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.
authors: Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045, USA.
authors: Li
authors: L
authors: Xu
authors: Eden P
authors: EP
authors: Go
authors: Joel
authors: J
authors: Finney
authors: HeeJung
authors: H
authors: Moon
authors: Mason
authors: M
authors: Lantz
authors: Kathryn
authors: K
authors: Rebecchi
authors: Heather
authors: H
authors: Desaire
authors: Minae
authors: M
authors: Mure
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D002241:Carbohydrates / Q000737:chemistry; D020134:Catalytic Domain; D002460:Cell Line; D004331:Drosophila melanogaster; D006031:Glycosylation; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011809:Quinones / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry
chemical_list: D002241:Carbohydrates; D010455:Peptides; D011134:Polysaccharides; D011809:Quinones; D011994:Recombinant Proteins; D012997:Solvents; C100796:lysine tyrosylquinone; D000594:Amino Acid Oxidoreductases; C119091:LOXL2 protein, human; D008239:Lysine