Articular cartilage corefucosylation regulates tissue resilience in osteoarthritis
- Department of Orthopaedic Surgery, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Japan
- Institute for Glyco‑core Research (iGCORE), Nagoya University, Japan
- Center for Sports Medicine, Hokkaido University Hospital, Japan
Figures
Figure 1 with 1 supplement
Effects of α-mannosidase injection in the knee joint in vivo.
(A) Time course of intra-articular α-mannosidase injection and unloading. Red arrows indicate the one-shot, intra-articular injection that was performed once a week. (B) Histological evaluation of …
Figure 1—figure supplement 1
Enzymatic activity under physiologic pH conditions in joint injection and cartilage culture systems.
The activity of mannosidase under saline (A) and DMEM (B) conditions at 37 °C (n=4). Data are shown as mean ± standard deviation. **p<0.01. the Welch t-test was used for statistical analysis.
Figure 2 with 2 supplements
Cartilage degradation induced by mannosidase stimulation.
(A) Schematic diagram of the procedures used to establish the early OA model before irreversible and progressive destruction of articular cartilage. (B) Mannosidase-treated cartilage stained with …
Figure 2—figure supplement 1
Histological effects of mannosidase on cartilage culture systems.
Mannosidase-treated cartilage was stained with HE, and immunohistochemical staining for Col2, Col10, and TUNEL. Overall, Col2 staining decreased in the IL-1β-treated cartilage. The superficial layer …
Figure 2—figure supplement 2
Impact of other glycoside hydrolases on NO production from cartilage.
NO release in articular cartilage explants. Explants were cultured under normal culture conditions (control) and under the effect of mannosidase (1.9 U/mL) and sialidase (1.9 U/mL). NO release was …
Figure 3 with 1 supplement
Cartilage N-glycomes.
(A) MALDI-TOF MS spectra showing the quantitative N-glycan profiles of cartilage before (upper) and after (lower) mannosidase treatment. (B) Scatter plot of the changes in m/z per protein abundance …
Figure 3—figure supplement 1
Glycoform present in the N-glycans of chondrocytes: core fucose.
N-glycan profiling of chondrocytes isolated from mouse knee cartilage. The released N-glycans using PNGase F are captured and labeled with aoWR using BlotGlyco beads (Sumitomo Bakelite). MALDI-TOF …
Figure 4 with 1 supplement
Loss of resilience due to FUT8 deficiency in cartilage.
(A) Biological reaction of FUT8. FUT8 transfers fucose to the innermost GlcNAc residue of complex N-glycans via α1,6-linkage (corefucosylation). (B) Targeted disruption of Fut8 locus. The Fut8 gene …
Figure 4—figure supplement 1
The proportion of high-mannose type N-glycans in the cartilage of Fut8 cKO mice.
Quantified high-mannose type N-glycans (pmol/cartilage) are stratified into HM5 [(Hex)2 + (Man)3(GlcNAc)2], HM6 [(Hex)3 + (Man)3(GlcNAc)2], HM7 [(Hex)4 + (Man)3(GlcNAc)2], HM8 [(Hex)5 + …
Figure 5 with 1 supplement
OA acceleration in Fut8 cKO mice.
(A) Double staining with alizarin red and alcian blue of the whole skeleton of wild-type and Col2a1-Cre;Fut8flox/flox cKO littermate embryos (newborn). Scale bars, 1 cm (left). Weight and body …
Figure 5—figure supplement 1
Expanded zone of hypertrophic chondrocytes in Fut8 cKO mice.
Quantification of hypertrophic zones of tibia from 4-week-old wild-type (100.1±23.4 μm, n=10) and Col2a1-Cre; Fut8flox/flox (146.1±17.9 μm, n=10) mice. The length of the hypertrophic zone at the …
Figure 6
Altered glycosylation of human Osteoarthritis (OA) cartilage based on comprehensive glycan analysis.
