Recombinant Human Lysyl Oxidase-like 2 Secreted from Human Embryonic Kidney Cells Displays Complex and Acidic Glycans at All Three N-Linked Glycosylation Sites

Human lysyl oxidase-like 2 (hLOXL2), a glycoprotein implicated in tumor progression and organ fibrosis, is a molecular target for anticancer and antifibrosis treatment. This glycoprotein contains three predicted N-linked glycosylation sites; one is near the protein's active site, and at least one more is known to facilitate the protein's secretion. Because the glycosylation impacts the protein's biology, we sought to characterize the native, mammalian glycosylation profile and to determine how closely this profile is recapitulated when the protein is expressed in insect cells. All three glycosylation sites on the protein, expressed in human embryonic kidney (HEK) cells, were characterized individually using a mass spectrometry-based glycopeptide analysis workflow. These data were compared to the glycosylation profile of the same protein expressed in insect cells. We found that the producer cell type imparts a substantial influence on the glycosylation of this important protein. The more-relevant version, expressed in HEK cells, contains large, acidic glycoforms; these glycans are not generated in insect cells. The glycosylation differences likely have structural and functional consequences, and these data should be considered when generating protein for functional studies or for high-throughput screening campaigns.