Research Highlights

PCR glycogene analysis: Medium throughput, higher sensitivity

Relative transcript abundance for processing steps involving N-glycan trimming and branching in the endoplasmic reticulum and Golgi complex. Detail of Fig. 3 of Nairn et al. which is free for full text access. Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.

Does the amount of glycogene messenger RNA determine the composition of cell surface glycans? Synchronous application of mass spectrometry analysis on glycans and glycogene transcription analysis by microarrays – introduced to allow a high-throughput determination of transcription levels – confirms this relationship by and large. However, a qRT-PCR strategy, which could support the microarray analysis and may allow for a higher analytical sensitivity, has not yet been developed. Nairn et al. now describe in the Journal of Biological Chemistry an experimental setup that allows a medium-throughput determination of glycogene transcription levels by qRT-PCR.

The authors compiled a list of 745 mouse genes involved in glycan synthesis by using various databases, such as CAZy (Carbohydrate-Active Enzymes), KEGG (Kyoto Encyclopedia of Genes and Genomes) and the CFG (Consortium for Functional Glycomics) databases. After performing qRT-PCR on these glycogenes in mouse organ samples, Nairn et al. found that in most cases their data did not contradict mass spectrometric (MS) analyses of glycans on mouse cells. A notable exception was the presence of oligomannose structures in the liver which implied a reduced trimming of the N-glycan mannose core by mannosidases or extension by acetylglucosaminyltransferases; however, their expression in the liver was similar to that of other organs. Nairn et al. explain this finding with the fact that the smooth endoplasmic reticulum (sER), where the transfer of the high-mannose N glycan to its protein acceptor and initial mannosidase reactions take place, is much more elaborate in the liver than in other organs. Thus, N-glycosylation products from sER are less likely to encounter the glycosylation machinery of the Golgi apparatus necessary to make more complex glycan structures.

Nairn et al. detected twice as many glycogene transcripts in the liver and 50% more in the mouse kidney than previously found by the second version of the Affymetrix glycogene microarray. They discovered an upregulation of the 2,8-sialyltransferase and its glycosylation target, the polysialylated NCAM (neural cell adhesion molecule) in the mouse testes. They also found increased ganglioside 2,8-sialyltransferase transcripts in testis, confirming previous hypotheses from other groups for a role for polysialylated gangliosides in spermatogenesis. Furthermore, the qRT-PCR approach allowed the authors to readily identify the enzyme isoform responsible for a specific glycosyltransferase reaction, such as in the case of the 5 fucosyltransferase isoforms performing 1,3 fucosylation in mouse tissues.

Taken together, the qRT-PCR approach provides higher sensitivity for the analysis of transcripts encoding approximately 745 glycogenes by comparison to microarray approaches. However it is more limited than high-throughput microarray approaches in the number of target genes that can be analyzed in one run. As the actual glycan composition depends also upon factors such as cell substructure and metabolic state, broad-based profiling of transcript levels may provide an effective complement to established MS-based glycan analysis methods to examine regulation of glycan structures in biological systems. Thus, glycogene qRT-PCR analysis adds a new powerful approach to the current glycomic analysis toolbox.

Author: Mirko von Elstermann