by Pamela Stanley, Ph.D., Department of Cell Biology, Albert Einstein College of Medicine in New York. Stanley presented this report at the Glycobiology Interest Group's annual "Glycoday" on May 30, 1995, in Annapolis, Md.


To identify biological functions of mammalian carbohydrates, members of my lab have isolated a panel of Chinese Hamster Ovary (CHO) mutants with altered glycosylation of proteins and lipids and, consequently, a novel array of carbohydrates at the cell surface. Many mutant cell lines are phenotypically recessive, represent loss-of-function mutations, and display immature, or biosynthetic intermediate, carbohydrate structures on glycoconjugates. Other mutants are phenotypically dominant and represent gain-of-function glycosylation mutants. This means that the gain-of-function mutants express more complicated carbohydrates than do wild-type cells. The first examples of this that we discovered in CHO cells were the mutants LEC11 and LEC12 (see figure). Both of these cell lines express the Lex trisaccharide determinant on cell-surface glycoconjugates, but only LEC11 cells express the sialylated version, known as SLex (see figure). The latter structure, unlike the Lex trisaccharide, is an excellent ligand for the selectin group of cell-adhesion molecules, when it is presented on an appropriate cell-surface glycoprotein or glycolipid. The presence of the Lex and SLex structures on the surface of LEC11 or of Lex on LEC12 CHO cells is due to the de novo expression of two distinct [[alpha]](1,3)fucosyltransferases that are developmentally regulated in mammals. We are now attempting to define the mutation-like event that activates transcription of these usually quiescent genes, allowing CHO cells to express the developmentally regulated carbohydrates. Additional mutants that express the Lex determinant have been isolated, and each possesses an [[alpha]](1,3)fucosyltransferase with distinctive properties, suggesting they are the products of different genes. Another gain-of-function mutant that we identified about 10 years ago is LEC10, which expresses the bisecting GlcNAc (see figure). The existence of carbohydrates with Lex, Slex, or the bisGn residue was known before the CHO mutants were isolated. However, the most recent gain-of-function mutants to be characterized possess proposed structures that have never previously been described from any source. The novel modifications were discovered on pure carbohydrates unique to the LEC18 and LEC14 CHO dominant mutants (see figure), and both represent novel core structures of N-linked glycans, presumably reflecting the activation of quiescent genes encoding new GlcNAc-transferases. Our preliminary results are consistent with this. The properties of LEC14 and LEC18 CHO cells reveal a potentially large reservoir of developmentally regulated glycosylation genes that are awaiting discovery through gain-of-function CHO mutants.


Q: What was your starting point in this research, and how have your questions evolved?

A: The starting point for this work was the discovery in the early 1970s that toxic plant lectins are useful as selective agents for isolating cells with mutations in enzymes that glycosylate cell-surface macromolecules. The first CHO mutant to be characterized biochemically lacked a transferase (GlcNAc-TI), which is required for the synthesis of complex and hybrid N-glycans. Surprisingly, the absence of GlcNAc-TI and the major truncation of cell-surface carbohydrates that resulted did not have any effect on cell growth or viability in culture. Our lab and another group have now shown, however, that mice lacking GlcNAc-TI are severely affected and die at midgestation. This evidence supports the long-standing suspicion that specific cell-surface carbohydrates, though not important for basic somatic-cell functioning in culture, are absolutely required for mammalian development. Exploring the developmental role of these molecules led us to pursue gain-of-function glycosylation mutants. Our discovery that CHO cells express the developmentally regulated carbohydrates Lex and SLex, as revealed by the LEC11 and LEC12 mutants, was tremendously exciting, and the nature of these mutants made us realize that biologically functional new molecules could be discovered by identifying the biochemical basis of these dominant mutations.

Q: Which findings have been most surprising to you or to other scientists?

A: The most surprising and exciting realization was that by characterizing gain-of-function glycosylation mutants, we could discover completely new molecules that would be very hard--even impossible--to discover by any other approach. The proposed new carbohydrate structures synthesized by LEC14 and LEC18 cells have not been found in secreted or membrane glycoproteins from any source to date. In vivo, N-glycans with such modifications may be synthesized only in one cell type during a brief stage development and may thus be virtually impossible to detect via current technologies. Also, the enzymes that synthesize these glycans may be very difficult to assay in a complex background.

