Glycobiology Research and Training Opportunities are Plentiful

glycans on cell surface

Artist’s rendering of asymmetrically-branched carbohydrates on cell surface proteins.

Glycobiology is the study of glycans, the carbohydrate molecules that cover the surface of most human cells. Glycans attach to cell surface proteins and lipids, in a process called glycosylation. These cell surface structures are responsible for processes as varied at protein folding, cell signaling and cell-cell recognition, including sperm-egg recognition and immune cell interactions. Glycans play important roles in the red blood cell antigens that distinguish blood types O, A and B.

Opportunities in Glycomics Research
As more is learned about the role of glycans in cell communication, they are becoming important disease research targets, particularly the role of glycans in cancer and inflammatory diseases (2).

Some of the open questions surrounding glycans and glycosylation include glycan structural diversity. While some carbohydrates exist as straight or symmetrically branched chains, those populating the human glycome are asymmetrically branched, making them difficult to create and study in the laboratory (3). Continue reading

Glycosyltransferases: What’s New in GT Assays?

In his 2014 blog, “Why We Care About Glycosyltransferases” Michael Curtin, Promega Global Product Manager for Cell Signaling, wrote:

“Glycobiology is the study of carbohydrates and their role in biology. Glycans, defined as ‘compounds consisting of a large number of monosaccharides linked glycosidically’ are present in all living cells; They coat cell membranes and are integral components of cell walls. They play diverse roles, including critical functions in cell signaling, molecular recognition, immunity and inflammation. They are the cell-surface molecules that define the ABO blood groups and must be taken into consideration to ensure successful blood transfusions.

The process by which a sugar moiety is attached to a biological compound is referred to as glycosylation. Protein glycosylation is a form of post-translational modification, which is important for many biological processes and often serves as an analog switch that modulates protein activity. The class of enzymes responsible for transferring the sugar moiety onto proteins is called a glycosyltransferase (GT).”

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Improved Characterization and Quantification of Complex Cell Surface N-Glycans

MSextractcroppedN-Glycosylation is a common protein post-translational modification occurring on asparagine residues of the consensus sequence asparagine-X-serine/threonine, where X may be any amino acid except proline. Protein N-glycosylation takes place in the endoplasmic reticulum (ER) as well as in the Golgi apparatus.

Approximately half of all proteins typically expressed in a cell undergo this modification, which entails the covalent addition of sugar moieties to specific amino acids. There are many potential functions of glycosylation. For instance, physical properties include: folding, trafficking, packing, stabilization and protease protection. N-glycans present at the cell surface are directly involved in cell−cell or cell−protein interactions that trigger various biological responses.

The standard method used to profile the N-glycosylation pattern of cells is glycoprotein isolation followed by denaturation and/or tryptic digestion of the glycoproteins and an enzymatic release of the N-glycans using PNGase F followed by analysis mass spec. This method has been reported to yield high levels of high-mannose N-glycans that stem from both membrane proteins as well as proteins from the ER.(1,2)

For those researchers interested in characterizing only cell surface glycans (i.e.,  complex N-glycans)  a recent reference has developed a model system using HEK-292 cells that demonstrates a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells (3). The method involves standard centrifugation followed by enzymatic release of cell surface N-glycans. When compared to the standard methods the detection and quantification of complex-type N-glycans by increased their relative amount from 14 to 85%.

  1. North, S. J. et al. (2012) Glycomic analysis of human mast cells, eosinophils and basophils. Glycobiology. 2012, 22, 12–22.
  2. Reinke, S. O. et al. (2011) Analysis of cell surface N-glycosylation of the human embryonic
    kidney 293T cell line.
    J. Carbohydr. Chem.  30, 218–232.
  3. Hamouda, H. et al. (2014) Rapid Analysis of Cell Surface N‑Glycosylation from Living Cells Using Mass Spectrometry. J of Proteome Res. 13, 6144–51.

Why We Care About Glycosyltransferases

Today’s post is a guest blog from Michael Curtin in the cellular analysis and proteomics group at Promega.

Glycobiology is the study of carbohydrates and their role in biology. Glycans, defined as “compounds consisting of a large number of monosaccharides linked glycosidically” are present in all living cells and coat cell membranes and are integral components of cell walls (1). They play diverse roles, including critical functions in cell signaling, molecular recognition, immunity and inflammation. They are the cell-surface molecules that define the ABO blood groups and must be taken into consideration to ensure successful blood transfusions. (2).The process by which a sugar moiety is attached to a biological compound is referred to as glycosylation. Protein glycosylation is a form of post-translational modification, which is important for many biological processes and often serves as an analog switch that modulates protein activity.The class of enzymes responsible for transferring the sugar moiety onto proteins is called a glycosyltransferase (GT).

GTs can be divided into three major types based on their roles:

  • Oligosaccharide elongation for peptidoglycan biosynthesis
  • Regulation of protein activities by post-translational modification
  • Small molecule glucuronidation as means of drug metabolism

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