title: Proteinase 3 interacts with a 111-kD membrane molecule of human umbilical vein endothelial cells.
abstract: Proteinase 3 (PR3) is the major autoantigen of antineutrophil cytoplasmic antibodies in Wegener's granulomatosis. Previously, it was demonstrated that PR3 induces apoptosis of human endothelial cells and that PR3 contributes to endothelial cell activation by enhancing interleukin-8 production. The present study demonstrates that PR3 binds specifically to human umbilical vein endothelial cells (HUVEC). Digoxigenin (DIG)-labeled PR3 bound readily to HUVEC cultured on coverslips. By fluorescence-activated cell sorter analysis, a homogeneous binding of PR3 to HUVEC, using either DIG-labeled or unlabeled PR3, was observed. No detectable membrane expression of PR3 was observed after either tumor necrosis factor-alpha stimulation or in nonstimulated HUVEC. The binding of PR3-DIG to HUVEC was dose-dependent and was inhibited by unlabeled PR3. Scatchard analysis revealed 2000 binding sites per cell, with a Kd of 0.1 microM. Affinity precipitation of biotin-labeled HUVEC membrane proteins with protein G-Sepharose bearing PR3 resulted in specific precipitation of a membrane molecule with a molecular weight of 111 kD under nonreducing conditions and 52 and 63 kD under reducing conditions. It is hypothesized that PR3, either released systemically or locally at inflammatory sites following activation of primed polymorphonuclear neutrophils, may lead to endothelial cell injury and activation of endothelial cells.
authors.affiliation: Department of Nephrology, Leiden University Medical Centre, The Netherlands.
authors.forename: Miriam E J
authors.initials: MEJ
authors.lastname: Taekema-Roelvink
authors.affiliation: Department of Nephrology, Leiden University Medical Centre, The Netherlands.
authors.forename: Cees
authors.initials: C
authors.lastname: VAN Kooten
authors.affiliation: Department of Nephrology, Leiden University Medical Centre, The Netherlands.
authors.forename: Evert
authors.initials: E
authors.lastname: Heemskerk
authors.affiliation: Department of Nephrology, Leiden University Medical Centre, The Netherlands.
authors.forename: Willeke
authors.initials: W
authors.lastname: Schroeijers
authors.affiliation: Department of Nephrology, Leiden University Medical Centre, The Netherlands.
authors.forename: Mohamed R
authors.initials: MR
authors.lastname: Daha
mesh_terms: D002469:Cell Separation; D002478:Cells, Cultured; D004076:Digoxigenin; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D053146:Myeloblastin; D012697:Serine Endopeptidases / Q000008:administration & dosage / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D012697:Serine Endopeptidases; D053146:Myeloblastin; D004076:Digoxigenin