The HNK-1 epitope expressed on GluA2 enhances cell surface expression of GluA1.

<p>A cell biotinylation assay was applied to HEK293 cells expressing GluA1 with (+) or without (-) GluA2 or HNK-1-synthesizing enzymes (GlcAT-P and HNK-1ST) as indicated. Biotinylated GluA1 and GluA2 were immunoblotted with anti-GluA1 and anti-GluA2/3 polyclonal antibodies, respectively (Surface) (A). The cell lysates were also immunoblotted with the same polyclonal antibodies (Total) and an HNK-1 monoclonal antibody (B). (C) Relative intensities of surface expression levels of GluA2 or GluA1 (surface/total) were calculated and normalized with or without the HNK-1 epitope.</p

The HNK-1 epitope on N-glycan at N413 enhances cell surface expression of GluA2.

<p>(A) A cell biotinylation assay was applied to HEK293 cells expressing GluA2 (WT, N256S, or N413S) with (+) or without (-) HNK-1-synthesizing enzymes (GlcAT-P and HNK-1ST). Biotinylated GluA2 was immunoblotted with anti-GluA2/3 polyclonal antibody (Surface). The cell lysates were also immunoblotted with the same polyclonal antibody (Total) and an HNK-1 monoclonal antibody. (B) Relative intensities of surface expression levels of GluA2 (surface/total) were calculated and normalized with that of WT without the HNK-1 epitope.</p

N-glycan at N370 of GluA2 regulates the intracellular trafficking of GluA1.

<p>(A) GluA1 and GluA2 (WT, N256S, N370S, or N413S) were transfected into HEK293 cells. GluA1 and GluA2 were immunoprecipitated with anti-GluA1 (right) and anti-GluA2/3 (middle) polyclonal antibodies, respectively, and then immunoblotted with each antibody. The cell lysates were also immunoblotted for loading control (Input) (left). (B) A cell biotinylation assay was applied to HEK293 cells expressing GluA1 and/or GluA2 under several conditions: GluA1 alone, GluA2WT alone, GluA1 and GluA2WT, GluA1 and GluA2N256S, or GluA1 and GluA2N370S. Biotinylated GluA1 and GluA2 were immunoblotted with anti-GluA1 and anti-GluA2/3 polyclonal antibodies, respectively (Surface). The cell lysates were also immunoblotted for loading control (Total).</p

N-glycan at N370 is essential for cell surface expression of GluA2.

<p>(A) GluA2 is composed of NTD (pink), LBD (blue), transmembrane domains, and a cytoplasmic domain. NTD includes two N-glycosylation sites (N256 and N370), and N406 and N413 are located in the linker between NTD and LBD. The amino acid number was counted from the first methionine of the signal sequence. (B) A cell biotinylation assay was applied to HEK293 cells expressing GluA2 wild-type (WT) or N-glycosylation site mutants (N256S, N370S, N406S, or N413S). Biotinylated GluA2 was immunoblotted with anti-GluA2/3 polyclonal antibody (Surface). The lysates were also immunoblotted for loading control (Total). (C) HEK293 cells expressing WT or mutants were doubly immunostained. Cell surface GluA2 was stained with anti-GluA2 N-terminal monoclonal antibody (red) under nonpermeabilizing conditons. Intracellular GluA2 was subsequently stained with anti-GluA2/3 polyclonal antibody (green) after cell permeabilization.</p

The HNK-1 epitope is preferentially expressed on N-glycan at N413.

<p>GluA2 (WT, N256S, N370S, N406S, or N413S) was transfected into HEK293 cells with HNK-1-synthesizing enzymes (GlcAT-P and HNK-1ST). After immunoprecipitation (IP) with anti-GluA2/3 polyclonal antibody, immunoprecipitates were immunoblotted with an HNK-1 monoclonal antibody and anti-GluA2/3 polyclonal antibody (A). The cell lysates were immunoblotted with an HNK-1 monoclonal antibody (Input) (B).</p

Engineering a Low-Immunogenic Mirror-Image VHH against Vascular Endothelial Growth Factor

