title: 10mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin.
abstract: 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.
  
  
  METHODS
  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 5-10mM 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, Asn(387) and Asn(440), 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.
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: McCulloch
authors.forename: J D
authors.initials: JD
authors.lastname: Wylie
authors.forename: J-M
authors.initials: JM
authors.lastname: Longpre
authors.forename: R
authors.initials: R
authors.lastname: Leduc
authors.forename: S S
authors.initials: SS
authors.lastname: Apte
mesh_terms: D051722:ADAM Proteins / Q000187:drug effects*; D000071116:ADAMTS5 Protein; D015153:Blotting, Western; D002478:Cells, Cultured; D045683:Furin / Q000187:drug effects*; D005944:Glucosamine / Q000378:metabolism*; D006801:Humans; D011499:Protein Processing, Post-Translational; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013223:Statistics as Topic
chemical_list: D045683:Furin; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D005944:Glucosamine