Specific Proteolysis of Human Plasminogen by a 24 kDa Endopeptidase from a Novel Chryseobacterium Sp.

• Published in: Biochemistry (Easton) 2000-01, Vol.39 (2), p.479-488
• Main Authors: Lijnen, H. Roger, Van Hoef, Berthe, Ugwu, Francesca, Collen, Désiré, Roelants, Ivo
• Format: Article
• Language: English
• Subjects: alpha-Macroglobulins - chemistry; Amino Acid Sequence; Amino Acids - analysis; Angiostatins; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Chromatography; Chryseobacterium; Electrophoresis, Polyacrylamide Gel; endopeptidase; Endopeptidases - chemistry; Endopeptidases - genetics; Endopeptidases - metabolism; Humans; Molecular Sequence Data; Peptide Fragments - metabolism; Plasminogen - chemistry; Plasminogen - genetics; Plasminogen - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi992014r

A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300−400 μg/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 NH2-terminal amino acid sequence (Val-Ala-Thr-Pro-Asn-Leu-Glu-...) was not found in the availabe databases. The 24k-endopeptidase specifically hydrolyzed the Ser441−Val442 peptide bond in human plasmin(ogen), with additional cleavage of the Lys78−Val79 and Pro447−Val448 peptide bonds, and a secondary cleavage at Lys615−Val616. 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 μg/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 α2-macroglobulin. Interaction of this novel 24k-endopeptidase with plasminogen thus yields an angiostatin-like fragment and affects plasmin-mediated cellular proteolytic activity.