The second surprising finding was the enormous effect that the addition of one sugar residue has on the lectin-binding properties of cell-surface carbohydrates. In the case of LEC10 cells, the presence of the bisecting GlcNAc residue causes cells to become 20 times more resistant to the toxin ricin and about 10 times more hypersensitive to another toxic lectin, the erythroagglutinin called E-PHA. Both of these lectins bind to galactose (Gal) residues, and the Gal residues they bind to are still present in LEC10 cells. In LEC10 cells, however, the bisecting GlcNAc changes the conformation of the carbohydrate to make the Gal residues markedly less accessible to ricin and more accessible to E-PHA. This paradigm holds true for the single-sugar changes found in the other dominant CHO glycosylation mutants: LEC11, LEC12, LEC29, and LEC30, which have varying and characteristic degrees of increased resistance to wheat germ agglutinin (WGA) and increased sensitivity to ricin, and LEC14 and LEC18, which are resistant to the pea lectin (PSA) and Lens culinaris agglutinin (LCA). Thus, the regulated expression of a single glycosyltransferase gene can dramatically alter lectin-recognition specificities at the cell surface.

Q: What were the greatest stumbling blocks, and what new observations, techniques, reagents, or insights helped you get past them?

A: The most difficult aspect of carbohydrate work is proving that you have a precise structure. We were fortunate that 1H-NMR spectroscopy of complex carbohydrates was developed in the mid 1970s. For assigned structures present in the NMR database, it was then possible to determine a structure for an unknown from the spectrum of approximately 200 ug of chemically pure oligosaccharide or glycopeptide. In that manner, we were able to deduce the new carbohydrate structures characteristic of LEC10, LEC11, and LEC12 cells. The major difficulty was working out how to obtain enough of the relevant carbohydrate in extremely pure form. Although the SLex determinant was not in the NMR literature, we were able to assign new resonances by obtaining spectra of the carbohydrates before and after removal of the terminal sialic acid. Unfortunately, this approach yielded no structures comparable to the glycopeptides unique to LEC14 and LEC18 in the Sugabase database which lists known structures of carbohydrates. Our lifesaver in this case was the use of electro-spray mass spectrometry (ES/MS) and mass spectrometry, mass spectrometry (MS/MS) of the major molecular ions to generate fragment ions that could only have arisen from the specific structures we propose. The key here was the realization that if the sample was carefully prepared and the experiment performed under certain controlled conditions, the resulting ES/MS spectra were interpretable and reproducible. Composition and linkage analyses by GC/MS were also critical for arriving at our postulated structures.

Q: How can clinical scientists capitalize on this research?

A: Right now, no biological roles for the proposed new carbohydrates of LEC14 or LEC18 are known, and, therefore, further work will be required before clinically relevant uses become apparent. However, the carbohydrates characterized by LEC10 (bisGn), LEC11 (Lex and SLex), and LEC12 (Lex) CHO cells are all expressed at high levels in some tumors. It is known that the presence of even a single sugar residue, such as bisGn, has a profound effect on the conformation of the structure to which it is added. Thus, we think that the changed properties of a cancer cell that expresses these structures may have functional consequences on tumor progression or, more likely, on metastasis. It has been shown by others that certain mouse tumor cells do not metastasize if they do not add the [[beta]]1,6GlcNAc branch to complex N-glycans.

Many of the cell-surface carbohydrates we study are also antigenic and thus can be used to aid in cancer diagnosis, although this can be complicated by the blood-group genotype of an individual. To date, no carbohydrate antigen that is completely diagnostic has been described, but expression of SLex is characteristic of certain cancers.

Q: How are you following up on this work, and what questions would you ultimately like to answer?

A: We would now like to know the role that the unique cell-surface carbohydrates we have discovered play in development. In those cases characterized to date, the novel carbohydrates expressed by gain-of-function CHO mutants are the product of developmentally regulated transferases. We are following up on this knowledge by using CHO cells to expression-clone these and related transferase genes as well as others that regulate the expression of glycosyltransferase genes. Once we clone these genes, we will characterize their expression pattern in the mouse and construct mutants with a null mutation in the transferase gene. This approach would appear to be the fastest way to uncover biological functions of these carbohydrates. Because the mutations generated will only change particular structures by eliminating one sugar residue, they should not be embryonically lethal and may yield interesting phenotypes. Even if the null mutation does not produce an altered phenotype, however, the mice may be useful in cancer studies because their cells cannot add the sugars that normally generate tumor antigens. Such animals could be tested for response to carcinogens, and their metastatic patterns could be compared with those of other strains of mice. These are complex, multifaceted questions, and we seek collaborators with established carcinogenic or metastatic protocols that could yield insights into cancer biology using our mouse glycosyltransferase mutants. --J.E.S.

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