Angiostatin binds ATP synthase on the surface of human endothelial cells

Abstract

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and͞or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins

Angiostatin's molecular mechanism: Aspects of specificity and regulation elucidated

“…Sharma's group investigated whether purified human angiostatin could bind any proteins in bovine aortic EC extracts using a ligand blotting method [Tuszynski et al, 2002] In contrast to the data of Tuszynski et al [2002], we could find no evidence of angiostatin binding to annexin II on HUVEC [Moser et al, 1999] discussed in ''ATP Synthase: A Cell Surface Receptor for Angiostatin''). Possible explanations for this discrepancy include the use of different cell lines (HUVEC vs. BAE), the use of a homologous vs. a heterologous system, inappropriate binding to denatured proteins on Western blots, and differences in the preparation of angiostatin.…”

“…A number of cell surface receptors had previously been identified for plasminogenthe parent molecule from which angiostatin is derived-as well as other plasminogen cleavage products such as plasmin [Moser et al, 1999[Moser et al, , 2002. However, none of these other plasminogen products exhibited angiostatic activity, suggesting that angiostatin bound to a different site.…”

“…A single previous publication had reported detection of F 1 F O ATP synthase on the surface of the tumor cell line A549 [Das et al, 1994]; however, this observation was also met with skepticism and was regarded to be the likely consequence of aberrant trafficking in a genetically unstable tumor cell line or perhaps an artifact of cell culture. Since Das [1998] and Moser and co-authors [Moser et al, 1999[Moser et al, , 2001] first presented data that mammalian cells may express certain mitochondrial proteins on the plasma membrane, other investigators have described a wide variety of mitochondrial matrix proteins that have been definitively localized to the plasma membrane surface of certain cells [Soltys and Gupta, 1999]. Moreover, in many cases such matrix proteins have been shown to play functional roles in their plasma membrane locations, including P32 protein, also known as the gC1q receptor for complement protein C1q [Soltys et al, 2000].…”

“…To further characterize the relevance of cell surface F 1 F O ATP synthase, Moser and co-authors [Moser et al, 1999[Moser et al, , 2001] tested two hypotheses. First, they asked whether non-inhibitory antibodies against F 1 F O ATP synthase could block angiostatin binding and activity.…”

“…Another important point is that many experiments performed in commercial media contain 14 mM bicarbonate, which gives a pH of 7.0-7.1. Thus, it is relevant that in studies using such commercial media, angiostatin was reported to decrease EC viability (40%) at ''normal'' extracellular pH [Moser et al, 1999], whereas experiments using M199 media with 26 mM bicarbonate, which has a pH of 7.3, showed no angiostatin effect on EC viability [Wahl et al, , 2002a. Another difference between these two studies was that the former was done using recombinant angiostatin obtained from Entremed, Inc. (Rockville, MD), whereas the latter study utilized angiostatin in its native state, purified from plasminogen cleavage reactions.…”