Bovine α2-antiplasmin N-Terminal and reactive site sequence

AbstractBovine α2-antiplasmin (α2AP) has been purified and partially characterized. The amino acid composition is very similar to that of human α2AP, and the N-terminal (23 residues determined) and reactive site loop sequences (42 residues determined) are highly homologous to those of the human protein. Compared with human α2AP, bovine α2AP has an 18-residue N-terminal extension, homologous with part of the pre-sequence of human α2AP. A re-investigation of the N-terminal sequence of freshly prepared human α2AP reveals a new form extended by 12 residues

Crystallization and preliminary X-ray diffraction analysis of eukaryotic α2-macroglobulin family members modified by methylamine, proteases and glycosidases

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Summary: α2-Macroglobulin (α2M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2M (hα2M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.European, Spanish, and Catalan Agencies. Grant Number: FP7-HEALTH-2010-261460; Gums&Joints. Grant Number: FP7-PEOPLE-2011-ITN-290246; RAPID. Grant Number: FP7-HEALTH-2012-306029-2; TRIGGER. Grant Numbers: BFU2012-32862, CSD2006-00015; La Marató de TV3. Grant Numbers: 2009-100732, 2009SGR1036; ESRFPeer Reviewe

Crystallisation and preliminary X-ray analysis of the receptor-binding domain of human and bovine α2-macroglobulin

AbstractThe receptor-binding domains (RBDs) of human and bovine α2-macroglobulin (α2M) have been isolated after limited proteolysis of methylamine-treated α2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine α2M give diffraction patterns suitable for X-ray analysis, and a complete dataset with a maximum resolution of 2.3 Å has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3121 or P3221 with cell parameters a = b = 106.8 Å, c = 72.2 Å

Generation of angiostatin-like fragments from plasminogen by prostate-specific antigen

Angiostatin, a potent inhibitor of angiogenesis, tumour growth and metastasis, is a biologically active fragment of plasminogen, containing the kringle domains 1–4. It is generated from plasminogen by limited proteolysis. We show that prostate-specific antigen (PSA), a serine proteinase secreted by human prostate and human prostate cancer cells, is able to convert Lys-plasminogen to biologically active angiostatin-like fragments, containing kringles 1–4, by limited proteolysis of peptide bond Glu439–Ala440 in vitro. In an in vitro morphogenesis assay, the purified angiostatin-like fragments inhibited proliferation and tubular formation of human umbilical vein endothelial cells with the same efficacy as angiostatin. This finding might help to understand growth characteristics of prostate cancer, which usually has low microvessel density and slow proliferation. © 1999 Cancer Research Campaig

Generation of active fragments from human zymogens in the bradykinin-generating cascade by extracellular proteases from Vibrio vulnificus and V. parahaemolyticus

Vibrio vulnificus is an opportunistic human pathogen causing septicemia, and the infection is characterized by formation of the edematous skin lesions on limbs. This pathogenic species secretes a thermolysin-like metalloprotease as a virulence determinant. The metalloprotease was confirmed to activate human factor XII-plasma kallikrein-kinin cascade that results in liberation of bradykinin, a chemical mediator enhancing the vascular permeability, from high-molecular weight kininogen. Namely, the metalloprotease showed to generate active fragments by cleavage of Arg-Ile, Arg-Val or Gly-Leu peptide bond in human zymogens (plasma prekallikrein and factor XII). In spite of induction of the sufficient vascular permeability-enhancing and edema-forming reaction in the guinea pig model, a serine protease from V. parahaemolyticus, a human pathogen causing primarily watery diarrhea, showed far less ability to activate and to cleave the human zymogens. These results in part may explain why only V. vulnificus often causes serious edematous skin damages in humans.</p

One-step purification of murine IgM and human [alpha]2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 methyl [alpha]--mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl [alpha]--mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. [alpha]2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man-([alpha]1,3)Man units are bound to the immobilized lectin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27051/1/0000041.pd

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

Binding of plasminogen to fibrin and cell surfaces is essential for fibrinolysis and pericellular proteolysis. We used surface plasmon resonance and enzyme kinetic analyses to study the effect of two mAbs (A10.2, CPL15) on plasminogen binding and activation at fibrin surfaces. A10.2 is directed against the lysine-binding site (LBS) of kringle 4, whereas CPL15 recognises a region in kringle 1 outside the LBS. In the presence of CPL15 and A10.2 mAbs, binding of plasminogen (K(d)=1.16+/-0.22 micromol/l) to fibrin was characterised by a mAb concentration-dependent bell-shaped isotherm. A progressive increase in the concentration of mAbs at the surface was also detected, and reached a plateau corresponding to the maximum of plasminogen bound. These data indicated that at low mAb concentration, bivalent plasminogen-mAb-plasminogen ternary complexes are formed, whereas at high mAb concentration, a progressive shift to monovalent plasminogen-mAb binary complexes is observed. Plasmin formation in the presence of mAbs followed a similar bell-shaped profile. Monovalent Fab fragments of mAb A10.2 showed no effect on the binding of plasminogen, confirming the notion that a bivalent mAb interaction is essential to increase plasminogen binding and activation at the surface of fibrin