(A) Total glycome profiling of human OA cartilage. Pie charts at the vertices of the pentagon correspond to the glycan expression profiles of N-glycans, O-glycans, GSL-glycans, free oligosaccharides …
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Figure 6—source data 1
List of glycans quantified in healthy and osteoarthritic human cartilage.
- https://cdn.elifesciences.org/articles/92275/elife-92275-fig6-data1-v1.zip
Figure 7
Graphical abstract.
The corefucosylation of N-glycans is associated with the reduction of high-mannose type N-glycans in articular cartilage functions to maintain the formed vitreous cartilage as cartilage. Failure of …
Tables
Table 1
N-glycans affected by α-mannosidase.
Nineteen of 78 glycans underwent significant changes on either day 3 or 6.
| Average | SD | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| day 3 | day 6 | day 3 | day 6 | p-value | ||||||
| man- | man+ | man- | man+ | man- | man+ | man- | man+ | day 3 | day 6 | |
| (Hex)2 (HexNAc)2 | 0.792357 | 0.338717 | 1.698703 | 0.456702 | 0.13679 | 0.050814 | 0.535795 | 0.06191 | 0.005751 | 0.016286 |
| (Hex)2 (HexNAc)2 (Fuc)1 | 3.234707 | 0.961793 | 8.220451 | 0.924848 | 0.674962 | 0.171418 | 2.152247 | 0.065893 | 0.004824 | 0.00421 |
| (Hex)3 (HexNAc)2 (Fuc)1 | 1.490549 | 0.803672 | 2.709981 | 1.15013 | 0.296407 | 0.119276 | 0.737097 | 0.183631 | 0.020411 | 0.023657 |
| (Hex)4 (HexNAc)2 | 0.835177 | 1.59262 | 1.387137 | 1.927679 | 0.149341 | 0.226128 | 0.39749 | 0.261157 | 0.008393 | 0.120372 |
| (Hex)4 (HexNAc)2 (Fuc)1 | 0.376984 | 0.283877 | 0.54256 | 0.352081 | 0.044946 | 0.018371 | 0.106303 | 0.060982 | 0.029345 | 0.054541 |
| (Hex)2 + (Man)3(GlcNAc)2 | 13.90417 | 17.95947 | 15.15816 | 23.33117 | 1.234223 | 1.006214 | 1.724106 | 1.244995 | 0.011592 | 0.002645 |
| (HexNAc)1 (Fuc)1 + (Man)3(GlcNAc)2 | 1.301597 | 1.570904 | 0.815818 | 2.076143 | 0.207081 | 0.202172 | 0.050657 | 0.208993 | 0.182306 | 0.00053 |
| (Hex)2 (Fuc)1 + (Man)3(GlcNAc)2 | 0.322479 | 0.213651 | 0.423122 | 0.268242 | 0.046893 | 0.010074 | 0.112891 | 0.049903 | 0.017103 | 0.095448 |
| (Hex)3 + (Man)3(GlcNAc)2 | 4.861187 | 5.775936 | 6.654761 | 5.079474 | 0.305603 | 0.3547 | 1.417819 | 1.179548 | 0.027682 | 0.213128 |
| (Hex)1 (HexNAc)1 (Fuc)1 + (Man)3(GlcNAc)2 | 0.750266 | 0.722315 | 0.514881 | 0.608877 | 0.102088 | 0.016991 | 0.017088 | 0.036114 | 0.664285 | 0.015161 |
| (HexNAc)2 (Fuc)1 + (Man)3(GlcNAc)2 | 1.652909 | 3.456976 | 1.353847 | 3.715004 | 0.132951 | 0.654484 | 0.010019 | 0.352926 | 0.009456 | 0.000317 |
| (Hex)1 (HexNAc)2 + (Man)3(GlcNAc)2 | 0.876123 | 0.772922 | 0.646969 | 0.817259 | 0.129494 | 0.084843 | 0.088617 | 0.055944 | 0.312527 | 0.048099 |
