J. Michael Pierce

The Challenge and Promise of Glycomics

Glycomics is a broad and emerging scientific discipline focused on defining the structures and functional roles of glycans in biological systems. The staggering complexity of the glycome, minimally defined as the repertoire of glycans expressed in a cell or organism, has resulted in many challenges that must be overcome; these are being addressed by new advances in mass spectrometry as well as by the expansion of genetic and cell biology studies. Conversely, identifying the specific glycan recognition determinants of glycan-binding proteins by employing the new technology of glycan microarrays is providing insights into how glycans function in recognition and signaling within an organism and with microbes and pathogens. The promises of a more complete knowledge of glycomes are immense in that glycan modifications of intracellular and extracellular proteins have critical functions in almost all biological pathways.

Regulation of Glycan Structures in Animal Tissues TRANSCRIPT PROFILING OF GLYCAN-RELATED GENES

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes.

Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase.

Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked β(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.

Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural analysis

Background: Glycans contribute to vertebrate development, but regulatory mechanisms are unknown. Results: Glycans and transcripts encoding the glycosylation machinery were profiled during stem cell differentiation. Conclusion: Changes in glycans frequently correlated with changes in transcripts, supporting a significant role for transcriptional regulation. Significance: Knowledge of the mechanisms that regulate glycan expression provides insight into the roles of glycosylation in development. The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development.

N-glycosylation alters cadherin-mediated intercellular binding kinetics.

We present direct evidence that the N-glycosylation state of neural cadherin impacts the intrinsic kinetics of cadherin-mediated intercellular binding. Micropipette manipulation measurements quantified the effect of N-glycosylation mutations on intercellular binding dynamics. The wild-type protein exhibits a two-stage binding process in which a fast, initial binding step is followed by a short lag and second, slower transition to the final binding stage. Mutations that ablate N-glycosylation at three sites on the extracellular domains 2 and 3 of neural cadherin alter this kinetic fingerprint. Glycosylation does not affect the affinities between the adhesive N-terminal domains, but instead modulates additional cadherin interactions, which govern the dynamics of intercellular binding. These results, together with previous findings that these hypo-glycosylation mutations increase the prevalence of cis dimers on cell membranes, suggest a binding mechanism in which initial adhesion is followed by additional cadherin interactions, which enhance binding but are modulated by N-glycosylation. Given that oncogene expression drives specific changes in N-glycosylation, these results provide insight into possible mechanisms altering cadherin function during tumor progression.

Developmental Expression of the Neuron-specific N-Acetylglucosaminyltransferase Vb (GnT-Vb/IX) and Identification of Its in Vivo Glycan Products in Comparison with Those of Its Paralog, GnT-V

Background: GnT-Vb(IX) branches the α-mannose to the O-Manβ(1,2)-GlcNac in brain. Results: GnT-Vb does not synthesize N-linked structures in vivo. GnT-V, however, can compensate for GnT-Vb activity in vivo. Conclusion: GnT-Vb is predominantly involved in synthesizing branched O-mannosyl glycans in mouse brain. Significance: It is shown for the first time in vivo that GnT-Vb and -V have different activity in the synthesis of N- and O-linked glycans. The severe phenotypic effects of altered glycosylation in the congenital muscular dystrophies, including Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, and congenital muscular dystrophy 1D, are caused by mutations resulting in altered glycans linked to proteins through O-linked mannose. A glycosyltransferase that branches O-Man, N-acetylglucosaminyltransferase Vb (GnT-Vb), is highly expressed in neural tissues. To understand the expression and function of GnT-Vb, we studied its expression during neuromorphogenesis and generated GnT-Vb null mice. A paralog of GnT-Vb, N-acetylglucosaminyltransferase (GnT-V), is expressed in many tissues and brain, synthesizing N-linked, β1,6-branched glycans, but its ability to synthesize O-mannosyl-branched glycans is unknown; conversely, although GnT-Vb can synthesize N-linked glycans in vitro, its contribution to their synthesis in vivo is unknown. Our results showed that deleting both GnT-V and GnT-Vb results in the total loss of both N-linked and O-Man-linked β1,6-branched glycans. GnT-V null brains lacked N-linked, β1,6-glycans but had normal levels of O-Man β1,6-branched structures, showing that GnT-Vb could not compensate for the loss of GnT-V. By contrast, GnT-Vb null brains contained normal levels of N-linked β1,6-glycans but low levels of some O-Man β1,6-branched glycans. Therefore, GnT-V could partially compensate for GnT-Vb activity in vivo. We found no apparent change in α-dystroglycan binding of glycan-specific antibody IIH6C4 or binding to laminin in GnT-Vb null mice. These results demonstrate that GnT-V is involved in synthesizing branched O-mannosyl glycans in brain, but the function of these branched O-mannosyl structures is unresolved using mice that lack these glycosyltransferases.

Lectin-based glycoproteomic techniques for the enrichment and identification of potential biomarkers.

