BioPEGylation of polyhydroxybutyrate promotes nerve cell health and migration.

This study reports on the superior suitability of Polyhydroxybutyrate-polyethylene glycol hybrid polymers biosynthesised by Cupriavidus necator over PHB as biomaterials for tissue engineering. Incorporation of PEG106 (DEG) during PHB biosynthesis reduced crystallinity, molecular weight, and hydrophobicity while improving mechanical properties. In vitro olfactory ensheathing cell (OEC) proliferation was enhanced by cultivation on PHB-b-DEG films. Cultivation on PHB and PHB-b-DEG films showed no cytotoxic responses and cell viability and membrane integrity was sustained. PHB-b-DEG films promoted OECs entering into the DNA replication (S) phase and mitotic (G2-M) phase during the cell growth cycle and apoptosis was low. This study also confirmed an association between the level of neurite-outgrowth inhibitory protein (Nogo) and receptor pair Ig-like receptor B (PirB) expression and cell proliferation, both being down-regulated in cells grown on hybrid films when compared with PHB and asynchronous growth. Thus, DEG-terminated PHB-based biomaterials have great potential as biological scaffolds supporting nerve repair.

Glycosylation in a mammalian expression system is critical for the production of functionally active leukocyte immunoglobulin-like receptor A3 protein.

The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2-4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by ß-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions.

The role of lysine amino nitrogen in the biosynthesis of mousy off-flavor compounds by Dekkera anomala.

Mousy off-flavor is an insidious and economically disastrous microbiologically derived spoilage characteristic of wine and other fermented beverages. Tainted wines are rendered unpalatable and there is currently no satisfactory procedure for removal of the off-flavor. Here we report the confirmation of that both d- and l-lysine can act as a precursor for the formation of mousy off-flavor N-heterocycles. Further, through the use of stable isotope feeding experiments, we could establish that a pentylamine group from lysine is incorporated into the piperideine moiety of two off-flavor N-heterocycles. A biochemical pathway for the formation of mousy off-flavor compounds is proposed.