Research Highlights

N-glycomics: Of apicomplexa and algorithms

Effects of tunicamycin treatment on T. gondii morphology, shown by immunostaining of two marker proteins. Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.

N-glycosylation regulates protein sorting, folding and extracellular function, and N-glycomics provides information on the entirety of glycan structures present on proteins in a specific cell type or organism. Now, in Glycobiology, Renkonen et al. describe an automated approach to combined N-glycoproteomics/ N-glycomics and in Molecular and Cellular Proteomics, Fauquenoy et al. report a study on Toxoplasma gondii N-glycosylation.

Renkonen et al. present a novel accelerated approach to N-glycomics/N-glycoproteomics that integrates MS, database mining and statistical methods to allow researchers to determine glycan composition and amino acid sequence simultaneously. The tryptic digest of the protein sample is analyzed by MS after N-glycopeptide enrichment and the MS data then subjected to in silico analysis. Using database comparison, the authors assign amino acid sequences to the glycopeptides and validate the results statistically. The glycopeptide's N-glycan composition is then determined and scored by applying an algorithm to the MS data. This algorithm builds N-glycan structures de novo and finally delivers a group of the statistically most likely glycan compositions, which can be further downsized by methods such as lectin staining. Renkonen et al. validated their strategy using a set of tryptic transferrin digests of known composition. They then analyzed blood serum glycopeptides to generate a list of the amino acid sequences and N-glycan compositions of 80 glycopeptides found in the sample. The method presented by Renkonen et al. is a promising step toward combined high-throughput glycoproteomics that targets native glycans and proteins in a single run.

T. gondii is a protozoan parasite that causes severe health problems in congenitally infected children. Using mass spectrometry (MS) Fauquenoy et al. showed that the majority of the parasite's N-glycans possess 3-8 mannose molecules that lack both lactose elongation and terminal fucosylation or sialylation. Thus, T. gondii N-glycans resemble the earlier stages of N-glycosylation seen in higher eukaryotes. N-glycans were mainly found in the secreted protein complex known as the the glideosome and in proteins of the late secretory organelle named rhoptry, both aiding T. gondii movement toward, and invasion into, the host cell. Treating the cells with the N-glycosylation inhibitor tunicamycin reduced T.gondii replication by 90%; the treated cells lacked the inner membrane complex, suggesting that in T. gondii the formation of this complex is supported by N-glycosylation. These findings may settle the dispute over N-glycan presence in T. gondii, and lead to further analysis into the function of individual N-glycans in parasite motility and host cell interaction.

Author: Mirko von Elstermann