Copper incorporation into lysyl oxidase

T. Kosonen, J. Y. Imu-Hare, M. S. Cleyy, Carl L Keen, R. B. Rucker

Research output: Contribution to journalArticle

Abstract

Lysyl oxidase (L.Ox.), a copper(Cu)-dependent enzyme, is essential for collagen and elastin crosslinking. Although it is established that L.Ox. activity is responsive to Cu deficiency, few details are known about the incorporation of Cu into the protein. To this end, we have used cell-free transcription / translation and skin fibroblast culture systems to examine possible sites and conditions important to Cu incorporation. In our cell-free translation experiments utilizing a full-length L.Ox. cDNA as a template, prolysyl oxidase translation products bound Cu weakly independent of their glycosylation. When confluent human skin fibroblasts were incubated in the presence of 50-300 uCi Cu and inhibitors, cycloheximide (1 ng/ml or 10 ug/ml), tunicamycin (10 ug/ml), or brefeldin A (10 ug/ml), the following was observed regarding 67Cu secretion into medium as putative L.Ox. New protein synthesis of lysyl oxidase is needed for 67Cu incorporation and secretion. Tunicamycin, a glycosylation inhibitor, only modestly influenced 67Cu secretion as L.Ox. Importantly, brefeldin A, a protein secretion inhibitor, did not inhibit cellular Cu incorporation, but did inhibit L.Ox. secretion to the cell medium. Lysyl oxidase was separated on a Superdex 75 HPLC column, by PAGE, and elastin affinity columns. Fractions containing lysyl oxidase were identified in ELISA utilizing L.Ox. antibodies, and by functional assays based on Hj02 production using purified tropoelastin as substrate. Based on our findings, Cu can be inserted into prolysyl oxidase, possibly at the level of Golgi or at a post-Golgi step. Our results parallel data on Cu incorporation into ceruloplasmin, and Aus reveal a common theme for the post-translational insertion of metal cofactors.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number3
StatePublished - 1996

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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    Kosonen, T., Imu-Hare, J. Y., Cleyy, M. S., Keen, C. L., & Rucker, R. B. (1996). Copper incorporation into lysyl oxidase. FASEB Journal, 10(3).