Toward an experimental system for the examination of protein mannosylation in Actinobacteria

The Actinobacterial species Cellulomonas fimi ATCC484 has long been known to secrete mannose-containing proteins, but a closer examination of glycoproteins associated with the cell has never been reported. Using ConA lectin chromatography and mass spectrometry, we have surveyed the cell-associated glycoproteome from C. fimi and collected detailed information on the glycosylation sites of 19 cell-associated glycoproteins. In addition, we have expressed a previously known C. fimi secreted cellulase, Celf_3184 (formerly CenA), a putative peptide prolyl-isomerase, Celf_2022, and a penicillin-binding protein, Celf_0189, in the mannosylation capable host, Corynebacterium glutamicum. We found that the glycosylation machinery in C. glutamicum was able to use the recombinant C. fimi proteins as substrates and that the glycosylation matched closely that found in the native proteins when expressed in C. fimi. We are pursuing this observation as a prelude to dissecting the biosynthetic machinery and biological consequences of this protein mannosylation.

Automated summary

We have investigated the glycoproteome of the Actinobacterial species Cellulomonas fimi ATCC484 and identified 19 cell-associated glycoproteins using ConA lectin chromatography and mass spectrometry. Furthermore, we expressed selected C. fimi proteins in a mannosylation capable host and found that the glycosylation machinery in the host could glycosylate the recombinant proteins matching the native glycosylation in C. fimi. This observation sets the stage for further dissection of the biosynthetic machinery and biological consequences of protein mannosylation.