Immunogenicity is a major caveat of protein therapeutics. In particular, the long-term administration of protein therapeutic agents leads to the generation of antidrug antibodies (ADAs), which reduce drug efficacy while eliciting adverse events. One promising solution to this issue is the use of mirror-image proteins consisting of d-amino acids, which are resistant to proteolytic degradation in immune cells. We have recently reported the chemical synthesis of the enantiomeric form of the variable domain of the antibody heavy chain (d-VHH). However, identifying mirror-image antibodies capable of binding to natural ligands remains challenging. In this study, we developed a novel screening platform to identify a d-VHH specific for vascular endothelial growth factor A (VEGF-A). We performed mirror-image screening of two newly constructed synthetic VHH libraries displayed on T7 phage and identified VHH sequences that effectively bound to the mirror-image VEGF-A target (d-VEGF-A). We subsequently synthesized a d-VHH candidate that preferentially bound the native VEGF-A (l-VEGF-A) with submicromolar affinity. Furthermore, immunization studies in mice demonstrated that this d-VHH elicited no ADAs, unlike its corresponding l-VHH. Our findings highlight the utility of this novel d-VHH screening platform in the development of protein therapeutics exhibiting both reduced immunogenicity and improved efficacy

Biosynthetic model for HNK-1 epitope on aggrecan in PNNs.

<p>GlcAT-I is responsible for synthesis of a linkage region of GAG, which is usually further elongated into a long GAG chain (e.g., CS chain). HNK-1ST transfers a sulfate group to GlcA of the linkage region of aggrecan, which likely results in the expression of the linkage type HNK-1 epitope, HSO₃-GlcA-Gal-Gal-Xyl, in PNNs. The expression of the HNK-1 epitope on aggrecan suppresses the CS polymerization that starts from GlcA of the linkage region.</p

HNK-1 carbohydrate epitope expressed in GlcAT-P-deficient mice.

<p>(A, B) Soluble fractions prepared from 2- and 6-week-old mouse brains (WT and GlcAT-P-deficient mice) were treated with chABC with or without PNGase F, subjected to SDS-PAGE and then blotted using anti-HNK-1 mAb, 6B4 mAb, Cat-315 mAb, phosphacan pAb and aggrecan pAb. To compare the molecular weight of HNK-1 immunoreactive band with aggrecan or phosphacan, an HNK-1 mAb blot of 6-week-old PKO mice treated with chABC and PNGase F is shown on the right of the aggrecan panel. (C) Using urea-soluble fractions from 6-week-old PKO mouse brains (input), aggrecan was immunoprecipitated using aggrecan pAb (IP: aggrecan) or normal rabbit IgG (IP: IgG). The precipitated aggrecan was subjected to SDS-PAGE and western blotting with HNK-1, 6B4, Cat-315 and aggrecan antibodies.</p

LC/MSⁿ structural analysis of HNK-1 epitope on aggrecan.

<p>(A) PA-labeled <i>O</i>-linked glycans were prepared from aggrecan-Fc co-expressed with HNK-1ST in COS-1 cells. The base peak chromatogram of the glycans was obtained using selected ion monitoring (SIM) (<i>m/z</i> 782.5–832.5) in the negative ion mode (<i>upper panel</i>). An extracted ion chromatogram (EIC) of the ion at <i>m/z</i> 807.0–807.4 (<i>lower panel</i>). (B) MS/MS spectra (<i>upper panel</i>) of the ion [M-H]^- (<i>m/z</i> 807.2) detected in peak A and MS/MS/MS spectra (<i>lower panel</i>) of the predominant product ion (<i>m/z</i> 727.2) in the MS/MS. S, sulfate group; HexA, hexuronic acid; Hex, hexose; Xyl-PA, xylose labeled with 2-aminopyridine.</p

HNK-1 carbohydrate and aggrecan are expressed in the PNNs.

<p>Sagittal sections of cerebral cortex from 6-week-old WT (A-C) and PKO mice (D-L) were singly (for WT) or doubly (for PKO) immunostained with HNK-1 mAb (A, D), 6B4 mAb (B, G), or Cat-315 mAb (C, J), and aggrecan pAb (E, H, K). F, I and L are overlaid images. High magnification images of HNK-1-, 6B4- or Cat-315- and aggrecan-positive PNNs are shown in the insets. Scale bars, 200 μm (A-C), 100 μm (D-L) and 20 μm (insets).</p