichment of bifidobacteria in the gastrointestinal tract of breast fed infants. In addition, we will translate these findings to structurally and functionally equivalent bovine milk oligosaccharides, a commercially accessible substrate that can be easily delivered into CAM foods and therapies aimed at gut health. The significance of this application is a greater mechanistic understanding of the beneficial effects of synbiotic (milk oligosaccahrides plus cognate bifidobacteria) applications thereby providing both strategies and diagnostic measures to better address a variety of disorders marked by intestinal dysbiosis. PUBLIC HEALTH RELEVANCE: Probiotics and prebiotics (complex carbohydrates preferred by probiotics) are frequently used by the American public to promote intestinal health and wellness. These probiotics have been linked to positive health effects including reduction of gut inflammation, diarrhea and allergic reactions. Using prebiotics that naturally evolved in human and bovine milk, the proposed research will study the underlying mechanisms whereby milk prebiotics (milk oligosaccharides) enrich probiotic bacterial growth and interaction with cells in the mammalian gut.

DESCRIPTION (provided by applicant): Glycomics is rapidly emerging as a new paradigm for biomarker discovery. Diseases as diverse as infection and cancer are known to involve changes in glycosylation. Glycans on cell surfaces are important for understanding nearly all cell surface interactions. They are key targets for drugs and may yield cell-specific therapeutics. In addition, they are also shed and can give indications of the changes in glycosylation associated with the disease. The study of glycosylation of cell surfaces is still in its infancy. The majority of the research has employed fluorescently labeled lectins providing few structural details. In this proposal, we will develop techniques to study surface glycans by using methods that release them specifically and examine them with high sensitivity. In the process, we will develop comprehensive methods to determine glycan structures and micoheterogeneity. To achieve these tasks, we will develop a high throughput method for glycomics analysis that will rapidly identify glycan structures. This goal would have seemed impossible given the complexity and the heterogeneity of glycan structures. However, we will develop a method with a constructed database at its core that will serve as template with descriptors including liquid chromatography retention time, accurate mass, and tandem mass spectrometry to identify individual glycan (or oligosaccharide) structures. The creation of this database will form the kernel of a comprehensive database will allow routine analysis of oligosaccharide possible. The development of methods for the rapid identification will significantly advance glycobiology research. Additionally, the analysis of glycans by liquid chromatography will provide a new method for biomarker discovery by allowing the analysis of structural isomers thereby increasing the richness of the compound pool.

DESCRIPTION (provided by applicant): Complex oligosaccharides have been proposed to exert important biological properties for human health. However, despite advances in the commercialization of simple plant oligosaccharides and polysaccharides, these products of plant metabolism are unable to substantially alter the human gut microbial ecology nor its metabolism nor the physiology and disease risks of humans. Human milk, on the other hand, is an attractive source of oligosaccharides precisely because human milk produces a unique microbiota whose persistence yields scientifically established evidence of benefits to human infants. The free oligosaccharides in human milk have emerged through evolution in remarkable abundance and with significant structural diversity. Research in the collaborating laboratories has demonstrated that these oligosaccharides constitute a symbiotic system in human infants stimulating the competitive growth of commensal bifidobacteria. In this project we propose to build the analytic, genetic and biological tools to establish the structures and functions of the oligosaccharides in human milk as thematic principles to guide the development, research and industrialization of bioactive oligosaccharides for human health. To achieve this goal, we will: (A) elucidate in precise chemical detail the entire human milk glycome and categorize a diverse pool of human lactating subjects differing in the composition of expressed oligosaccharides, (B) develop analytical tools to rapidly determine human secretor versus non- secretor status based on the oligosaccharide profile and link maternal secretor status to milk oligosaccharide composition and microbiota diversity, and (C) establish the genetic basis of bifidobacterial selection by the specific structures in the ensemble of human milk oligosaccharides using whole genomic analysis of a range of bifidobacterial strains, relating specific oligosaccharide consumption by bacteria to functional analysis of genes and proteins in and on bacterial surfaces.

