Screening And Identification Of Proteins Interacting To Vasostatin

Calreticulin, a ubiquitous and highly conserved protein, was originally identified in the sarcoplasmic reticulum of skeletal muscle and served as one of the major storage depots for calcium ions within the endoplasmic reticulum and participates in calcium signaling. An endothelial inhibitor was purified from the supernatant of an Epstein-Barr virus transformed cell line and identified as a fragments of calreticulin in 1998. The purified recombinant N-terminal domain of calreticulin inhibited the proliferation of endothelial cells in vitro as well as of angiogenesis in vivo, while no inhibitions to other lines. This N--terminus of calreticulin was named as vasostatin which includes 1-180 aa of the whole molecule. It is the most conserved domain among the calreticulins so far cloned and has no homology to other protein sequences. When inoculated into athymic mice, vasostatin significantly reduced the growth of the transplanted human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenic inhibitor that specifically targets the proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.In addition, several potential mechanisms are proposed for this angiogenic inhibitor, including the generation of free radicals such as super anions and nitric oxide, and up-regulation of the Fas/FasL system. Although it does not bind calcium, it can bind the cytoplasmic domain of subunits of integrins regulating cell attachment, can interact with the nuclear receptors for glucocorticoid, androgen, and retinoic acid, regulating their binding to DNA. Vasostatin directly and specifically inhibited the growth of the endothelial cells while had minimal effect on the growth of other cells. Previously, calreticulin was reported to bind specifically and reversibly to endothelial cells in vitro, and to localize selectively to the vascular endothelium in vivo. It was also found to promote nitric oxide release from endothelial cells. Although its role in angiogenesis is controversial, nitric oxide was reported to suppress angiogenesis and endothelial cell migration.We do not know the mechanism by which vasostatin inhibits endothelial cell growth.Preliminary experiments in vitro have failed to support a role of nitric oxide as a mediator of growth inhibition by vasostatin. However, we believe that inhibition of endothelial cell proliferation is central to suppression of angiogenesis and tumor growth by vasostatin. Tumor cells were not growth inhibited by vasostatin in vitro, and tumor tissues from vasostatin-treated mice were histologically similar to the controls in vivo, suggesting that vasostatin acts indirectly on tumor cells. In vasostatin-treated animals, vessels distant from the tumor appeared normal, and even the established tumor vasculature had limited evidence of vasostatin-induced damage. This suggests that the antirumor effects of vasostatin are related to inhibition of new vessel formation rather than to a toxic effect on established tumor vascular structures.Laminins are multifunctional glycoproteins of the ECM that contribute to the architecture of the basement membranes and play a crucial role in cell adhesion, growth, migration, and differentiation.The mechanisms underlying these effects are unclear. We show that endothelial cells express the extracellular matrix protein laminin, including chains cc5yl which is the target for that vasostatin binding.When added to endothelial cell cultures, vasostatin specifically inhibits endothelial cell attachment to laminin and by this mechanism, it reduce the subsequent endothelial cell growth induced by basic fibroblast growth factor. As an angiogenic inhibitor that specifically disrupts endothelial cell attachment to components of the extracellular matrix. Thereof vasostatin has a unique potential as a cancer therapeutic.Looking for proteins interacting to vasostatin may provide a new mechanism to inhibite the proliferation of endothelial cells elicited by vasostatin.1. Application of the bacterial two-hybrid system for the validation of extracellular protein-protein interactionsTo explore the possibility and feasibility in the analyzing extracellular protein-protein interactions using the bacterial two-hybrid system, the interactions of TNF-related apoptosis-inducing ligand (TRAIL) and its three receptors, DR4, DR5 and osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) were identified in the system of Stratagene BacterioMatch. A fused expression plasmid was constructed betweenpTRG with tetracycline (T) resistance