The major vault protein (MVP) is the predominant component of a large cytoplasmic ribonucleoprotein particle, the vault complex [1,2]. The vault particle was originally identified as a barrel shaped body in preparations of clathrin-coated vesicles and named after its morphology reminiscent of the vaulted ceilings of cathedrals [3]. Vaults exist in thousands of copies per cell and are widely expressed in all eukaryotic organisms [4][5][6][7][8]. In both structure and composition vaults are highly conserved throughout evolution in diverse phylogenetic lineages including mammals, avians, amphibians and slime moulds [9]. They represent multimeric protein complexes with one predominant member, the MVP which constitutes more than 70% of the total complex. The remaining mass comprises vault RNA and two high molecular weight proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1) [10,11] Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.Abbreviations EGF, epidermal growth factor; GST, glutathione S-transferase; MVP, major vault protein; PAP, potato acid phosphatase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SH2, Src homology 2; TCL, total cell lysate; TEP1, telomerase-associated protein 1; VPARP, vault poly(ADP-ribose) polymerase.

Although a severe shortage of organs in transplantation can be overcome by using xenotransplantation of porcine donor organs, profound immune rejection to xenogeneic antigens remains a main obstacle. To elucidate the role of hydrogen peroxide (H(2)O(2)) on xenogeneic immune responses, we investigated its effects on porcine aortic endothelial cells (PAECs). We found that H(2)O(2) can specifically induce vascular cell adhesion molecule-1 (VCAM-1) expression on PAECs, but little on human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs). Furthermore, we further confirmed that H(2)O(2) induces activation of NFkappaB in PAECs, but not in HAECs. Interestingly, cell adhesion assay showed that U937, human promonocytic leukocyte, can adhere to PAECs in an H(2)O(2)-dependent manner and by using a neutralizing assay with anti-VCAM-1-specific antibodies, we also found that the interaction is mediated primarily by VCAM-1. Finally, we also demonstrated that up-regulation of VCAM-1 expression on PAECs by reactive oxygen species-producing HL-60, human leukemic neutrophil cells, could be significantly diminished by over-expressing an H(2)O(2)-removing catalase. In summary, our results suggest that NFkappaB-dependent porcine VCAM-1 expression by H(2)O(2) may promote interaction of human leukocyte to PAECs, and thus may play an important role on inducing xenogeneic immune responses.

The CEA 79 antibody has been used in bone marrow scintigraphy for the differential diagnosis of skeletal tumors and the evaluation of the bone marrow status of patients with various hematological disorders. The specific localization of radio-labeled CEA 79 antibody in bone marrow depends on its reactivity with NCA-95 (nonspecific cross-reacting antigen-95) present on the surface and in the cytosol of human granulocytes and myelopoietic cells. To make a CEA 79 scFv molecule that would be less immunogenic and more penetrating than the intact mouse immunoglobulin, we constructed a pRSET Sfi I/Not I expression vector. The scFv gene was then excised from a pCANTAB 5 E phage display vector by digestion with Sfi I and Not I and inserted into the pRSET Sfi I/Not I expression vector. Upon transformation of a BL21(DE3)pLysS strain of E. coli, CEA 79 scFv became expressed in inclusion bodies requiring a renaturation process for solubilization. The final yield of CEA 79 scFv was 5 mg per a liter of culture. The refolded CEA 79 scFv exhibited an affinity (Kd = 2.1 x 10(-9) M) equivalent to that of the original CEA 79 antibody (K(d) = 3.3 x 10(-9) M) and the same immunoreactivity to CEA and NCA-95 in Western blots and in immunohistochemical staining experiments.

An epitope-mimetic peptide of anti-JL1 mAb was found using combinatorial peptide phage display libraries. It induced strong humoral response against itself, but only a limited fraction of this humoral response was cross-reactive with the original JL1 antigen.