Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain.
We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.
Laboratoire de médecine moléculaire, Hôpital Sainte-Justine-UQAM, Centre de cancérologie, Charles-Bruneau, Centre de Recherche de l'Hôpital Sainte-Justine, 3175, Chemin Côte-Sainte-Catherine, Montreal, Que., Canada H3T 1C5.
Dominique
D
Boivin
Mathieu
M
Provençal
Sébastien
S
Gendron
David
D
Ratel
Michel
M
Demeule
Denis
D
Gingras
Richard
R
Béliveau
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005341:Fibrinolysin / Q000096:biosynthesis*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D020539:Sequence Analysis, Protein; D020349:Surface Plasmon Resonance; D014020:Tissue Extracts / Q000737:chemistry*
D020533:Angiogenesis Inhibitors; D007145:Immunoglobulin kappa-Chains; D014020:Tissue Extracts; C111481:shark cartilage extract; D005337:Fibrin; D010958:Plasminogen; D005341:Fibrinolysin