Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Terry H. Y. Lee, Ainslie Mitchell, Sydney Liu Lau, Hongyan An, Poornima Rajeaskariah, Valerie Wasinger, Mark Raftery, Katherine Bryant*, Nicodemus Tedla

*Corresponding author for this work

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: LILRA3 is a soluble receptor with unknown functions that is abundantly present in human serum. Results: Optimally glycosylated recombinant LILRA3 protein produced only in a mammalian system binds potential ligands and suppresses monocyte function. Conclusion: LILRA3 suppresses LPS-mediated TNF production, suggesting that it is a new anti-inflammatory protein. Significance: This work provides first insight into the biochemical characteristics and functions of LILRA3.

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 TNF production by monocytes. Binding to monocytes was partially blocked by -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.

Original languageEnglish
Pages (from-to)32873-32885
Number of pages13
JournalJournal of Biological Chemistry
Volume288
Issue number46
DOIs
Publication statusPublished - 15 Nov 2013

Keywords

  • Glycoprotein
  • Glycosylation
  • Leukocyte
  • Mass Spectrometry (MS)
  • Monocytes
  • Protein Deamidation
  • Protein Expression
  • Protein Purification
  • Recombinant Protein Expression
  • Tumor Necrosis Factor (TNF)
  • PICHIA-PASTORIS
  • RHEUMATOID-ARTHRITIS
  • MULTIPLE-SCLEROSIS
  • CRYSTAL-STRUCTURE
  • N-GLYCOSYLATION
  • LIGAND-BINDING
  • ASSOCIATION
  • STABILITY
  • DISEASE
  • CELLS

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