Glycoprotein Synthesis and Biochemistry of the Secretory Pathway

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Analysis of the glycoproteins in cell and animal models allows exploration of the rules of glycoprotein synthesis in the ER/Golgi secretory compartments and production of the glycoproteins for functional studies. We use most commonly HEK293F cells for the expression of human glycoproteins because of the efficiency of this cell culture model and its common use in other laboratories or biopharmaceutical industry. Genetically engineered or pharmaceutically modified cell-lines provide a set of models which we use in combination with the in house established analytical methods to define glycoforms of proteins of interest but also the rules that govern their synthesis. The regulatory mechanisms of protein glycosylation in the ER are reasonably well understood but what defines the types of glycosylation observed on glycoproteins and their impact on protein secretion needs further study. This area of intense investigation has important implication for human pathophysiology but also for the production of biopharmaceuticals.

One example relevant to the studies of immune responses are our studies of immune checkpoint proteins whose function is regulated by their glycosylation. Our studies show that glycosylation of the therapeutically targeted PD-L1 protein depends on the cell type it is expressed in but some structural features of the protein determine preference of specific sites of glycosylation for certain types of glycoforms (e.g. high mannose glycans or polyLacNAc structural motives) expected to regulate interactions and function of the checkpoints.

Figure 1: MD simulation of the glycosylated PD-L1/PD-1 complex. Results show that N116 and N74 glycans of PD-1 and the N219 glycan of PD-L1 interact and that the N74 glycan contacts the PD-L1 protein (from Oliver Grant and Rob Woods, CCRC). The table shows number of identified glycoforms (GP IDs), site occupancy, and most common glycans found at each site of attachment for PD-L1 expressed in MD-MBA-231 breast cancer cells and enriched by cell-surface biotinylation.

Various signaling pathways or structural adjustments of the Golgi secretory compartment under stress conditions determine changes in the glycosylation of proteins and, ultimately, how the cells respond to the signals from the environment. We study the rules governing these processes and types of processes they regulate. One application we follow involves studies of the glycosylation of neurotrophins, a family of neurostimulators essential for the development of the nervous system. The glycosylated pro-neurotrophins mature by proteolytic processing in the Golgi compartment. Our studies of the HEK293F expresssed proBDNF show that the protein carries sulfated LacdiNAc glycoforms which regulate specific functions of the glycoprotein. We want to understand the regulatory mechanisms and ways to alter the secretory pathway.

Figure 2. ProBDNF carried sulfated LacdiNAc N-glycans whose function in the maturation of the protein is investigated by genetic engineering and pharmacological interventions in combination with optimized LC-MS/MS analytical methods. Benicky et al (2019) J Biol Chem. 8;294(45):16816-16830.