Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas

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.

Mycoplasma pneumoniae from the Respiratory Tract and Beyond

“…Control of phosphorylation of many of these proteins by the kinase and phosphatase pair PrkC and PrpC is poorly understood at the regulatory level but essential for function (102). However, phosphorylation of M. pneumoniae proteins is widespread even in the absence of PrkC, suggesting the existence of other means by which proteins become phosphorylated (103). Although undiscovered protein kinases may exist, at least some proteins, such as enolase, are labeled with phosphate because of their covalent linkage to phospholipids, representing a recently discovered means by which proteins may be targeted to the plasma membrane.…”

Moonlighting Proteins at the Candidal Cell Surface

“…Such putative secretory routes include the direct translocation of proteins through the cell membrane by dedicated membrane-associated transporters, modulation of the activity of plasma membrane flippases, membrane flipping together with the adhered cytoplasmic proteins exhibiting the affinity for phospholipids and the escape of cytoplasmic proteins during cell division through the fragmentation zone engaging a lock-type mechanism [ 62 , 63 , 64 , 65 , 66 ]. In the case of some human and bacterial moonlighting proteins, it has been suggested that particular post-translational modifications, such as 2-phosphoglycerate-dependent automodification, rhamnosylation or monomethylation, might support their extracellular transport or membrane localization [ 67 , 68 , 69 ]. This issue has yet to be investigated for C. albicans proteins however.…”

Protein moonlighting: what is it, and why is it important?

“…The mechanism of cell surface attachment could potentially be a part of a secretion mechanism, although some recombinantly expressed and purified intracellular/surface moonlighting proteins are capable of reattaching to the cell surface. With a few exceptions [115] most of the ICSPs do not contain known amino acid sequence or structural motifs for attaching to the cell surface. Cell surface attachment could involve a new version of a known mechanism or it may involve an as-yet-unknown mechanism.…”