title: Specific proteolysis of human plasminogen by a 24 kDa endopeptidase from a novel Chryseobacterium Sp.
abstract: A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300-400 microg/L from conditioned medium of a bacterial strain which was identified as a new species in the genus Chryseobacterium Sp. on the basis of its 16S rDNA sequence and DNA:DNA hybridizations. The NH(2)-terminal amino acid sequence (Val-Ala-Thr-Pro-Asn-Leu-Glu-.) was not found in the availabe databases. The 24k-endopeptidase specifically hydrolyzed the Ser(441)-Val(442) peptide bond in human plasmin(ogen), with additional cleavage of the Lys(78)-Val(79) and Pro(447)-Val(448) peptide bonds, and a secondary cleavage at Lys(615)-Val(616). Thereby, plasminogen is converted into an angiostatin-like fragment containing kringles 1-4 (K1-4) and miniplasminogen (kringle 5 and the serine proteinase domain). The purified K1-4 fragment showed a comparable cytotoxicity toward endothelial cells as the elastase-derived K1-3 fragment (12.7% versus 10.6% at a concentration of 10 microg/mL). Plasminogen, bound to monocytoid THP-1 cells, was also cleaved by the 24k-endopeptidase, resulting in generation of an angiostatin-like fragment and in a decreased capacity to generate cell-associated plasmin following activation by urokinase. The 24k-endopeptidase was not efficiently neutralized by specific inhibitors against the serine, cysteine, aspartic, or matrix metalloproteinase classes of enzymes. In human plasma or serum, however, it induced only very limited plasminogen degradation, apparently due to neutralization of its activity by alpha(2)-macroglobulin. Interaction of this novel 24k-endopeptidase with plasminogen thus yields an angiostatin-like fragment and affects plasmin-mediated cellular proteolytic activity.
authors.affiliation: Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium. roger.lijnen@med.kuleuven.ac.be
authors.forename: H R
authors.initials: HR
authors.lastname: Lijnen
authors.forename: B
authors.initials: B
authors.lastname: Van Hoef
authors.forename: F
authors.initials: F
authors.lastname: Ugwu
authors.forename: D
authors.initials: D
authors.lastname: Collen
authors.forename: I
authors.initials: I
authors.lastname: Roelants
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D043168:Angiostatins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000737:chemistry
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D010446:Peptide Fragments; D000511:alpha-Macroglobulins; D043168:Angiostatins; D010958:Plasminogen; D010450:Endopeptidases