Role of Glycosyltransferase 25 Domain 1 in Type I Collagen Glycosylation and Molecular Phenotypes

Glycosylation in type I collagen occurs as O-linked galactosyl- (G-) lesser and glucosylgalactosyl-hydroxylysine (GG-Hyl); however, its biological significance is still not well understood. To investigate the function of this modification in bone, we have generated preosteoblast MC3T3-E1 (MC)-derived clones, short hairpin (Sh) clones, in which Glt25d1 gene expression was stably suppressed. In Sh clones, the GLT25D1 protein levels were markedly diminished in comparison to controls (MC and those transfected with the empty vector). In Sh collagen, levels of both G- and GG-Hyl were significantly diminished with a concomitant increase in the level of free-Hyl. In addition, the level of immature divalent cross-links significantly diminished while the level of the mature trivalent cross-link increased. As determined by mass spectrometric analysis, seven glycosylation sites were identified in type I collagen and the most predominant site was at the helical cross-linking site, α1–87. At all of the glycosylation sites, the relative levels of G- and GG-Hyl were markedly diminished, i.e., by ∼50–75%, in Sh collagen, and at five of these sites, the level of Lys hydroxylation was significantly increased. The collagen fibrils in Sh clones were larger, and mineralization was impaired. These results indicate that GLT25D1 catalyzes galactosylation of Hyl throughout the type I collagen molecule and that this modification may regulate maturation of collagen cross-linking, fibrillogenesis, and mineralization

Effect of low cross-linked matrices on osteoblast and osteoclast.

(a) The ablation of cellular components by DOC was confirmed by DAPI staining. Bar: 50 μm. (b)The Lox activity of prepared matrices before and after the DOC treatment was analyzed. The Lox activity was significantly decreased in the matrices produced by BAPN-treated cells. DOC treatment did not significantly affect the Lox activity of prepared matrices, regardless of the BAPN concentration. (c) The proliferation of MC cells was significantly increased in the low cross-link density matrices. (d) Alkaline phosphatase (ALP) activity increased in cells seeded on low cross-linked matrices. (e) Gene expression of Cbfa1/Runx2, type I collagen α2 chain (Col1a2), alkaline phosphatase (Alpl), and osteocalcin (Spp1) significantly increased in these matrices at 3 and 7 days of culture. (f) Osteoclasts were cultured on differentially cross-linked matrices for 6 days under the differentiation condition. The number of multi-nuclear tartrate-resistant acid phosphatase (TRAP)-positive cells increased in the low cross-link density matrices. (g) Gene expression of cathepsin K (Ctsk), nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1), and dendritic cell-specific transmembrane protein (DCstamp) increased in low cross-link density matrices. *p < 0.05, compared to the control.</p

Morphometric analysis of femur.

Morphometric analysis of femur.</p

Histological and histomorphometric analysis.

(a) Hematoxylin and eosin, and tartrate-resistant acid phosphatase (TRAP)-stained histological sections of the distal femur epiphysis at 0 week (after 8-week of the BAPN consumption). (b) No differences of osteoblast and osteoclasts activities were observed at 0 and 2 weeks after replacing to the control diet. After 4 weeks, number of osteoblasts per bone surface (N.Ob/BS) and osteoblast surface per bone surface (Ob.S/BS), representing osteoblast activity, significantly increased, while number of osteoclasts per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS), representing osteoclast activity did not change. *p < 0.05, compared to the control. Bar: 50 μm. (c) Picrosirius red-stained samples at 16 weeks of age (after 8-week of the BAPN consumption followed by 4-week of control diet) are analyzed under polarized light. After 4-week of control diet, immature/irregular collagen matrix still detected both in cortical and cancellous bone. Bar: 50 μm. Quantitative data also confirmed that the immature collagens, detected in green, retained high value after 4-week of control diet in cancellous bone.</p

Effect of β-aminopropionitrile (BAPN) on matrix formation and collagen components produced by MC3T3-E1 cells.

