Title : Post-translational modifications of recombinant human
lysyl oxidase-like 2 (
rhLOXL2 ) secreted from Drosophila S2 cells
Abstract :
- Human lysyl oxidase-like 2 ( hLOXL2 ) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion
- hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase ( LOX ) catalytic domain
- Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells
- The recombinant proteins are soluble, in contrast to LOX , which is consistently reported to require 2-6 m urea for solubilization
- The recombinant proteins also show activity in tropoelastin oxidation
- After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689 , as well as N-linked glycans at Asn-455 and Asn-644
- Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected
- The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX ( hLOX ), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2
- The catalytic domain of hLOX was not secreted from S2 cells using the same expression system
- These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain
Output (sent_index, trigger,
protein,
sugar,
site):
- 10. hLOXL2, , hLOXL2, the N-glycan at Asn-644, -
- 6. Asn-455, , -, N-linked glycans, Asn-455 and Asn-644
- 6. Asn-644, , -, N-linked glycans, Asn-455 and Asn-644
- 8. N-glycosylation, , -, -, Asn-644
- 8. N-glycosylation, , -, -, site
Output(Part-Of) (sent_index,
protein,
site):
- 10. LOX, domain
- 2. LOX, domain
- 2. lysyl oxidase, domain
- 8. LOX, domain
- 8. hLOX, domain
- 9. hLOX, domain
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 6. Human lysyl oxidase-like 2, N-linked glycans, Asn-455 and Asn-644
- 8. Human lysyl oxidase-like 2, -, Asn-644