Title : Glycosylation in a mammalian expression system is critical for the production of functionally active
leukocyte immunoglobulin-like receptor A3 protein
Abstract :
- 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 β-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
Output (sent_index, trigger,
protein,
sugar,
site):
- 11. glycosylated, , LILRA3, -, -
- 9. N-glycosylation, , -, -, Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 9. leukocyte immunoglobulin-like receptor A3 protein, -, Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites