Understanding bacterial protein N-rhamnosylation

• Applicant: Privatdozent Dr. Jürgen Lassak
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Analytical Chemistry
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 356237927

One of the most common forms of protein posttranslational modification is coupling of a sugar unit to an amino acid side chain, known as glycosylation. The modification is accomplished by a class of enzymes known as glycosyltransferases. When the sugar is covalently linked to the nitrogen of the carbamide group of asparagine or the guanidino group of arginine, one speaks of N-glycosylation. Recent discoveries of new N-glycosyltransferases in bacteria and the development of modification-specific antibodies indicate for a large variety of previously unknown prokaryotic N-glycoproteins. Based on our findings on protein monorhamnosylation, we now want to investigate their distribution in microorganisms in more detail. In the first part of the project, we focus on the actinobacterium Mycobacterium phlei, which possesses a complex and dynamic rhamnoproteome. Our goal is to develop a methodology for the enrichment monorhamnosylated proteins and their efficient analysis via mass spectrometry, to gain for the first time a comprehensive and site-resolved picture of the importance of rhamnosylation in M. phlei. To achieve the highest possible selectivity and sensitivity of the analysis, we will, in addition to liquid chromatography and high-resolution time-of-flight mass spectrometry, employ trapped ion mobility spectrometry as an additional dimension of separation and characterize it for this new application. First, we will investigate the utility of the collision cross section to identify rhamnosylated peptides, and second, we will develop methods to prioritize modified peptides during data acquisition based on their relative ion mobility. In the second part of the project, we will focus on monorhamnosylations catalyzed by the glycosyltransferase EarP. Contrary to initial assumptions, its acceptor substrate spectrum is not limited to the translation factor EF-P. Accordingly, our goal is to fully understand EarP-mediated glycosylation through biochemical studies and mass spectrometry, and to describe the target protein spectrum in Pseudomonas putida and Shewanella oneidensis comprehensively. Ultimately, we will apply our gained knowledge about the EarP acceptor substrate promiscuity to biotechnologically exploit the enzyme as glycosynthase.

• DFG Programme: Research Grants