Chondroitin sulfate N-acetylgalactosaminyltransferase-1 is required for normal cartilage development

Abstract

CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.

Figure 1. CSGalNAcT1-null mice did not express the CSGalNAcT1 enzyme

(A) Construct of the targeting vector for producing the CSGalNAcT1-null mice. The numbers represent the exon number (exon 1 is defined as the first exon of this gene where the transcription starts). RV, EcoRV site; S, ScaI site. (B) Immunoblot analysis using an anti-CSGalNAcT1 antibody to probe head homogenate from wild-type mice (lane 2), heterozygous (CSGalNAcT1^+/−) mice (lane 3) and null mice (CSGalNAcT1^−/−) at E18.5. Lane 1 shows the negative control (wild-type homogenate after absorption by the recombinant CSGalNAcT1 protein). Note that no reactivity was evident in the homogenate from null mice (lane 4). (C) RT–PCR analysis of genes encoding several enzymes involved in CS synthesis. The expression level is shown as a ratio of each gene to G3pdh for normalization. Fam20B is an enzyme that phosphorylates xylose [23]. Note that no other specific enzymes showed elevated expression to compensate for the loss of CSGalNAcT1 protein.

Figure 2. CS production in cartilage is reduced in CSGalNAcT1-null mice

(A) The total amount and disaccharide analysis of the cartilage in wild-type (Wt), heterozygous (+/−; CSGalNAcT1^+/−) and null (−/−; CSGalNAcT1^−/−) mice. Student's t test was performed to compare the disaccharide amount derived from CS in the null mice with that in the wild-type or the heterozygous mice. Results are shown as mean±S.E.M. *P<0.05; **P<0.01 (n=6). Δdi-0S, ΔHexAα1-3GalNAc; Δdi-4S, ΔHexAα1-3GalNAc(4S); Δdi-6S, HexAα1-3GalNAc(6S). (B) Toluidine Blue staining of the epiphyseal cartilage from E18.5 wild-type (Wt; left) and CSGalNAcT1-null (−/−) fetuses. Metachromasia of Toluidine Blue (purple colour) can be seen in the wild-type cartilage, whereas no metachromasia is discernible in the null mice cartilage. Note the reduced size of the epiphyseal cartilage in the null mice, compared with their wild-type counterpart (bidirectional arrows, upper panels). The extracellular spaces in the cartilage of null mice have many spicules (arrows, lower panels), whereas the intercellular regions from wild-type mice do not. pro, proliferative zone; hyp, hypertrophic zone. Scale bars: in upper panels, 200 μm; in lower panels, 50 μm. (C) Gel-filtration analysis of the length of CS sugar chains in the E18.5 cartilage in wild-type (●), heterozygous (CSGalNAcT1^+/−; ○) and null (CSGalNAcT1^−/−; ▲) mice. There are no significant differences in the total amount of CS loaded on to the gels among groups. Note that the second peak between fraction numbers 30 and 35 is present both in wild-type and heterozygous mice, but not in CSGalNAcT1-null mice, indicating that the size of the GAG chains of CS changed in CSGalNAcT1-null mice. Arrowheads indicate the size of the molecular-mass-marker standards [mean molecular masses (K, kDa): 200, 65.5, 37.5 and 18.1 respectively; all from Sigma]. The calibration of the Superdex 200 column was performed using a series of size-defined commercial dextran polysaccharides. The results shown represent one of three series of independent experiments, where the three series of experiments gave essentially identical results.

Figure 3. CSGalNAcT1-null mice have reduced skeletal growth

(A and B) The body weight (A) and the body length (B) in the wild-type (●) and CSGalNAcT1-null mice (○) during postnatal development. At 4 weeks after birth, the body mass and the body length of the null mice were slightly reduced compared with those of the wild-type.*P<0.05 (Student's t test). (C) The fetal body (top panel) and skeleton (bottom panel) of wild-type (Wt), heterozygous (+/−) and null (−/−) mice. (D) Various bone segments of the fetal mice. Note that only forelimb bones of the null mice are shorter than those of wild-type mice. (E) Measurements of femur length. The femurs at E18.5 of the null mice are significantly shorter than those of wild-type and heterozygous mice (Student's t test; P<0.00001). The number of the femurs measured for each genotype is shown in parentheses. The results are shown as means±S.E.M.

Figure 4. The thickness of the growth plate is reduced in CSGalNAcT1-null mice

(A) Histological views of the epiphyses of E18.5 wild-type (Wt) and CSGalNAcT1-null (−/−) mice. Upper panels: hind limbs (f, femur; t, tibia), middle panels: the femurs of these littermates; lower panels: higher magnification views of the middle panels focusing on the epiphyseal cartilage. Consistent with the decreased size of femurs and tibiae of the null mice, the femoral epiphyseal cartilage (epi) was reduced in size in null mice compared with that of their wild-type counterparts. Scale bar: upper panels, 1 mm; middle panels, 500 μm; lower panels, 300 μm. (B) Histological findings on the growth plates of 4-week-old wild-type (Wt) and null (−/−) mice. Upper and lower panels show lower and higher magnification respectively. The longitudinal length of the growth plates (GP) was shortened in the null mice (bidirectional arrows, lower panels). Scale bars: upper panels, 800 μm; lower panels, 200 μm. (C and D) Immunodetection of type-II (II; upper panels) and type-X (X; lower panels) collagen in the epiphyses of the wild-type (Wt) and null (−/−) E18.5 fetuses (C) and 4-week-old mice (D). In (C), the type-II collagen-positive area (brown) was seen throughout the epiphyses of both Wt and null mice, despite the finding that cartilage area of the null mice was reduced (upper panels). Even in (D), the reduced size of the type-II collagen reactive-growth plate in the null mice was observed. Judging from the hypertrophic zone marker type-X collagen immunostaining, cartilage from E18.5 (C) and four-week-old (D) animals had a reduced hypertrophic zone. Scale bars: (C), 300 μm; (D), 100 μm. (E) Quantitative measurement of the epiphyseal cartilage in area. Both E18.5 and 4-week-old (4w) femurs were collected. (Mann–Whitney U test; P<0.05). The results are shown as means±S.E.M.

Figure 5. Abnormal ultrastructure of the type-II collagen is frequently observed in cartilage and chondrocytes of CSGalNAcT1-null mice

(A, C and E) Wild-type (Wt) mice; (B, D and F) CSGalNAcT1-null (−/−) mice; at E18.5. Micrographs (A) and (B) were obtained from the resting zone, and (C) and (D) from the proliferative zones. Note that many electron-dense extracellular fibrils (arrows) are seen connecting chondrocytes in the resting (B) and proliferative (D) zones of the epiphysis from null mice, whereas only a few such fibrils are observed in both zones (A; resting zone, and C; proliferative zone) of wild-type mice. At a higher magnification, the wild-type cartilage matrix had collagen fibrils that spread radially (E), whereas cartilage of the null mice had a fine meshwork of twisted cartilaginous fibrils (F). Ch, chondrocytes. Scale bars: (A)–(D), 10 μm; (E)–(F), 1 μm.