Development and characterisation of an assay for furin activity.
Furin is a serine endoprotease that is responsible for the proteolytic processing of proteins within the secretory pathway, including cytokines, hormones, integrins, other proteases, and also pathogen-derived proteins. It is likely that the level of furin activity determines the extent of processing of these substrates. Furin is ubiquitously expressed across all tissues, at low levels, but can be induced in response to environmental cues such as hypoxia and cytokine stimulation. However, all studies to date that have investigated furin expression have been limited to analysis of furin mRNA; there has been no assay sensitive enough to quantify endogenous furin. Though activity-based assays have been described for furin-like enzyme activity, we demonstrate that these assays are dominated by the activity of other enzymes and cannot be used to approximate furin activity. A sensitive and specific assay for furin activity was therefore developed and characterised, using an antibody capture step to immobilise furin from whole cell lysates. Furin activity is quantified relative to that of recombinant active furin protein, to allow estimation of active furin protein concentration. The assay has a minimum detection limit of 0.006 nM; sensitive enough to determine the furin activity of many of the cell lines tested. The specificity of the assay was demonstrated by genetic modulation of furin expression. Furthermore, the assay was used to demonstrate that the cytokine transforming growth factor beta (TGF-β) stimulates increased furin activity in HepG2 cells, confirming and extending previous reports that TGF-β increases furin expression, and adding to the mounting body of evidence that cellular furin activity can be modulated.
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, UK. glb38@cam.ac.uk
Gemma L
GL
Bourne
David J
DJ
Grainger
D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D002457:Cell Extracts / Q000737:chemistry; D045683:Furin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D056945:Hep G2 Cells; D006801:Humans; D000860:Hypoxia / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D056747:Immunomodulation; D007163:Immunosorbent Techniques; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D002457:Cell Extracts; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D045683:Furin