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

Monoclonal antibodies targeting the disintegrin-like domain of ADAMDEC1 modulates the proteolytic activity and enables quantification of ADAMDEC1 protein in human plasma

Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease with unknown biological function and a short domain structure. ADAMDEC1 mRNA has previously been demonstrated primarily in macrophages and mature dendritic cells. Here, we generated monoclonal antibodies (mAbs) against the mature ADAMDEC1 protein, as well as mAbs specific for the ADAMDEC1 pro-form, enabling further investigations of the metalloprotease. The generated mAbs bind ADAMDEC1 with varying affinity and represent at least six different epitope bins. Binding of mAbs to one epitope bin in the C-terminal disintegrin-like domain efficiently reduces the proteolytic activity of ADAMDEC1. A unique mAb, also recognizing the disintegrin-like domain, stimulates the caseinolytic activity of ADAMDEC1 while having no significant effect on the proteolysis of carboxymethylated transferrin. Using two different mAbs binding the disintegrin-like domain, we developed a robust, quantitative sandwich ELISA and demonstrate secretion of mature ADAMDEC1 protein by primary human macrophages. Surprisingly, we also found ADAMDEC1 present in human plasma with an approximate concentration of 0.5 nM. The presence of ADAMDEC1 both in human plasma and in macrophage cell culture supernatant were biochemically validated using immunoprecipitation and Western blot analysis demonstrating that ADAMDEC1 is secreted in a mature form

Alpha2 Macroglobulin-Like Is Essential for Liver Development in Zebrafish

Background: Alpha 2 Macroglobulin family members have been studied extensively with respect to their roles in physiology and human disease including innate immunity and Alzheimer’s disease, but little is known about a possible role in liver development loss-of-function in model systems. Principal Findings: We report the isolation of the zebrafish a2 macroglobulin-like (A2ML) gene and its specific expression in the liver during differentiation. Morpholino-based knock-down of A2ML did not block the initial formation of the liver primordium, but inhibited liver growth and differentiation. Significance: This report on A2ML function in zebrafish development provides the first evidence for a specific role of an A2M family gene in liver formation during early embryogenesis in a vertebrate

A novel IgE antibody targeting the prostate-specific antigen as a potential prostate cancer therapy

Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 andCD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa.Fil: Daniels-Wells, Tracy R. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Helguera, Gustavo Fernando. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Tecnologia Farmaceutica; Argentina; University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Leuchter, Richard K. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Quintero, Rafael. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Kozman, Maggie. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Rodríguez, José A.. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América; University of California. The Molecular Biology Institute; Estados Unidos de América;Fil: Ortiz-Sánchez, E. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América; Biomedical Research in Cancer. Basic Research Division. National Institute of Cancerology; Mexico.;Fil: Martínez-Maza, Otonel. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Schultes, Brigit C.. Advanced Immune Therapeutics; Estados Unidos de América;Fil: Nicodemus Christopher. Advanced Immune Therapeutics; Estados Unidos de América;Fil: Penichet, Manuel. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América; University of California. The Molecular Biology Institute; Estados Unidos de América

α2-Macroglobulin can crosslink multiple plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules and may facilitate adhesion of parasitized erythrocytes

Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M--(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies.

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.Early Career Fellowship from the National Health and Medical Research Council GNT1012521(A.R.W.); Wellcome Trust Programme Grant (J.R.K., C.M.D.) 094425/Z/10/Z; Samsung GRO Grant (M.R.W.)This is the final version of the article. It first appeared from PLoS via http://dx.doi.org/10.1371/journal.pone.013003

Genomic view of the evolution of the complement system

The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA

Rat plasma α1-inhibitor3 binds to receptors for α2-macroglobulin

AbstractThe cellular binding and uptake was studied for α1-inhibitor3, a monomeric 200 kDa proteinase inhibitor present in rat plasma. After intravenous injection in the rat the inhibitor disappeared from the circulation with a half-time of 2.5 min when complexed with chymotrypsin, whereas the half-time for uncomplexed inhibitor was more than 60 min. 6 min after the injection of labelled complex, 83% was in the liver and 2.5% in the spleen. In vitro experiments at 4°C with isolated hepatocytes and peritoneal macrophages showed binding to the previously described receptors which bind and internalize the tetrameric rat and human α2-macroglobulin-proteinase complexes. The binding affinities were similar for the two types of complexes and binding was followed by uptake and degradation of the labelled complex when the cells were warmed to 37°C. The binding of uncomplexed α1-inhibitor3 was low and did not increase following treatment with methylamine in spite of cleavage of the internal thiol ester. α1-Inhibitor3-methylamine was changed to the receptor binding form when treated with chymotrypsin which caused the cleavage of at least one peptide bond in the bait region