Glycan structures on glycoproteins are controlled by several factors such as regulated expression of glycosyltransferases and glycosylhydrolases, as well as regulation of glycoprotein expression, folding, and transport through the ER and Golgi. In cancer, for example, the glycosylation of glycoproteins can be significantly altered due to changes in the expression levels of glycosyltransferases as a result of oncogene activated signaling pathways coupled with gain or loss in chromosome copy number. Cumulatively these changes result in glycoproteins exported to the cell surface and extracellular region with altered glycan structures that can lead to significant changes in cell phenotype. Therefore, it is advantageous to be able to capture and identify proteins that express particular glycans or classes of glycans. In this report, we discuss extraction methods and lectin capture methodology that can be used to enrich and identify by mass spectrometry glycoproteins that express specific glycans that change in response to disorders or diseases, such as the presence of malignancies.

Discrimination between Adenocarcinoma and Normal Pancreatic Ductal Fluid by Proteomic and Glycomic Analysis

Sensitive and specific biomarkers for pancreatic cancer are currently unavailable. The high mortality associated with adenocarcinoma of the pancreatic epithelium justifies the broadest possible search for new biomarkers that can facilitate early detection or monitor treatment efficacy. Protein glycosylation is altered in many cancers, leading many to propose that glycoproteomic changes may provide suitable biomarkers. In order to assess this possibility for pancreatic cancer, we have performed an in-depth LC-MS/MS analysis of the proteome and MS(n)-based characterization of the N-linked glycome of a small set of pancreatic ductal fluid obtained from normal, pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma patients. Our results identify a set of seven proteins that were consistently increased in cancer ductal fluid compared to normal (AMYP, PRSS1, GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was consistently decreased (LIPR2). These proteins are all directly or indirectly associated with the secretory pathway in normal pancreatic cells. Validation of these changes in abundance by Western blotting revealed increased REG protein glycoform diversity in cancer. Characterization of the total N-linked glycome of normal, IPMN, and adenocarcinoma ductal fluid clustered samples into three discrete groups based on the prevalence of six dominant glycans. Within each group, the profiles of less prevalent glycans were able to distinguish normal from cancer on this small set of samples. Our results emphasize that individual variation in protein glycosylation must be considered when assessing the value of a glycoproteomic marker, but also indicate that glycosylation diversity across human subjects can be reduced to simpler clusters of individuals whose N-linked glycans share structural features.

Chapter 16 – Cancer Glycomics

Publisher Summary New technologies are continuously evolving to identify specific glycan, glycopeptide, and glycoprotein differences found in cancer patient sera when compared to non-diseased, control sera. This chapter emphasizes recent studies that have focused on using analytical technologies to characterize altered glycan and glycoconjugate expression in cancer cells, tissues, and fluids such as serum from patients with cancer. Particular emphasis is placed on those that seek to exploit these aberrant glycan changes to develop potential tumor markers that could be used to direct chemotherapeutic agents or serve as diagnostic or prognostic markers of cancer or cancer risk. With the possibility of utilizing actual tumor-derived glycans and glycopeptides immobilized in arrays on slides, the odds of detecting antibodies to cancer-specific glycopeptides appear to significantly increase. There is new promise in the generation of robust immune responses to glycopeptides. Applying glycomic technologies for the identification of cancer-specific glycopeptides could, therefore, lead to the generation of efficacious anti-cancer vaccines. A formidable challenge that impacts all glycomics marker discoveries is to develop reliable clinical assay systems for detection of specific cancer glycoprotein glycoform such that large numbers of samples can be assayed during clinical trials. The results show that complicated sample preparation and time-consuming analytical procedures must be reduced and adapted so that analyses can be performed in a specialized clinical laboratory.

7th HUPO World Congress: The Human Disease Glycomics/Proteomics Initiative (HGPI) Session 17 August 2008, Amsterdam, The Netherlands

The Human Disease Glycomics/Proteomics Initiative (HGPI) Session was held on August 17, 2008, at the HUPO World Congress in Amsterdam. Reports were made on the progress of the first and second analytical pilot studies to profile N‐ and O‐linked glycan structures of standard glycoproteins utilizing laboratories from around the world. In addition, recent advances in glycan structural analyses were presented, including the use of O‐linked glycan libraries of standards, use of negative mode nano‐LC‐MS for O‐linked glycan analysis, and identification of aberrant O‐glycosylation of IgA1 as a cause of IgA nephropathy. A report was made of a newly discovered lectin, malectin, which appears to function in the folding/quality control of glycoproteins with N‐linked glycans and may regulate several human disorders whose etiology involves protein quality control in the ER. The major glycan ligand for malectin was identified using a novel printed glycan microarray. Advances in the analysis of the genes that are associated with glycan expression and recognition – the glycotranscriptome – were described, as well as technologies to determine the relative quantitation of N‐ and O‐linked glycans from as few as 2×106 cells. These technologies are being applied to identify potential biomarkers of stem and cancer cells.