A yeast glycoprotein shows high-affinity binding to the broadly neutralizing human immunodeficiency virus antibody 2G12 and inhibits gp120 interactions with 2G12 and DC-SIGN

Abstract

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a Delta pmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the Delta mnn1 deletion to the Delta pmr1 background, ensuring the exposure of terminal alpha1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man(8)GlcNAc(2) structures in Delta och1 Delta mnn1 Delta mnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies.

FIG. 1.

Critical proteins involved in S. cerevisiae N-linked glycosylation. In the ER, Mns1 cleaves mannose from core Man₉GlcNAc₂ on nascent polypeptides to form Man₈GlcNAc₂. In the Golgi apparatus, S. cerevisiae adds mannose residues to this oligosaccharide to form core-type and polymannose-type glycans with up to 200 mannoses per structure. Mnn1 adds terminal α1,3-linked mannose caps (open boxes), Och1 adds the initial α1,6-linked mannose of the polymannose side chain (dark-gray boxes), and Mnn9 elongates the α1,6-linked backbone (light-gray box). The deletion of PMR1 results in aberrant glycosylation with severely truncated polymannose side chains so that the N-glycans are similar to those found in the Δmnn9 mutant (35), like yeast core-type N glycosylation (curved arrow). Throughout processing in the Golgi apparatus, the ER-secreted Man₈GlcNAc₂ remains intact (trapezoidal boxes). Phosphomannose residues were omitted for simplicity.

FIG. 2.

2G12 detection of a yeast protein in Δpmr1-containing mutants. (A) Equal amounts of yeast cell lysates from single-mutant and WT yeast were screened for 2G12 binding by Western blotting, with 50 ng of 293-expressed ADA gp120 as a control. (B) Cell lysate samples from the three Δpmr1 Δmnn1 clones were tested for 2G12 binding by Western blotting. (C) Culture medium samples from Δpmr1 and Δpmr1 Δmnn1-clone 5 (Clone 5) were blotted with 2G12 (top) and an antibody specific to the unknown protein, later identified as Pst1 (bottom). The migration positions of molecular mass markers, in kilodaltons, are indicated to the left of the blots.

FIG. 3.

Identification and verification of the yeast glycoprotein that cross-reacts with MAb 2G12. (A) The amino acid sequence of the glycoprotein Pst1, isolated from Δpmr1 Δmnn1 yeast and identified by nano-LC/MS/MS, is depicted. The detected peptides are underlined, with each being identified at least twice; the boldface letters at the N and C termini depict the signaling peptide and GPI anchor signal, respectively; and the highlighted letters are potential N-linked glycosylation sites. The amino acid sequence was obtained from GenBank under the accession number NP_010340 (23). (B) Pst1 was precipitated from Δpmr1 and Δpmr1 Δmnn1 culture media by 2G12 and detected by Western blotting with anti-Pst1. Starting material (S), flowthrough (F), wash (W), and eluate (E) samples are indicated.

FIG. 4.

Analysis of Pst1 glycans. (A) MALDI-TOF mass spectrogram of N-linked glycans released from purified Δpmr1 Δmnn1-Pst1. (B) Δpmr1 Δmnn1-Pst1 digested with Endo H or with jack bean or A. saitoi mannosidase was analyzed by Western blotting using anti-Pst1 (left) or 2G12 (right), with mock-digested and untreated Pst1 used as controls.

FIG. 5.

Analysis of 2G12 reactivity between Pst1 and gp120. (A) MALDI-TOF mass spectrogram of N-linked glycans released from purified TM-Pst1. (B) A comparison of purified Δpmr1 Δmnn1-Pst1, TM-Pst1, JR-FL gp120, and Yu2 gp120 is shown with 500 ng of each protein stained by Coomassie blue (top) or 250 ng detected by Western blotting with anti-Pst1 (middle) and anti-gp120 (bottom). (C) Western blot comparing 2G12 (1 μg/ml) binding to purified Δpmr1 Δmnn1-Pst1, TM-Pst1, JR-FL gp120, and Yu2 gp120 loaded at 1 μg (top), 0.5 μg (middle), and 0.25 μg (bottom). (D) Comparison of 2G12 binding to 100 ng of purified Δpmr1 Δmnn1-Pst1 (Δp/m-Pst1), TM-Pst1, JR-FL gp120, and Yu2 gp120 proteins by ELISA. The values are the averages ± standard errors of the means (error bars) of three independent experiments. The gp120 proteins were produced in 293T cells. OD, optical density.

FIG. 6.

TM-Pst1 binding to 2G12 by SPR. (A) Real-time SPR RU of purified TM-Pst1 (left) and Yu2 gp120 (right) from 12.5 nM to 800 nM on a 2G12-immobilized sensor chip. (B) Steady-state fitting of TM-Pst1 (left) and Yu2 gp120 (right) with the indicated K_D values.

FIG. 7.

TM-Pst1 inhibition of 2G12-gp120 interaction. (A) The percent 2G12 binding to 100 ng of immobilized JR-FL gp120 was analyzed by ELISA in the presence of TM-Pst1, Pst1-Endo H, Yu2 gp120, and JR-FL gp120. There was no preincubation of inhibitors with 2G12, and 100% represents 2G12 binding in the absence of inhibitor. The values are the averages ± standard errors of the means (error bars) of three independent experiments. Molar IC₅₀s were calculated by nonlinear regression using GraphPad Prism 5.0, using the apparent molecular masses of TM-Pst1, Yu2, and JR-FL gp120 at 100 kDa (Fig. 5B). (B) (Top) Neutralization activity of MAb 2G12 against HxB-pseudotyped virus in the presence of Pst1-Endo H and TM-Pst1, with MAb 2F5 as a control. The results are representative of three separate experiments. (Bottom) Neutralization activity of 2G12 assessed against SF162-pseudotyped virus in the presence of Pst1-Endo H and TM-Pst1. The results are representative of two separate experiments.

FIG. 8.

TM-Pst1 inhibition of DC-SIGN-gp120 interaction. (A) Graded concentrations of TM-Pst1, mannan, and BSA were evaluated for direct inhibition of soluble JR-FL gp120 binding to immobilized DC-SIGN by ELISA. The results are shown relative to 100% gp120 binding, calculated in the absence of inhibitor. The values are the averages ± standard errors of the means (error bars) of three independent experiments for Pst1 and mannan and two for BSA. IC₅₀s were calculated by nonlinear regression using GraphPad Prism 5. (B) (Top) Inhibition of JRFL gp120 (1 μg) binding to THP-DC-SIGN by 10 μg of mannan, BSA, or TM-Pst1. (Bottom) Dose-dependent inhibition of JR-FL gp120 (1 μg) binding to THP-DC-SIGN cells by TM-Pst1. As controls, cells were incubated with only antibodies (no gp120) or gp120 without inhibitors (gp120). The results are representative of two independent experiments.