| (Hex)4 + (Man)3(GlcNAc)2 | 2.450873 | 2.895117 | 3.097092 | 2.245556 | 0.253136 | 0.10024 | 0.766259 | 0.409485 | 0.04753 | 0.164815 |
| (Hex)2 (HexNAc)1 (Fuc)1 + (Man)3(GlcNAc)2 | 0.819582 | 0.867903 | 0.64262 | 0.728035 | 0.073132 | 0.069334 | 0.04725 | 0.006566 | 0.452941 | 0.036177 |
| (HexNAc)3 (Fuc)1 + (Man)3(GlcNAc)2 | 2.046827 | 2.744773 | 1.385735 | 2.486179 | 0.256854 | 0.248038 | 0.122548 | 0.205141 | 0.027643 | 0.001339 |
| (Hex)5 + (Man)3(GlcNAc)2 | 3.384827 | 2.579308 | 4.207472 | 2.518858 | 0.330947 | 0.236353 | 1.087182 | 0.392432 | 0.026518 | 0.064636 |
| (HexNAc)4 + (Man)3(GlcNAc)2 | 0.498715 | 0.340168 | 0.283597 | 0.26723 | 0.062833 | 0.027843 | 0.245731 | 0.233142 | 0.016187 | 0.937326 |
| (Hex)6 + (Man)3(GlcNAc)2 | 6.567266 | 2.574261 | 7.830155 | 3.542284 | 0.705065 | 0.227602 | 0.927784 | 0.289577 | 0.000733 | 0.001576 |
| (Hex)2 (HexNAc)1 (Fuc)1 (NeuAc)1 + (Man)3(GlcNAc)2 | 0.635073 | 0.537187 | 0.515992 | 0.507301 | 0.037168 | 0.044506 | 0.061995 | 0.051956 | 0.043073 | 0.861418 |
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(m/z protein).
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SD; standard deviation, man-; control, man+; cartilage stimulated by α-mannosidase.
Table 2
List of the top 10 deviations from the y=x line.
The values are expressed as relative values of the amount of substance (pmol) calculated based on internal standards to the total amount of N-glycan.
| Glycan structure | Control | Mannosidase | Dissociation |
|---|---|---|---|
| (Hex)2 + (Man)3(GlcNAc)2 | 0.13433877 | 0.17518289 | 0.04084413 |
| (HexNAc)2 (Fuc)1 + (Man)3(GlcNAc)2 | 0.01345174 | 0.03953942 | 0.02608767 |
| (Hex)5 + (Man)3(GlcNAc)2 | 0.02873745 | 0.01473236 | 0.01400509 |
| (Hex)6 + (Man)3(GlcNAc)2 | 0.03127904 | 0.01857732 | 0.01270172 |
| (HexNAc)3 (Fuc)1 + (Man)3(GlcNAc)2 | 0.01922672 | 0.03108068 | 0.01185396 |
| (Hex)2 (HexNAc)2 (Fuc)1 | 0.01876579 | 0.00792915 | 0.01083663 |
| (Hex)3 + (Man)3(GlcNAc)2 | 0.03565567 | 0.02670789 | 0.00894778 |
| (HexNAc)1 (Fuc)1 + (Man)3(GlcNAc)2 | 0.01320084 | 0.01968082 | 0.00647997 |
| (HexNAc)4 (Fuc)2 + (Man)3(GlcNAc)2 | 0.05688225 | 0.05042987 | 0.00645238 |
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p mol relative (%).
Additional files
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Supplementary file 1
A primer list of qRT-PCR.
- https://cdn.elifesciences.org/articles/92275/elife-92275-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92275/elife-92275-mdarchecklist1-v1.docx
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Source data 1
Raw data for all figures.
- https://cdn.elifesciences.org/articles/92275/elife-92275-data1-v1.xlsx