Program Director/Principal Investigator (Last, First, Middle): MIIIS, D a v l d , A . PROJECT SUMMARY (See instmctions): The use of prebiotics and probiotics to restore a healthy gut microbiota represent a desirable target, but the lack of mechanistically relevant signatures of how specific bacteria interact with the intestinal environment and the host has hindered the development of effective and well-characterized prebiotic and probiotic treatments. The long-term goal is to translate the successful strategy of mammalian lactation, shown using human milk glycans, to the development of targeted, effective synbiotics by using plentiful and available bovine milk glycan streams. The overarching hypothesis to be tested is that the evolutionary relationship between infant-borne bifidobacteria and bovine milk glycans and glycoconjugates produce a synergistic human milk glycan-like phenotype that can effectively colonize, restore a healthy gut microbiota and induce host response to better protect epithelial barrier function and thus improve health outcomes. First, the team will address whether in infant-borne bifidobacteria species, complex milk glycoconjugates induce specific glycosyl hydrolases and transporters that are necessary to consume these complex substrates. Milk glycoconjugate catabolism by infant-borne bifidobacteria will be examined by detailed transcriptomics, specific enzymatic and transporter analysis, and glycoprofiling to identify precise links between glycan components and their cognate bifidobacterial processing mechanisms. Second, the research team will determine whether select infant-borne bifidobacteria that consume complex milk glycoconjugates compared to simple sugar substrates are more effective in inducing a protective response within the host epithelium. Measurements of bifidobacterial adherence, improved barrier function, release of inflammatory mediators and activation of enteroendocrine cells will be obtained from gut epithelial and enteroendocrine cells in vitro and ex vivo in rat small and large intestinal tissue. Finally, the team will determine whether modulation of intestinal function by application of synbiotic milk glycan- and glycoconjugate-consuming bifidobacteria improves outcomes in a rodent model of intestinal and metabolic disease. The significance of this project is that it will take a systematic and mechanistic approach to understanding the synbiotic relationship. RELEVANCE (See instmctions): The gut microbiome is a crucial component of human health. Safe and effective approaches for correcting, maintaining, and guiding establishment of a healthy gut microbiota, particularly in infancy and early childhood, are needed. The project is relevant to NCCAM's mission because it supports a portfolio of synbiotic interventions for improving health, with mechanistic signatures of biological effects.

RESEARCH SUMMARY The role that diet plays in brain development will be probed by determining the components of the brain that are directly impacted by nutrition. Human milk is highly glycosylated containing short oligosaccharide chains that are more abundant than proteins. In early nutrition, there is a big burden on diet to accommodate the rapidly developing brain. The brain is rich in sialic acids, which are involved in neuronal outgrowth thereby creating synaptic connectivity through cell-cell interactions that in turn create memory. Human milk oligosaccharides are rich in sialic acids. It has long been proposed that sialic acids in human milk are an important source of sialic acids in the brain. However, the compounds in the brain that incorporate the exogenous sialic acids are not known, although they are generally believed to include proteins and lipids. This research will determine with high specificity the glycoconjugates in the brain that incorporate exogenous monosaccharides, particularly those from human milk oligosaccharides during the normal course of breast- feeding. A method combining advanced separation, mass spectrometry and chemical biology will be developed to determine the glycan structures, the glycoprotein, and the glycolipid involved in the incorporation of glycans from breast milk. Structural heterogeneity will be determined with lipid-specific characterization. Glycans in glycoproteins will be determined with structural heterogeneity at the site-specific level for both N- and O- glycosylation sites. We will develop deep glycomics tools to probe the glycoconjugates as glycoproteins and glycolipids that incorporate components from human milk. Determining the specific protein and lipid glycoconjugates that incorporate milk oligosaccharides would provide the most direct link between nutrition and brain development. 1

Project Summary Alzheimer?s disease (AD) is a debilitating neurodegenerative disease that is growing in prevalence despite decades of investment in the research and development of diagnostics and therapeutics. Because of the high healthcare burden and devastating effects of AD on cognitive function and quality of life, early biomarkers of disease and interventions to prevent and treat AD are a public health priority. The search for biomarkers and treatments has turned to glycobiology, the study of complex sugars attached to proteins and lipids. The post- translational glycosylation of proteins and lipids plays a role in a number of critical biological processes including cell-to-cell communication and signaling, and protein structure and function. Recently, major alterations in glycosylation have been documented in the brains, cerebrospinal fluid, and blood of AD patients even before the onset of advanced cognitive decline. Glycosylation is likely to be causally involved in AD, thus measuring glycosylation in blood is a promising strategy for discovering early biomarkers. Apolipoprotein E (ApoE) is the single greatest genetic risk factor for AD. Carriers of the ApoE4 isoform are 4-12 times more likely to develop AD depending on the number of copies of ApoE4, and develop the disease as many as 20 years earlier. ApoE is a glycosylated protein, and nearly all of the proteins, carriers, and receptors involved in its metabolism are also glycosylated. The structure of ApoE heavily influences its functionality. Glycosylation is known to alter protein structure yet neither the glycosylation of ApoE, nor the overall glycosylation in the brain and in the blood have been adequately characterized in Alzheimer?s disease patients because the lack of precise analytical tools for measuring glycosylation has been a technological barrier to progress. Our group has pioneered the development of advanced and sensitive liquid chromatography-mass spectrometry methods as tools to precisely measure glycosylation alterations across hundreds of lipids and proteins simultaneously. Our method is rapid-throughput and can monitor the site-specificity, structure specificity, and linkage specificity of all attached glycans in hundreds of clinical samples with high reproducibility, accuracy and sensitivity. The three specific aims of this project are to map the global glycosylation alterations in the brains and blood of Alzheimer?s disease patients compared with controls, to characterize the site-specific changes in glycosylation of ApoE, and to document the effects of glycosylation changes on critical pathways involved in Alzheimer?s disease pathology. Importantly, the differences in glycosylation in AD will be mapped within each ApoE genotype, which will enable the discovery of precision medicine based solutions. Successful completion of this project will lead to the development of new glycosylation-based biomarkers for the early detection of Alzheimer?s disease and ApoE-specific targets for the development of novel therapeutics in AD.

