Abstract
Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria.
Highlights
The genus Mycoplasma is composed of minimalist wall-less bacteria that are obligate parasites
We found that a rabbit polyclonal antibody against amino acids 1–300 of human alpha enolase reacted with enolase from M. pulmonis (Fig 1B)
We find here that rhamnose serves to link proteins of M. pulmonis to phospholipid
Summary
The genus Mycoplasma is composed of minimalist wall-less bacteria that are obligate parasites. Mycoplasma genitalium has the distinction of having the smallest genome known for a free-living eubacteria, 470 predicted coding regions [1]. One consequence of this minimalism is host specificity and dependency. Mycoplasmas are dependent on their host for cholesterol and lipids Despite such a limited genome, this genus synthesizes polysaccharides, glycosylates proteins, forms biofilms, and has a multi-component, sophisticated host immune avoidance system [2,3]. It has all the systems required for a free-living, self-replicating bacterium.
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