Detecting the Sweet Biomarker on Cancer Cells.

The ability to detect cancer biomarkers (i.e., characteristic biomolecules appearing on cancer cells but not on normal cells) is crucial for early diagnosis of cancers. Now, a research team led by Liuqing Wen and Peng George Wang at Georgia State University provides a chemical tool for labeling and visualizing an antigen that is highly expressed on the surfaces of various cancer cells.[1]

Cells are coated with a dense layer of sugars (also called carbohydrates or glycans), and the cancer cells are no exception. Quite often, cancer cells carry specific glycan epitopes that either are not expressed, or only minimally so, in normal tissues. One of these cancer-specific glycans is sialylated Thomsen-Freidenreich, or sialyl-T, a trisaccharide found on mucin-type O-linked glycoproteins (Figure A). Accumulating evidence indicates that the presence of sialyl-T is correlated strongly with tumor development and progression.[2] Unfortunately, detecting sialyl-T on live cells has been difficult until now due to the lack of an effective traditional testing method, such as an antibody or lectin (Figure A). In addition, antibodies and lectins often suffer from relatively low specificity and affinity.

Figure 1

One-step CeGL labeling of sialyl-T. (A) Chemical structure of sialyl-T α-linked to the serine or threonine residue of cell-surface proteins (purple diamond–yellow circle–yellow square). (B) Living cells were treated with ST6GalNAc-IV and a CMP-sialic acid analogue containing a biotin moiety. ST6GalNAc-IV specifically recognizes sialyl-T on the cell surface and transfers biotin-functionalized sialic acid onto the GalNAc (yellow circle) of sialyl-T. The biotin can then be stained with streptavidin-fluorophore for imaging or streptavidin beads for enrichment and glycoproteomic profiling.

Figure 2

Three methods for labeling cell-surface glycans. (A) Antibodies and lectins conjugates with fluorescent probes can be used to directly detect glycans on cell surfaces. (B) MGL exploits the cellular machinery to metabolically incorporate monosaccharide analogues containing a bioorthogonal functional group, X, into cell-surface glycans. In the second step, a probe containing a complementary bioorthogonal group, Y, is reacted with X to label the glycans. (C) CeGL uses glycosyltransferases that have stringent acceptor specificity but can transfer monosaccharide analogues from the corresponding nucleotide sugars.

An alternative approach involves using cellular metabolism to label glycans with a monosaccharide analogue containing a bioorthogonal functional group such as an azide or alkyne, which can be subsequently conjugated with imaging probes or affinity tags using click chemistry (Figure B). This two-step chemical method has become a primary tool in the detection of glycans on live cells and in living organisms.[3,4] However, this sort of metabolic labeling can only target monosaccharides, such as sialic acid. Although sialyl-T contains a sialic acid, other glycans, including those found on the surface of healthy cells, also bear this monosaccharide. A recently emerged technique, chemoenzymatic glycan labeling (CeGL), addresses this limitation.[5,6] The CeGL strategy exploits a class of enzymes called glycosyltransferases that can specifically recognize a glycan acceptor and transfer on it a monosaccharide analogue via its corresponding nucleotide sugar donor (Figure C). When the monosaccharide analogue contains a bioorthogonal group, a second-step click reaction can again be performed to install various probes. Several glycan epitopes have been probed using CeGL, including LacNAc, Fucα2Gal, and TF antigen.[6]

In this issue of ACS Central Science,[1] Wen and co-workers used CeGL to detect sialyl-T by searching for a glycosyltransferase that only recognizes sialyl-T as the acceptor, but with relaxed standards for the donor. They found a human α-N-acetylgalactosaminide sialyltransferase (ST6GalNAc IV) that has high specificity for sialyl-T and is able to transfer biotin-functionalized sialic acid analogues (Figure B).

The use of biotin, rather than a chemical handle, makes this a one-step labeling process, which is an appealing feature of ST6GalNAc. The key is ST6GalNAc’s broad tolerance for large modifications on sugar donors. Mbua et al. earlier reported that another sialyltransferase, ST6Gal-I, can also transfer sialic acid analogues bearing large functional groups and be used for one-step CeGL labeling of N-linked glycans.[7] Wen and coauthors suggest that the tolerance of sugar donors with large modification groups might be a common feature shared by sialyltransferases.[1]

By using the ST6GalNAc IV-based CeGL, the authors visualized the expression of sialyl-T on a variety of cancer cells by fluorescence microscopy and quantitatively compared the expression level by flow cytometry. Furthermore, by using mass spectrometry-based proteomic analysis, they identified 78 cell-surface proteins in human breast cancer cells and 43 proteins in human colon cancer cells that are modified with sialyl-T.[1] These results provide valuable information for understanding the biological function of sialyl-T in cancer progression.

Further evaluation is needed to determine whether CeGL can be used in living animals. Encouragingly, engineered glycotransferases have recently been successfully injected into living mice to enzymatically modulate glycosylation of endogenous antibodies.[8]