The Cloning, Fusion Expression And Biological Characterization Of Tumstatin And Screening For Its Interacting Proteins

The development of new blood vessels from pre-existing ones is generally referred to as angiogenesis. In the adult, new blood vessels arise via angiogenesis, a process critical for normal physiological events such as wound repair, the ovarian cycle, and endometrium remodeling. However, uncontrolled neovascularization is associated with a number of pathological disorders including diabetic retinopathy. rheumatoid arthritis, as well as tumor growth and metastasis. Tumor growth and metastasis require angiogenesis, and this process is pivotal to the survival and subsequent growth of solid tumors beyond a few mm~3 in size. Expansion of tumor mass occurs not only by perfusion of blood through the tumor but also by paracrine stimulation of tumor cells by several growth factors and matrix proteins produced by the new capillary endothelium. Studies have shown that tumor growth is dependent on angiogenesis. This provides the rationale for antiangiogenic therapy in cancer. Basement membranes are thin layers of a specialized extracellular matrix that provide the supporting structure on which epithelial and endothelial cells grow and that surround muscle, fat. etc. It has been well demonstrated that basement membranes do not only provide a mechanical support but also influence cellular behaviors such as differentiation and proliferation. The one of major macromolecular constituents of basement membranes is type IV collagen. In general, type IV collagen promotes cell adhesion, migration, differentiation, and growth. Type IV collagen is expressed as six distinct a-chains, namely, al-a6 , assembles into triple helices, and further forms a network to provide a scaffold for other macromolecules in basement membranes. These a-chains are composed of three domains, the N-terminal 7 S domain, the middle triple helical domain, and the C-terminal globular noncollagenous domain 1 (NCl). The al and α2 isoforms are ubiquitously present in human basement membranes. The other four isoforms exhibit more tissue and organ-specific distributions. The distribution of the a3 (IV) chain is limited to certain basement membranes, such as glomerular basement membrane, several basement membranes of the cochlea, ocular basement membrane of the anterior lens capsule. Descemet's membrane, ovarian and testicular basementmembrane, and alveolar capillary basement membrane. This chain is absent from epidermal basement membranes of the skin and the vascular basement membrane of liver. The a3 (IV) NC 1 domain has been shown to bind and inhibit the proliferation of melanoma and other epithelial tumor cell lines in vitro. Studies demonstrate the pivotal role of the NC 1 domain of the a3 chain of human type IV collagen produced as a recombinant protein, in inhibiting the proliferation of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angiogenesis and tumor growth and also in inducing endothelial cell-specific apoptosis. This domain was named "tumstatin". Tumstatin is a novel anti-angiogenic protein domain derived from the a3 chain of type IV collagen. To date, a number of angiogenesis inhibitors have been identified, and certain factors such as angiostatin, endostatin, canstatin, and arrestin are tumor-associated angiogenesis inhibitors which are potentially generated in vivo. Among the six NCI domains of type IV collagen, three exhibited promising anti-angiogenic activity with distinct mechanisms of action. The NCI domain of the ot3 chain (tumstatin) was most potent in inhibiting the proliferation of endothelial cells and causing apoptosis when compared with the other a (IV) chain NCI domain. This newly discovered anti-angiogenic property of tumstatin, coupled with the the 185-203 aa a3 (IV) NCI antitumor cell activity, makes tumstatin a potentially useful therapeutic molecule in inhibiting tumor growth. The contents of the thesis following aspects: (1) Cloning, expression and bioassay of human tumstatin in E.coli in order to lay the foundation of further studies and applications of tumstatin. (2) A fusing protein of an antiagiogenic inhibitor (tumstatin45-132) and a cytotoxic factor (human tumor necrosis factor-a), tumstatin45-132-TNF, was constructed in order to improve its anti-tumor activity. (3) The novel interacting proteins to tumstatin were screened in order to provide the new clues for identification of the new functions and mechanisms oftumstatin45-132.Part One: Cloning, expression and bioassay of human tumstatinTo amplify the coding sequence for mature protein of tumastatin, express