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.

Fig 1. Heterogeneous expression of glycosylated recombinant human PDGF-BB in Pichia pastoris.

(A) Purified rhPDGF-BB proteins from six independent expression and purification experiments were analyzed by SDS-PAGE. Heterogeneous rhPDGF-BB bands could be observed under reducing conditions. (B) Purified rhPDGF-BB and rhIFN-ω produced by P. pastoris were treated with PNGase F to hydrolyze N-glycan residues. Two gels loaded with the same amount of each protein (5 μg) were simultaneously subjected to SDS-PAGE, and analyzed by Coomassie Blue staining (left panel) and glycosylation staining (right panel), respectively. There were no differences between rhPDGF-BB samples treated or not with PNGase F.

Fig 2. Identification of rhPDGF-BB isoforms.

(A) Five isoforms detected by SDS-PAGE were transferred to a PVDF membrane for protein N-terminal sequencing. (B) Schematic representation of N-terminal sequencing results. Bands I, II, and III represent intact PDGF-BB, while bands IV and V are truncated isoforms generated by the cleavage at the Arg 27-Thr 28 site. (C) rhPDGF-BB (5 μg) was subjected to SDS-PAGE under reducing conditions and analyzed by western blotting, Coomassie Blue staining, and glycosylation staining, respectively. Two bands corresponding to isoforms III and IV stained by Coomassie Blue could be detected by antibody staining (marked with red lines). Glycosylation staining revealed another two bands corresponding to isoforms I and II (marked with red lines). (D) SDS-PAGE and subsequent glycosylation staining of higher rhPDGF-BB load (10 μg and 20 μg) revealed three bands; the third band with the lowest molecular weight was assumed to correspond to isoform IV.

Fig 3. Mutation of the putative O-glycosylation sites in rhPDGF-BB.

(A) Schematic representation of point mutations in PDGF-BB. (B) Purified wild-type PDGF-BB and PDGF-BB^ΔGly mutant were separated by reducing SDS-PAGE and analyzed by Coomassie Blue staining (left panel), western blotting (middle panel), and glycosylation staining (right panel). A single homogeneous band could be observed after western blotting and Coomassie Blue staining, while no band was detected by glycosylation staining of the mutant.

Fig 4. Identification of O-glycosylation sites in rhPDGF-BB using LC/MS.

(A) Schematic representation of PDGF-B mutants. Monoisotopic mass of every mutant is shown. (B) Deconvoluted mass spectra of the wild-type PDGF and its mutants are presented with monoisotopic peaks; glycosylated isoforms are annotated.

Fig 5. Mitogenic activity of rhPDGF-BB and PDGF-BB^ΔGly.

(A) Proliferation of BALB/C 3T3 cells stimulated with different concentrations of rhPDGF-BB (black line) or PDGF-BB^ΔGly (red line) was analyzed by the WST-1 assay. Dose-response curves of a representative assay are shown. (B) EC₅₀ values were calculated based on three independent experiments and expressed as the mean ± SD (P = 0.0117).

Fig 6. Mouse immunization with rhPDGF-BB and PDGF-BB^ΔGly.

PDGF-BB- and PDGF-BB^ΔGly-specific antibody titers presented as the mean ± SD are defined as the reciprocal endpoint dilution. (A) Schematic representation of the immunization procedure. (B) Serum antibody titers against PDGF-BB and PDGF-BB^ΔGly in mice boosted at week 1, week 3, and week 4 after the first immunization (week 1, P = 0.25; week 3, P = 0.51; week 4, P = 0.68). (C, D) The titers of antisera against PDGF-BB and PDGF-BB^ΔGly at week 3 (C) and week 4 (D).