Fig 1.
Generation of GlcAT-S-deficient mice.
(A) Schematic diagram of the targeting construct. For targeted disruption of the mouse GlcAT-S gene (B3gat2), exon1 containing the initiation codon and transmembrane region were replaced with a neo/lacZ cassette. Hatched lines represent the fragments used for the construction of the targeting vector. PCR primers used for screening are shown in arrowheads. Black bars indicate probes for Southern blot analysis. (B) Southern blot analysis of targeted ES cell clones. Genomic DNA isolated from ES cell clones were digested with PstI or SpeI and EcoRV, blotted and then hybridized with the 3’ or 5’ probe, respectively. The expected sizes of the WT allele and targeted allele (KO) are indicated. The ES clones, 6, 29, 37, 62, 116, 127, 203 and 258 were positive when screening using PCR. ES clone 115 was used as a negative control. (C) Western blot analysis of SKO mice. The membrane proteins were prepared from 6-week-old mouse kidneys (WT; +/+, heterozygote; +/-, homozygote; -/-). A non-sulfated form of HNK-1 epitope was detected with M6749 mAb.
Fig 2.
HNK-1 carbohydrate epitope expressed in GlcAT-P-deficient mice.
(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.
Fig 3.
HNK-1 carbohydrate and aggrecan are expressed in the PNNs.
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).
Fig 4.
HNK-1 carbohydrate expressed in PKO and DKO mice.
(A) Soluble fractions from 6-week-old GlcAT-P-deficient mice (PKO) and GlcAT-P and -S double deficient mice (DKO) were treated with chABC and PNGase F, subjected to SDS-PAGE and blotted using HNK-1 mAb, 6B4 mAb, Cat-315 mAb, aggrecan pAb, phosphacan pAb, H28 (NCAM mAb) or actin mAb. The effect of PNGase F treatment was confirmed by western blotting with H28 mAb. Actin was used as the loading control. (B) Using soluble fractions from 6-week-old DKO mouse brains (input), aggrecan and phosphacan were immunoprecipitated with aggrecan pAb (IP: aggrecan), normal rabbit IgG (IP: IgG) or phosphacan pAb (IP: phosphacan). The precipitated aggrecan and phosphacan were subjected to SDS-PAGE and western blotting with HNK-1, aggrecan and phosphacan antibodies. (C-N) Sagittal sections of cerebral cortex from 6-week-old PKO (C-E) and DKO mice (F-N) were double-immunostained with HNK-1 mAb, 6B4 mAb or Cat-315 mAb (red) and WFA lectin (green). Scale bar, 100 μm.
Fig 5.
Expression of a novel HNK-1 epitope on aggrecan in COS-1 cells.
(A) Schematic diagram of the Fc-tagged short aggrecan used in this study. (B) Aggrecan-Fc with or without HNK-1ST was heterologously expressed into COS-1 cells. Aggrecan-Fc in the culture medium was purified using a Protein G Sepharose column and digested with 100 μU/ml chABC and then blotted with anti-Fc pAb, HNK-1 mAb and anti-CS antibodies (CS56 and clone IIH6). (C) Aggrecan-Fc and/or phosphacan-myc-Fc were transiently expressed with HNK-1ST-EGFP into COS-1 cells. Aggrecan-Fc and phosphacan-myc-Fc in the culture medium were precipitated with ethanol and digested with 200 μU/ml chABC, subjected to SDS-PAGE and then blotted with anti-Fc pAb, HNK-1 mAb, aggrecan pAb and phosphacan pAb.
Table 1.
Quantification of chondroitin sulfate on the purified recombinant aggrecan-Fc.
Fig 6.
LC/MSn structural analysis of HNK-1 epitope on aggrecan.
(A) PA-labeled O-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) (m/z 782.5–832.5) in the negative ion mode (upper panel). An extracted ion chromatogram (EIC) of the ion at m/z 807.0–807.4 (lower panel). (B) MS/MS spectra (upper panel) of the ion [M-H]- (m/z 807.2) detected in peak A and MS/MS/MS spectra (lower panel) of the predominant product ion (m/z 727.2) in the MS/MS. S, sulfate group; HexA, hexuronic acid; Hex, hexose; Xyl-PA, xylose labeled with 2-aminopyridine.
Fig 7.
Biosynthetic model for HNK-1 epitope on aggrecan in PNNs.
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.