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10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

McCulloch, D. R., Wiley, J. D., Longpre, J.-M., Leduc, R. and Apte, S. S. 2010, 10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin, Osteoarthritis and cartilage, vol. 18, no. 3, pp. 455-463, doi: 10.1016/j.joca.2009.10.014.

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Title 10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin
Author(s) McCulloch, D. R.
Wiley, J. D.
Longpre, J.-M.
Leduc, R.
Apte, S. S.
Journal name Osteoarthritis and cartilage
Volume number 18
Issue number 3
Start page 455
End page 463
Total pages 9
Publisher Elsevier
Place of publication London, England
Publication date 2010-03
ISSN 1063-4584
1522-9653
Keyword(s) aggrecanase
glucosamine
ADAMTS
furin
catabolism
Summary Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results

Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.
Language eng
DOI 10.1016/j.joca.2009.10.014
Field of Research 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics)
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1 Refereed article in a scholarly journal
ERA Research output type C Journal article
HERDC collection year 2010
Copyright notice ©2009, Osteoarthritis Research Society International
Persistent URL http://hdl.handle.net/10536/DRO/DU:30031460

Document type: Journal Article
Collection: School of Medicine
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Created: Mon, 06 Dec 2010, 14:20:20 EST by Penny Andrews

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