Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

Abstract

Purification of a2-plasmin inhibitor (a2PI) from human plasma by affinity chromatography on plasminogenSepharose resulted in copurification of a contaminating protein with MI 17000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified a2-PI preparation by several types of gel chromatography applied. The use of the kringle 1 -3 part of plasminogen, K(l + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an u2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1+2+3) or miniplasminogen.The KCbinding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34.The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (MI 17000), that are dissociated by 1 YO sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pl of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 f 0.03 pM (15 +_ 2 mg/l). The electrophoretic mobility of the KCbinding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(Dlysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.

Transforming growth factor‐β1 downregulates dexamethasone‐indunced tetranectin gene expression during the in vitro mineralization of the human osteoblastic cell line SV‐HFO

“…In the present study, we used this human osteoblastic in vitro model system to begin analysing the regulation of tetranectin gene expression during mineralization. Tetranectin is a protein originally isolated from plasma [26] that was found to be expressed in bone at a time and space coincident with mineralization in vivo and in vitro [27]. We report that dexamethasone treatment induced the expression of tetranectin mRNA and that TGF-fl~ inhibited mineralization and the induction of tetranectin mRNA.…”

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain

“…Tetranectin (TN) is a homotrimeric [1] Ca# + -and plasminogen (Plg)-binding protein found in plasma [2] and as a component of the extracellular matrix during muscle development and regeneration [3]. TN has been proposed to have a role in mineralization during osteogenesis [4,5].…”

“…TN binds specifically to Plg kringle 4 [2] and apolipoprotein (a) [10] in a lysine-sensitive way, and, dependent on Ca# + , to fibrin [11]. The mature TN monomer, of 181 amino acid residues and containing three internal disulphide bridges, is encoded by three exons [8,12], of which the polypeptide encoded by exon 2 is necessary and sufficient for trimerization [1].…”

Mutational Analysis of Affinity and Selectivity of Kringle-Tetranectin Interaction

“…TN binds to Plg K4, and binding is sensitive to both lysine and calcium (2,23). TN binding involves at least one amino acid * The present work was supported by Grant 9901966 from the Danish Natural Science Research Council.…”

“…1 is a homotrimeric (1) plasminogen (Plg) and calcium-binding plasma protein (2). TN is also found in cartilage (3) and is present in the extracellular matrix during development and regeneration of muscle (4).…”