and TRAIL'S soluble extracellular domain as the "bait", while the fused expression plasmids constructed between pBT with chloramphenicol (C) resistance and extracellular domain of DR4, DR5 or OCIF/OPG, respectively, as the "target". All the recombinant pTRG and pBT was co-transformed into bacterioMatch two-hybrid system reporter strain E. coli XLl-Blue MRF with kanamycin (K) resistance. The protein-protein interactions were detected by its carbenicillin (C)-resistant property on CTCK plate containing 250-500 p.g/ml of C (carbenicillin), 15 ug/ml of T (tetracyclin), 34 ug/ml of C (chloramphenicol) and 50 ug/ml of K, and validated by galactosidase activation on X-Gal-phenylethyl P-D-thiogalactoside (PETG) plate. Four groups, including that co-transformed by pTRG-TRAIL/pBT-DR4, pTRG-TRAIL/ pBT-DR5, controls (pBT/LGF2 and PTRG/Galll), and pTRG-TRAIL/pBT-OPG, grew as carbenicillin-resistant colonies on the plates. The pre-three groups showed higher resistance to carbenicillin (above 500 ug/ml) and the last one to lower (under 250 u.g/ml). The positive colonies were further validated by P-galactosidase activation on X-Gal-PETG plates. The others, including pTRG-TRAIL/pBT-LGF2, pBT-DR4 or pBT-DR5 or pBT-OPG/pTRG-Galll, or non-co-transformed plasmids themselves, were negative on carbenicillin or X-Gal-PETG plates.The results showed that the bacterial two-hybrid system is possible and efficacious in analyzing and identifying some extracellular protein-protein interactions, since the recognitions and interactions between TRAIL and their receptors have been proved by others chemical or biologic methods.2. Screening the ligand candidate interacting to vasostatin with the bacterial two-hybrid systemThe fused expression plasmid was constructed between pBT and vasostatin as the "bait", while the "target" used was the human liver cDNA library constructed into pTRG provided by Statagene. They were co-transformed and cultivated on CTCK plates under carbenicillin at the concentration of 500 ug/ml.Ten rounds were screened on total over 400 CTCK plates (15 cm), and transformation efficiency was 2x107~6*107. Over 1600 positive clones from the screening on CTCK with 500 ug/ml of carbenicillin, and about 260 positive clones from validation on X-Gal-PETG were obtained, respectively. They were sequenced andidentified as the coding regions for al-antitrpsin (26 times); unknown novel gene fragment (2 times); albumin (4 times); complement 1 y subunit (2 times); apolipoprotein A-II, apolipoprotein C, C reactive protein, orosomucoid 1, hemopexin, MFAP4 (microfibril -associated glycoprotein 4) (once for each). According to the present knowledge from the literatures, the most possible ligand candidate to vasostatin, MFAP4, a extracellular matrix protein was investigated further for its interaction to vasostatin. 3. Recombinant expression of MFAP4A full-length cDNA of MFAP4 was obtained from a human cDNA liver library and showed identity with the published sequence The coding region spanned 255 amino acids with an N-terminal region of 16 amino acids containing an Arg-Gly-Asp motif and one cysteine residue. The N-terminal region was followed by a single fibrinogen-like domain of 239 amino acids showing high homology to fibrinogen domains found in human ficolin An internal peptide sequence from the fibrinogen chain (GWTVFQKRLDGSV) has been shown to be involved in the binding to the leukocyte integrin Mac-1. This peptide sequence is highly conserved between the fibrinogen chain and hMFAP4. The cDNA of hMFAP4 was transformed into an E.coli strain K802 by means of the plasmid pMAL-c2 where it was expressed as a MBP fusion protein. After induction of protein expression the cells were lysed, and the fusion protein was purified on the Thiobond? resin affinity column, it migrates as a molecule of 45 ku on the SDS-PAGE.4 Identification of the interaction between vasostatin and MFAP4 with Western blottingWe examined whether the angiogenic inhibitor vasostatin can bind MFAP4. The mixture of purified MFAP4 and vasostatin was run on an electrophoresis and blotted onto nitrocellulose paper with single purified MFAP4 as control. After incubation and washing, vasostatin binding to the MFAP4 was revealed by HRP-labeled rabbit antihuman mAb directed to vasostatin .The result suggested that vasostatin can bind specifically to MFAP4 . In Yao's experiments, vasostatin bound minimally to fibronectin, vitronectin, collagen type IV, tenascin-C, thrombospondin, bFGF, and human VEGF. These experiments demonstrate that vasostatin can bind specifically to MFAP4.