Engineering mammalian mucin-type O-glycosylation in plants

Abstract

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.

FIGURE 1.

Depiction of the initiation process of mucin-type glycosylation in plants. The donor substrate UDP-GalNAc is synthesized from UDP-GlcNAc by P. aeruginosa C4-epimerase (WbpP) protein. UDP-GalNAc is transported into the Golgi lumen by endogenous plant sugar nucleotide transporter(s) where Golgi localized GalNAc-T2 and -T4 catalyze the transfer of GalNAc onto the target peptide(s) destined for secretion in the secretory pathway.

FIGURE 2.

O-Glycosylation of the MUC1 reporter construct. A, O-glycosylation target protein MUC1-YFP was N-terminally fused to a signal peptide for direction into the secretory pathway, followed by a His₆ tag. Gene constructs for introduction of O-glycosylation capacity into plants, comprising FLAG-tagged cytoplasmic, ER, or Golgi-targeted epimerase and native Golgi-targeted GalNAc-T2, either expressed as a single polycistronic protein, interspaced by the 2A self-splicing sequence (cleavage site indicated by an arrow), i.e. T2–2A-CytoEpi, CytoEpi-2A-T2, and GolgiEpi-2A-T2, or from separate promoters, i.e. EREpi-T2 and CytoEpi-T2. B, SDS-PAGE Western blot analysis of MUC1-YFP expressed alone (lane 1), together with cytoplasmic epimerase (lane 2), or with GalNAc-T2 (lane 3). MUC1-YFP co-expressed with either of the 2A-linked O-glycosylation machinery constructs (T2–2A-CytoEpi or CytoEpi-2A-T2) (lanes 4 and 5), 2A-linked Golgi-targeted epimerase, and GalNAc-T2 (lane 6), Golgi-targeted GalNAc-T2 with ER-targeted epimerase (EREpi) (lane 7), and cytoplasmic epimerase (lane 8) expressed from unlinked glycosylation machinery. Absence or presence of O-glycosylation machinery is indicated above the lanes with (−) or (+), respectively. Total protein extracts from transiently transformed N. benthamiana leaves were loaded and blots reacted with MUC1 specific MAbs 5E10 and 5E5, where 5E5 is specific for GalNAc-glycosylated MUC1 and does not react with unglycosylated MUC1. Approximately 30 μg of total protein was loaded in each lane.

FIGURE 3.

Structural analysis of MUC1-YFP expressed in N. benthamiana. A, purification of His-tagged MUC1-YFP. Left panel, SDS-PAGE Coomassie staining of MUC1-YFP expressed (−) alone and (+) together with 2A-linked glycosylation machinery (T2-2A-CytoEpi). Right panel, staining with Vicia villosa agglutinin (VVA) lectin for detection of GalNAc glycosylation. B, schematic illustration of endo-Asp-N digestion of O-glycosylated MUC1. C, analytical HPLC purification of endo-Asp-N-digested MUC1-YFP expressed alone (upper panel) or together with O-glycosylation machinery (lower panel) separating released 20-mer MUC1 repeat sequences with and without GalNAc-glycosylation. D–G, Orbitrap FT-MS analysis of purified endo-Asp-N MUC1-YFP products. D, MUC1-YFP expressed alone. E, MUC1-YFP co-expressed with the O-glycosylation machinery (T2-2A-CytoEpi). F, MUC1-YFP co-expressed with the inverse ordered glycosylation machinery (CytoEpi-2A-T2). G, MUC1-YFP co-expressed with 2A-linked Golgi targeted epimerase and GalNAc-T2 (GolgiEpi-2A-T2). Each spectrum is a deconvoluted average centroid m/z representation. Peaks consistent with the MUC1-YFP sequence are labeled with monoisotopic m/z. These correspond to DTRPAPGSTAPPAHGVTSAP (31–50, 51–70, or 71–90, calculated monoisotopic m/z 1886.9355; +1 × oxidation (i.e. Pro → Hyp), calculated monoisotopic m/z 1902.9304); DTLVNRIELKGIDFKE (220–235, calculated monoisotopic m/z 1890.0331); DGPVLLPDNHYLSYQSALSK (293–312, calculated monoisotopic m/z 2217.1186); DFFKSAMPEGYVQERTIFFK (185–204, +1 × oxidation (of Met or Pro), calculated monoisotopic m/z 2456.1955); DGNILGHKLEYNYNSHNVYITA (236–257, calculated monoisotopic m/z 2535.2263). Arrows denote peaks consistent with mono-, di-, and tri-glycosylated DTRPAPGSTAPPAHGVTSAP peptide (31–50, 51–70, or 71–90, calculated monoisotopic m/z 2090.0149, 2293.0943, and 2496.1736, respectively).

FIGURE 4.

Co-expression of GalNAc-T2 and -T4 completes GalNAc O-glycosylation of MUC1. A, Western blot analysis of target construct MUC1-YFP (lane 1) alone (−) or co-inoculated (+) with either of the 2A-linked glycosylation machinery constructs T2–2A-CytoEpi (lane 2) or T2–2A-CytoEpi and ectopically with GalNAc-T4 (lane 3). Co-expression of GalNAc-T4 with the 2A-linked glycosylation machinery (T2–2A-CytoEpi) conferred glycosylation of all five potential GalNAc O-glycosylation sites in MUC1–1TR. B, Orbitrap FT-MS analysis of endo-Asp-N-digested MUC1-YFP co-expressed with 2A-linked glycosylation machinery (T2–2A-CytoEpi) and GalNAc-T4, demonstrating glycosylation of the three GalNAc-T2-specific sites and the two additional GalNAc that are added by GalNAc-T4 (DT_T4RPAPGS_T2T_T2APPAHGVT_T2S_T4AP), i.e. complete GalNAc occupancy of sites.

FIGURE 5.

O-Glycosylation of human Mucin 16 (MUC16) reporter and interferon α2B. A, Western blot and lectin staining analysis of Mucin 16–1,2TR (MUC16); B, interferon α2B (INF-α2B), alone (−) or co-inoculated (+) with the 2A-linked glycosylation machinery construct T2–2A-CytoEpi. mAb M11 is a specific antibody to MUC16, and mAb T7 is a T7 tag antibody. Glycosylations of MUC16 and INF-α2B are shown by staining with VVA. 30 μg of total protein was loaded in each lane. The two constructs expressing MUC16 and INF-α2B are shown with the potential glycosylation sites (Ser or Thr) denoted by underlining.