the recombinant human tumstatin in E.coli and analyse its biological activities onendothelial cells. The cDNA fragment of tumstatin was obtained by a reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from 293 embryonic kidney cells. The RT-PCR product was cloned into pMD-18T vector, the resulted plasmid pMD-tumstatin was again cloned into pMAL-c2 expression vector. MBP-tumstatin fusion gene was efficiently expressed after IPTG induction. The expressed product accounted for more than 20% of the total bacterial proteins, as estimated by densitometry and existed mainly as soluble form. The purified MBP-tumstatin fusion protein was obtained by Sephacryl S-200. The MBP was cleaved from tumstatin by an enzymatic digestion with factor Xa according to New England Biolabs Inc protocols. Tumstatin was re-extracted from MBP by anion exchange chromatography using a pre-equilibrated (20 mmol/L Tris. pH 8.0. 25 mmol/L NaCl) Q Sepharose Fast Flow, and elution by a stepwise gradient during which MBP eluted at 125 mmol/L NaCl and tumstatin eluted at -225 mmol/L NaCl. The purity of tumstatin reached up 95%. Tumstatin shows as a 27 ku band on SDS-PAGE. The biological activity of tumstatin was detected with endothelial cell proliferation test, endothelial cell apoptosis test and in vitro tube formation inhibition test. Tumstatin showed significant inhibitory effect on endothelial cell proliferation (ED50 was about 15 (ig/ml). it also induced the apoptosis of endothelial cell and inhibited the tube formation in a dose dependent manner. The results suggest that tumstatin is an effective inhibitor of endothelial cells and has potential therapeutic usage on the treatment of the cancers.Part Two: Expression and biological characteration in vivo and in vitro of the fusion protein of tumstatin 45-132-TNFThe aim of this study is to fusing expression of an antiagiogenic inhibitor (tumstatin45-132) with a cytotoxic factor (human tumor necrosis factor-a, TNF) to yield a novel fusion protein. tumstatin45-132-TNF. in order to improve its anti-tumor activity. The idea was originated from the fact of the specific interaction between the tumstatin45-132 and its receptors (avp3), thus it is possible to directly target the fusion protein to the endothelial cells in the malignant tumor tissues that have previously been shown to contain high levels of avfo. The localized fusion protein is designed to killtumor cells as well as to inhibit angiogenesis within the tumor mass. A bacterial expression system was used to produce tumstatin45-132-TNF fusion protein. The cDNA fragment of tumstatin was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) with total RNA extracted from 293 embryonic kidney cells. The RT-PCR product was cloned into pMD-18T vector, the resulted plasmid pMD-tumstatin and pBV220-TNF were used as template to amplify tumstatin 45-132-TNF fusion gene by splicing by overlap extension (SOEing). Tumstatin 45-132-TNF fusion gene was efficiently expressed after IPTG induction as a 27 ku band on SDS-PAGE. The expressed product accounted for approximately 30% of the total bacterial protein, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the renaturated protein were purified by Sephacryl S-200 and Superdex 75 to a purity of 95%. Tumstatin 45-132-TNF fusion protein maintained both tumstatin45-132 and tumor necrosis factor-a activities as demonstrated by cell-based assays (ED5o= 5 ug/ml and 4.6><106 IU/mg. respectively). Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the tumstatin45-132 moiety interacts with cell adhesion receptor. avP3 integrin. In addition. tumstatin45-132-TNF induced cytotoxic effects in standard cytolytic assays, implying that tumstatin45-132-TNF can also bind TNF receptors and trigger death signals. The antitumor activities of tumstatin 45-132-TNF were demonstrated using liver cancer-bearing nude mice model. After daily injection (i.p.) of tumstatin45- 132-TNF (0.5 mg/kg/day) for 12 days, the tumor growth in the tumstatin45-132-TNF-treated group was only one-third in size as compared with that of the control group (118±16.6 mm3 vs 375±24.5 mm3). The growth rate in the tumstatin45-132-TNF-treated group is also significantly slower than that of the group treated with tumstatin45-132 alone (5 mg/kg/day). This decrease in tumor size was consistent with decrease in CD31-positive vasculature. More importantly, tumstatin45-132-TNF inhibits tumor growth at a dose much lower (0.5 mg/kg/day) than that reported for previous tumstatin45-132 treatments (5 mg/kg/day). We hope that this novel bifunctional protein will contribute significantly to human tumor therapy.