Andres Guerrero1; Yanhong Li1; Salem Alkanaimsh2; Lucas Arzola2; Bryce Hashimoto2; Minsook Hwang3; Aye Tu4; My Phu4; Abhaya Dandekar4; Bryce Falk3; Somen Nandi5; Raymond Rodriguez5; Karen McDonald2; Xi Chen1; Carlito Lebrilla1
1UC Davis, Chemistry Department, Davis, CA;2UC Davis, Chemical Engineering & Materials Science, Davis, CA; 3UC Davis, Plant Pathology, Davis, CA; 4UC Davis, Plant Science, Davis, CA; 5UC Davis, Molecular & Cellular Biology, Davis, CA
A method for accurate glycan analysis was developed to monitor the production of a recombinant protein with multiple glycosylation sites.
Glycan analysis of recombinant proteins containing multiple glycosylation sites is analytically challenging especially during the initial phases of production when sample amount and purity are major limitations. In this work we propose a glycoprotein analysis method that addresses these potential limitations, provide site-specific glycan information and facilitate the quantitative comparison between samples.
The method was successfully applied during the production of a glycoengineered recombinant protein: human butyrylcholinesterase (BuChE). BuChE produced in Nicotiana benthamiana was sialytated in vitro to increase its therapeutic potential and mimic the human glycosylation pattern. We describe the glycopeptide characterization of the BuChE glycoforms expressed in different plant subcellular localizations and the glycosylation product, after every step of glycan remodeling, up to the final desired glycosylated product.
The analytical method employed glycopeptide analysis by in-gel digestion using a non-specific protease followed by mass spectrometry. Briefly, samples were desalted by C8 solid phase extraction, denatured and run on a SDS-PAGE gel. Excised protein bands were digested with pronase E and the resulting lysates were extracted and purified for glycopeptides using graphitized solid phase extraction. Tandem-MS analysis was performed on a nanoESI-LC-Q-TOF using a graphitized chip nanoLC column. Glycopeptides were identified using GP-Finder 3 (in-house software) and the corresponding signals quantified by ion-counting. The procedure was initially optimized using another human recombinant plant-made glycoprotein, lactotransferrin produced in Oryza sativa.
The combination of gel electrophoresis and glycopeptide analysis facilitates the analysis of specific proteins in highly impure plant extracts. Additionally, the immobilization of the glycoprotein in the gel allows analysis on even very small amounts of sample. This analytical method was successfully applied to monitor glycosylation on intermediates and products through every step of the glycoprotein production.
The glycoprotein analysis showed that subcellular localization affects the N-glycan composition of the glycoprotein. The BuChE recovered from the plant endoplasmic reticulum (ER) was almost exclusively glycosylated with high-mannose, while the apoplast targeted extract (APO) was predominantly composed by the basic N-glycan plant core. Based on these results, ER BuChE would require an additional enzymatic step (trim off the high-mannose structures) to reach the desired sialylated structures compared to APO BuChE. Hence, APO BuChE was chosen as a substrate.
To obtain the desired glycoform from plant APO BuChE, sequential addition of N-acetylglucosamine, galactose and sialic acid is required using three enzymatic systems, namely N-acetylglucosaminyltransferase, β1,4-galactosyltransferase and α2,6-sialyltransferase. The activity of these enzymes was tested using plant recombinant lactotransferrin that, similarly to APO BuChE, exhibits basic plant core N-glycosylation. The analysis showed the systematic modification of the lactotransferrin N-glycosylation pattern after each redecoration step validating both, the in vitro glycan modification and our analytical approach.
As important as the glycan composition was to the product, the aim was product efficacy. A continuous feedback system was established between redecoration, glycoprotein analysis and protein bioactivity. APO BuChE N-glycan remodelation was performed under different conditions such as temperature and pH. At every step, product was analyzed using the analytical method to observe the progress. The final product contained at least 10%-30% of sialylation. Facilitated by our analytical method, further optimization of the redecoration process is expected to increase the degree of sialylation of the glycoprotein while keeping its activity.