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Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12-24 h. The amount of GCD in medium increased until 60-84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells.

Abstract. Autocrine motility factor (AMF), which is a secreted form of phosphoglucose isomerase, is mainly secreted by various tumors and has cytokine-like activity. AMF is known to stimulate proliferation, survival and metastasis of cancer cells, and angiogenesis within a tumor. The present study investigated whether inhibition of AMF using targeted-antibodies was able to suppress the growth of cancer. A migration assay using a Boyden chamber was utilized to measure the activity of AMF on the motility of cancer cells. A recombinant human AMF (rhAMF) prepared from E. coli transformed with the pET22b-AMF vector increased the motility of MDA-MB-231 and A549 cells, but it did not affect that of NCI-N87 or HepG2 cells, which exhibited the ability to secrete high amounts of their own endogenous AMF into the culture medium. The extent to which the AMF receptor was expressed on cancer cells did not correlate clearly with the cell motility stimulated by rhAMF. In A549-xenografted nude mice treated with sunitinib or cetuximab, a decrease in the plasma AMF concentration was accompanied by a reduction in tumor weight, suggesting an association between the plasma AMF concentration and anticancer activity. A monoclonal antibody (9A-4H), which revealed a high binding affinity for E. coli-derived rhAMF, significantly suppressed the growth of tumors in Balb/c nude mice transplanted with the human gastric cancer cell line NCI-N87, to the similar extent as trastuzumab, an anticancer antibody. The present study suggests, for the first time, that an antibody specific to AMF may be a therapeutic agent for gastric cancer. IntroductionPhosphoglucose isomerase (PGI), also termed phosphohexose isomerase (PHI), is a glycolytic enzyme that catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate in the glycolysis process. A secreted version of PGI/PHI, which has cytokine activity, was originally purified from the conditioned culture medium of human melanoma cells and was termed autocrine motility factor (AMF) owing to its ability to stimulate cell motility (1). Until the respective protein sequences were determined, AMF was termed neuroleukin, a maturation factor mediating differentiation of human myeloid leukemia cells, a myofibril-bound serine proteinase inhibitor, and sperm antigen-36 (2-5). Although the secretion of AMF by normal cells has not been observed, its secretion into culture medium has been identified in various cancer cells. Clinical studies measuring AMF in the serum or urine of patients with lung, gastrointestinal and renal cancer, have suggested that it may be a useful biomarker for certain types of cancer (6,7). In addition to the cell motility stimulating activity, AMF is involved in cellular proliferation (8), cellular survival (9), invasion of malignant cells (10), tumor metastasis (11) and the induction of angiogenesis (12).AMF binds to and stimulates the AMF receptor (AMFR), which is a 78 kDa glycoprotein (gp78) with seven transmembrane domains (13). When stimulated by AMF, AMFR activates signa...

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig(P)) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig(M)), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig(P) and hCTLA4Ig(M) had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig(P) and hCTLA4Ig(M) had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-gamma, but did not suppress the expression of macrophage-derived cytokines, including TNF-alpha and IL-1beta, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig(P) is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig(M).