Glycomics is a field that is a logical offshoot of the fields of genomics and proteomics and is considered as a sub-discipline of metabolomics. Glycobiology is the study of the structure, function and structure-function relationships of carbohydrates known to carry and store significant biological information crucial in virtually all physiological and pathophysiological processes.
The DNA blueprint of a cell, the genome, encodes the proteome. Intron and exon splicing in eukaryotes complicates the structural proteomics and the function of each protein (functional proteomics) is complicated by our inability to translate linear protein sequence to folded protein structure and to predict protein function from folded structure. The proteome is directly responsible for the synthesis of the metabolome (all natural products). The proteome is further complicated by posttranslational modification, the most frequent of which is glycosylation. Over 60% of human proteins are glycosylated yet the structure and function of this glycosylation is relatively unexplored. Moreover, every animal cell is surrounded by a complex coating of carbohydrates known as the glycocalyx that is critical in modulating various processes including signaling, cell-cell interaction, cell adhesion and migration (Figure 1). The fields of structural and functional glycomics are being studied in the BCME Constellation.
Figure 1. The glycocalyx of an animal cell containing membrane-bound glycolipids, glycoproteins and proteoglycans (Lanctot, P. M., et al, 2007, Curr. Opin. Chem. Bio)
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Stem Cell Glycomics: Embryonic stem cells (ESCs) are isolated from the inner cell mass of an embryo and are capable of differentiating into all types of cells making up an organism. Thus, the study of ESCs is important to understand the basic science of developmental biology and also for the practical application of creating cell lines, tissues and organs that can be used in the fields of tissue engineering, regenerative medicine and drug discovery. As a stem cell differentiates down various lineages into various cell types its communication tasks become more complex and so must its glycome to facilitate cell-cell and cell-extracellular matrix interactions. Furthermore, the glycans in extracellular matrix form a glyconiche that can serve to control the growth and differentiation of a stem cell. Work in the Biocatalysis and Metabolic Engineering (BCME) Constellation at Rensselaer is focused on understanding how the glycome changes as ESCs differentiate and how the glyconiche of a stem cells control their growth and differentiation. Changes in glycosaminoglycans (GAG) structure, which are polymer glycans with long, linear chain consisting of repeating disaccharide unit, are in particular interest. They found attached to protein core, comprising proteoglycans (PG) and found on cell surface and extracellular matrix.
Figure 2. Schematic plan of glycan analyses upon differentiation of several human Embryonic Stem Cells lines into representatives of mesoderm, endoderm and ectoderm.
Comprehensive study of glycomics of ESC is accomplished on different levels including transcriptomic, proteomic and structural analyses (Figure 2). Pluryipotent ESCs are driven to differentiate into representatives of different germ layers and GAGome changes are monitored along the differentiation pathways. Meanwhile, qRT-PCR is used to detect expression level of GAG biosynthetic enzymes (Figure 3), which are later confirmed by western blotting. LC-MS and CE electrophoresis are used to determine the structure of GAGs.
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Figure 3. Biosynthetic pathway of heparin/heparan sulfate and chondroitin sulfate/dermatan sulfate glycosaminoglycans. The Ser/Thr-containing polypeptide core protein is glycosylated and modified through the series of enzymatic reactions occurring in the endoplasmic reticulum and Golgi apparatus. Numbers on steps correspond to enzymes catalyzing relevant reactions as (1): XYLT1, XYLT2, (2): B4GALT7, (3): B3GALT6, (4): B3GALT3, (5): EXTL2, EXTL3, (6): EXT1, EXT2, (7): EXT1, EXT2, EXTL1, EXTL3, (8): CSGALNACT1, CSGALNACT2, (9): CHSY1, CHPF1, CHSY3, CHPF2, and (10): DSE1, DSE2 (Nairn, A. V., Linhardt, R. J. et al, 2007, J. Proteom. Res.)
The effect of glycan structure on stem cell fate is determined using cell-based microarray technologies, which allows high throughput analyses of ESCs. Cell-based microarray can be coupled with in-cell immunofluorescence to determine the effect of various signaling molecules on stem cell state
Figure 4. Three-dimensional cellular microarray for the study of murine stem cells. a. approach for microarray in-spot immunofluorescence for protein expression; b. Western blot for comparison of up-regulated HIF-1α expression to actin expression; c. in spot determination of HIF-1α expression to actin expression; and d. quantification of data. (Biotechnol Bioeng. 2010, 106:106-118.)
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Structural Glycomics in animal cells related to physiology/pathophysiology: The normal physiology of cells within an animal organism is regulated through signaling. This signaling can be autocrine, paracrine or endocrine, and it can be through growth factors, chemokines, hormones, or through cell-cell contact. This complex signaling frequently involves the participation of the gycocalyx and extracellular matrix and is critical for normal physiological function. In addition to normal physiological processes, much pathophysiology is associated with the subversion of the normal glycome or an abnormal glycome. For example, in malaria, the normal glycome of the mosquito and the human hosts are subverted by the parasite to gain entry and infect. An understanding of the similarity between the mosquito and human glycome could offer novel therapeutic approaches to the prevention of this disease.
Figure 5. The life cycle of Plasmodium in the mammalian host (A) and the mosquito (B). A. Sporozoites are injected during blood feeding by infected Anophelene mosquitoes, go to the liver and invade hepatocytes where they divide, rupture from the hepatocyte and enter erythrocytes. The released merozoites invade erythrocytes, mature, divide, rupture from the cell and enter new erythrocytes. The asexual erythrocytic cycle gives rise to the clinical symptoms associated with malaria. Some merozoites differentiate to gametocytes, which are infective for mosquitoes. Figure reprinted with permission from Miller et al., Science 234:1349, (1986). B. Mosquitoes become infected with Plasmodium when they ingest gametocytes (1) during blood feeding. In the midgut (MG) of the mosquito, these cells differentiate (2), fertilization occurs and the resultant zygote differentiates into an ookinete (3), which traverses the midgut wall (4) and comes to rest extracellulary between the basal lamina and the midgut (5). The ookinete then differentiates into an oocyst and sporozoites develop (6), and when mature, emerge into the hemocoel (HC) of the mosquito and invade salivary glands (7;SG). When the mosquito takes another blood meal, these salivary gland sporozoites are injected into the vertebrate host (8), thus continuing the cycle.
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