Exploring the fundamentals of bacterial protein O-mannosylation

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Protein mannosylation has been observed from bacteria to humans and appears to be a highly conserved protein modification that is essential in eukaryotes. The enzyme responsible for the protein mannosylation belongs to the highly conserved glycosyltransferase family (GT39), which are present as single copy genes in over 1300 species of bacteria, most of them in the actinobacterial group. Protein mannosylation in actinobacterial species was originally described 40 years ago, however we still lack a basic understanding of why these bacteria make this post-translational modification. We undertook a targeted enrichment of mannosylated proteins extracted from whole cell lysates using lectin chromatography with Concanavalin A and we were able to identify wide variety of mannosylated proteins. In addition to the known secreted mannosylated cellulases, we discovered that cytoplasmic and membrane associated proteins could also be mannosylated. Our data indicate the general nature of the protein mannosylation in Cellulomonas, as has been seen in Corynebacterium and Mycobacteria. However, the biochemistry of glycosylation and the biological significance of this common modification has not been adequately described for any of those proteins that bear the modification. To fully understand the biological role of this important (and conserved) post-translation modification system, we need to take this past global knock out phenotypes and probe this is in a more intimate way with selected targets to see how the carbohydrate influences protein function.    This proposal will examine the biochemistry of the glycosylation pathway using recombinant expression of the GT39 enzyme to determine its membrane topology, and structure-function in vivo. We will also express conserved mannosylated glycoproteins as targets for a more thorough analysis of the glycosylation pathway itself. Our program seeks to provide a more in-depth analysis of the molecular basis for the modification reaction itself, and the consequences of that modification using conserved proteins involved in basic growth and metabolism of Actinobacteria. Our survey of cellular glycoproteins will solidify the list of conserved proteins which have integral roles in bacterial physiology. Investigating this energy intensive modification of a subset of conserved proteins in diverse species will help us decipher the protein - carbohydrate interactions that control protein interactions, which in turn control cell growth and metabolism. Application of this knowledge to strain improvement of the industrially employed actinobacterial species could help product yields or perhaps expand the range of products they can provide. There is also potential to identify processes which could become new therapeutic targets in the pathogenic Actinobacteria.