Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

Abstract

Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.

Recombinant Protein Expression System in Corynebacterium glutamicum and Its Application

“…However, in contrast to the glycosylation system of mammalian cells, yeast cells have a mannose-rich glycosylation system; thus, they are often not suitable for use in medicine. In particular, post-translational modifications such as glycosylation in Pichia pastoris often lead to unexpected protein structure and function (Dai et al, 2015 ). Nevertheless, C. glutamicum may be advantageous as a host for the expression of antibody fragments such as the scFv and Fab (antigen-binding fragment), which do not require glycosylation (Yim et al, 2014 ).…”

The Degree and Length of O‐Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

“…[ 15 ] Other studies, however, suggest that P. pastoris can produce proteins with O‐glycans consisting of 6‐Man. [ 16,17 ] These mannose residues are mostly α‐1,2 or β‐linked, and they can also be phosphorylated. [ 4 ] β‐linkages on recombinant glycoproteins could cause an immunogenic response in humans due to interaction with the mannose receptor, or dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN).…”

“…The authors suggested that this could be either due to the absence of highly immunogenic terminal α‐1,3‐mannosyl groups, or due to lower complexity of mannose type O ‐glycans in P. pastoris compared to more complex ones in mammalian cells. [ 17 ]…”

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

“…In contrast to the absence of glycosylation in prokaryotic expression systems like Escherichia coli , glycosylation is common in eukaryotic host systems (i.e., Pichia pastoris ) and play important roles in proper folding, transport, and stability of proteins [ 23 , 34 – 38 ]. Yeast strains are widely used for recombinant protein production, during which both O - and N -glycosylations occur preceding protein secretion and have obvious effects on enzyme properties [ 39 – 41 ]. P. pastoris is capable of adding both O - and N -linked carbohydrate moieties to secreted proteins.…”