Biomarkers of food consumption are important indicators for clinical and feeding studies that establish our understanding of nutrition. Carbohydrates make up the largest component of a healthy diet. Biomarkers that are derived directly from carbohydrates are ideal. However, there is a fundamental research gap in that carbohydrate structures of specific foods that are commonly consumed are unknown. The long-term goal is to determine specific glycan biomarkers of dietary intake for all commonly consumed foods. The objective of the current application is to develop a detailed carbohydrate structure library of foods in terms of their monosaccharide and linkage compositions and to evaluate carbohydrate structures for their utility as biomarkers of dietary intake. Our central hypothesis is specific foods have unique carbohydrate structures that can serve as biomarkers of dietary intake. This hypothesis has been formulated on the basis of preliminary studies in which (a) detailed carbohydrate structures have been determined for hundreds of foods and (b) fecal glycans, microbes, and metabolites differed by dietary intervention of weaning foods in infants. Enzymes in the gut from human and their complement microbes are specific to structural carbohydrate features. Therefore, the specific aims are to (1) construct a glycomic library with paired analytical platform for food and feces, (2) conduct a microbial gene marker library using bioreactor studies and (3) test the predictive value of glycan and microbial biomarkers in a human feeding study. The approach is innovative, bringing unprecedented high-resolution carbohydrate content analysis?monosaccharide and linkage analysis of foods?with multiple omic measurements to biomarker prediction of dietary intake. The proposed research is significant and impactful because a high-resolution glycan library of foods together with glycan-microbe biomarker products of those foods will transform future clinical trials in which diet is an essential component.

Project Summary Alzheimer?s disease (AD) is a debilitating neurodegenerative disease that is growing in prevalence despite decades of investment in the research and development of diagnostics and therapeutics. Because of the high healthcare burden and devastating effects of AD on cognitive function and quality of life, early biomarkers of disease and interventions to prevent and treat AD are a public health priority. The search for biomarkers and treatments has turned to glycobiology, the study of complex sugars attached to proteins and lipids. The post- translational glycosylation of proteins and lipids plays a role in a number of critical biological processes including cell-to-cell communication and signaling, and protein structure and function. Recently, major alterations in glycosylation have been documented in the brains, cerebrospinal fluid, and blood of AD patients even before the onset of advanced cognitive decline. Glycosylation is likely to be causally involved in AD, thus measuring glycosylation in blood is a promising strategy for discovering early biomarkers. Apolipoprotein E (ApoE) is the single greatest genetic risk factor for AD. Carriers of the ApoE4 isoform are 4-12 times more likely to develop AD depending on the number of copies of ApoE4, and develop the disease as many as 20 years earlier. ApoE is a glycosylated protein, and nearly all of the proteins, carriers, and receptors involved in its metabolism are also glycosylated. The structure of ApoE heavily influences its functionality. Glycosylation is known to alter protein structure yet neither the glycosylation of ApoE, nor the overall glycosylation in the brain and in the blood have been adequately characterized in Alzheimer?s disease patients because the lack of precise analytical tools for measuring glycosylation has been a technological barrier to progress. Our group has pioneered the development of advanced and sensitive liquid chromatography-mass spectrometry methods as tools to precisely measure glycosylation alterations across hundreds of lipids and proteins simultaneously. Our method is rapid-throughput and can monitor the site-specificity, structure specificity, and linkage specificity of all attached glycans in hundreds of clinical samples with high reproducibility, accuracy and sensitivity. The three specific aims of this project are to map the global glycosylation alterations in the brains and blood of Alzheimer?s disease patients compared with controls, to characterize the site-specific changes in glycosylation of ApoE, and to document the effects of glycosylation changes on critical pathways involved in Alzheimer?s disease pathology. Importantly, the differences in glycosylation in AD will be mapped within each ApoE genotype, which will enable the discovery of precision medicine based solutions. Successful completion of this project will lead to the development of new glycosylation-based biomarkers for the early detection of Alzheimer?s disease and ApoE-specific targets for the development of novel therapeutics in AD.