The Glycosylation and in Vivo Stability of Human Granulocyte-Macrophage Colony-Stimulating Factor Produced in Rice Cells

Abstract

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been produced in several different cell types, and has different properties depending on the production process used. There has been no report of glycosylation ratio or of the role of glycans for plant cells-derived rhGM-CSF. We investigated the terminal sialylation of rhGM-CSF produced in rice cells (rrhGM-CSF) using lectins. Glycosylation ratios of rrhGM-CSF and yeast-derived rhGM-CSF (yrhGM-CSF) were evaluated by chemical deglycosylation. After intravenous administration to rats, the clearance rates of rrhGM-CSF and yrhGM-CSF as well as deglycosylated forms of them were evaluated. In vitro bioactivities of rrhGM-CSF and yrhGM-CSF were measured in dose-and time-dependent manners. Although rrhGM-CSF does not have terminal sialic acids, the glycosylation ratio was two-fold higher than that of yrhGM-CSF, and rrhGM-CSF sustained longer than yrhGM-CSF in the blood circulation. Moreover, yrhGM-CSF and rrhGM-CSF have almost same bioactivity via dose-and time-dependent manners. Deglycosylated rrhGM-CSF was cleared faster than native forms, while deglycosylated yrhGM-CSF and yrhGM-CSF were similarly cleared in blood circulation. These results suggest that the glycans on rrhGM-CSF are superior to the glycans on yrhGM-CSF in terms of in vivo stability.

A lectin-based approach to detecting carcinogenesis in breast tissue

“…Wells of a 96-well microplate (Greiner Bio-One, Frickenhausen, Germany) were coated with 1,600, 800, 400, 200 or 100 ng of tissue lysate protein per well at 4˚C, and blocked with 5% oxidized bovine serum albumin (BSA; GenDEPOT, Katy, TX, USA) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T; Duchefa Biochemie BV) for 4 h at room temperature. The cell lysates were mixed with carbonate buffer (pH 9.4) and then incubated for 20 h at 4˚C to coat the 96-well plate.. BSA treated with sodium meta-periodate (Sigma-Aldrich, St. Louis, MO, USA) was prepared as previously described (17). Subsequently, the wells were incubated with biotinylated concanavalin A (Con A; catalog no., B-1005), Ricinus communis Agglutinin I (RCA I; catalog no., B-1085), Aleuria aurantia lectin (AAL; catalog no., B-1395) or Maackia amurensis lectin II (MAL II; catalog no., B-1265) (Vector Laboratories, Inc., Burlingame, CA, USA) for 80 min at 37˚C, followed by poly-horseradish peroxidase (HRP)-conjugated streptavidin (catalog no., N200; Pierce™; Thermo Fisher Scientific, Inc.) for 40 min at 37˚C.…”

“…ELLA is the simplest method to analyze lectins; however lectin blotting and lectin microarrays may also be used (15). To date, ELLA systems have been used to investigate a limited number of materials, with purified glycoproteins analyzed most commonly (16,17). There are few previous studies that have analyzed glycan structures in clinical tissue biopsy specimens (18).…”

Effect of Rice Cell-Derived Human Granulocyte-Macrophage Colony-Stimulating Factor on 5-Fluorouracil-Induced Mucositis in Hamsters

“…25,26) It was founded that rice cell derived hGM-CSF has higher glycosylation ratio and its ratio influence a pharmaceutical potency of rhGM-CSF. 27) This study examined the healing effect of rhGM-CSF on 5-FU-induced oral mucositis. The severity and dimension of mucositis were reduced rapidly in the rhGM-CSF-treated groups from days 5 to 8, as measured by both gross and immunohistochemical evaluations.…”

“…Hence rhGM-CSF was more effective than yrhGM-CSF in recruiting leukocyte. 27) The present study was performed to evaluate the effect of plant-derived rhGM-CSF on wound healing in a hamster oral mucositis model.…”

Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) could accelerate burn wound healing in hamster skin

“…Other than human and mouse, sequences encoding for GM-CSF from a variety of species have been identified (Gasson 1991;Barreda et al 2004;Arellano and Lonial 2008). Meanwhile, recombinant human GM-CSF (rhGM-CSF), which contains more oligosaccharide side chains than any other types of GM-CSF, was specifically produced from rice cell culture through use of a recombinant technique in our laboratory (Kim et al 2008).…”