Establishment of N-Acetylmannosamine (ManNAc) Analogue-Resistant Cell Lines as Improved Hosts for Sialic Acid Engineering Applications

Abstract

Metabolic substrate-based sialic acid engineering techniques, where exogenously supplied N-acetylmannosamine (ManNAc) analogues are utilized by the sialic acid biosynthetic pathway, allow the cell surface to be endowed with novel physical and chemical properties and show promise for increasing the quality of recombinant glycoproteins. The in vitro toxicity of many ManNAc analogues, however, hinders the large-scale adoption of this technology. In this study, we used a selection strategy where cells were subjected to progressively higher levels of ManNAc analogues to establish novel cell lines that showed decreased sensitivity to analogue-induced in vitro toxicity. The decreased sensitivity to sugar analogue-induced apoptosis, demonstrated by the Annexin V-FITC detection method and DNA fragmentation assays, corresponded to increased sialic acid production in the resistant cell lines. The ManNAc analogue-resistant cell lines exhibited cross-resistance to apoptosis induced by staurosporine and an apoptosis-activating Fas antibody. We propose that the selection strategy employed to develop these novel cell lines, which serve as superior hosts for substrate-based sialic acid engineering applications, will generally apply to the development of host cell lines for biotechnology applications.

Roles for UDP-GlcNAc 2-Epimerase/ManNAc 6-Kinase outside of Sialic Acid Biosynthesis

“…Detection of Apoptosis by Annexin V Flow Cytometry AssaysThe Annexin V/propidium iodide flow cytometry method was used for the detection and quantification of apoptosis in transfected or GD3-treated HEK AD293 cells by following the procedure previously reported for Jurkat cells (39,40) with the added step of trypsinizing the adherent HEK cells (the previously tested Jurkat cells grow in suspension and did not require this step). After trypsinization and resuspension in complete medium, cells were counted with a Coulter Z2 instrument, and 1.0 ϫ 10 6 cells from each sample were pelleted by centrifugation, washed by gentle resuspension in Dulbecco's phosphate-FIGURE 1.…”

“…The full names and complete list of human sialyltransferases and pathway enzymes are given in Table 1. buffered saline, centrifuged again, and suspended in staining buffer. The cells were stained with fluorescein isothiocyanatelabeled Annexin V and propidium iodide and analyzed by flow cytometry as described (39,40).…”

Sialic Acid Glycoengineering Using an Unnatural Sialic Acid for the Detection of Sialoglycan Biosynthesis Defects and On-Cell Synthesis of Siglec Ligands

“…23,24 Some mannosamine analogues might not be recognized by these enzymes and, furthermore, several mannosamine analogues have been reported to be cytotoxic. 25,26 Sialic acid glycoengineering could become more efficient using unnatural sialic acids that can be directly incorporated into sialoglycans without extensive enzymatic conversion. So far, only a few unnatural sialic acid analogues have been developed, and data comparing the performance of these analogues with the corresponding mannosamine analogues in vitro and in vivo are scarce.…”

Development of delivery methods for carbohydrate-based drugs: controlled release of biologically-active short chain fatty acid-hexosamine analogs

“…In all cases, cell counts were compared with solvent-treated controls; in particular, because stock solutions of 1 were prepared in EtOH (to maintain sterility and assist solubility), the culture medium for all cell-based assays were adjusted to contain the same amount of ethanol as the test well with the highest level of 1 . It should be noted that this control was done simply as a precaution because the amount of EtOH used was less than 0.4 % (v/v), a level that previous studies have showed had no noticeable effect on the biological endpoints under evaluation [10, 20, 27]. …”

“…Accordingly, the sialic acid content was normalized against total protein concentration rather than against number of cells as is typically done in our experiments [7, 10, 27, 29]. Briefly, the cells from the harvested aliquots were washed with PBS to removed residual serum glycoprotein and re-suspended in PBS, lysed by freeze/thaw cycles, and centrifuged (16100 × g , 1.0 min).…”