(a) BAPN treatment did not affect the proliferation of MC cells, as demonstrated by MTS assay. (b) Expression of core-binding factor alpha 1/runt-related transcription factor 2 (Runx2/Cbfa1) and type I collagen α2 chain (Col1a2) did not change, while the levels of lysyl oxidase (Lox) were significantly elevated after BAPN treatment in a dose-dependent manner. (c) Collagen components were analyzed by electrophoresis. In the control, all chains (α, β, and γ) were clearly observable. However, BAPN treatment led to the inhibition of β- and γ-chain formation. Picrosirius Red staining was visualized under bright-field (d) and polarized light (e). Collagen quantity was not affected by BAPN, while the alignment of collagenous fibers and matrix maturation were impaired. Bar: 100 μm. (f) Collagen content was slightly increased following 0.5 mM BAPN treatment, but it decreased after the administration of 2 mM BAPN. Hyl/Hyp × 300 decreased after the treatment with 2 mM BAPN. Divalent DHLNL and HLNL levels decreased with 0.5 mM BAPN treatment and were not detectable following the treatment with 1.0 and 2.0 mM BAPN. Trivalent pyridinoline levels significantly decreased after the application of 0.5 mM BAPN, while they were undetectable following the treatment with 1.0 and 2.0 mM BAPN. *p < 0.05, compared to the control.</p

Effect of low cross-linked matrices on osteoblastic differentiation of BMSCs.

(a) BMSCs to the prepared matrices was shown. Cell nuclei were stained using DAPI (blue), and the cytoskeleton was stained with phalloidin (red). Initial adhesion of cells cultured on low cross-link density matrix increased. (b) Proliferation of BMSCs was significantly elevated in low cross-linked matrices. (c) ALP activity of BMSCs increased in low cross-link density matrices. (d) Gene expression of Col1a2, Alpl, and Spp1 was significantly increased, while that of Cbfa1/Runx2 did not change in low cross-link density matrices after 1 and 2 weeks. *p < 0.05, compared to the control. Bars: 20 μm.</p

Effects of β-aminopropionitrile (BAPN)-containing diet on body weight, bone volume, and collagen cross-linking in mice.

(a) The body weight of mice increased steadily throughout the experimental period, and no differences were observed between groups. (b) Histology of Picrosirius Red-stained samples at 12 weeks of age (after 8-week of the BAPN consumption) is shown. Under polarized light, positive pixel ratio of immature matrix (green) increased with BAPN administration, while that of mature matrix (red) did not change Bar: 200 μm. (c) Collagen Cross-links were analyzed at 12 weeks of age (after 8-week of the BAPN consumption). Collagen content and hydroxylysine (Hyl)/hydroxyproline (Hyp) × 300 were not affected by BAPN, but dehydrodihydroxylysinonorleucine (DHLNL) and dehydrohydroxylysinonorleucine (HLNL) levels decreased in a dose-dependent manner. Pyridinoline, deoxypyridinoline, and total aldehyde contents were significantly reduced in mice fed with BAPN-containing diet. *p < 0.05, compared to the control.</p

Typical chromatographic patterns of collagen cross-links in fractions purified by molecular sieve chromatography.

Molecular sieve elution profile of the acid hydrolysates of NaB3H4-reduced skin collagen. A, in WT, two peaks of radioactive content (R1 and R2) were collected. B, in KO, two peaks of radioactive content (R3 and R4) were collected. C and D, molecular sieve purifications of peaks R1 and R2 in WT, resulting in typical chromatographic patterns (C) and (D), respectively. E and F, molecular sieve purifications of peaks R3 and R4 in KO, resulting in typical chromatographic patterns (E) and (F), respectively. These chromatographic patterns were obtained by cross-link analysis of fractions purified by molecular sieve chromatography. HLNL, hydroxylysinonorleucine; HHMD, histidinohydroxymerodesmosine.</p

Summary of site-specific modification analysis by mass spectrometry of non-cross-linked, hydroxylated, and glycosylated residues in mice skin type I collagen.

Summary of site-specific modification analysis by mass spectrometry of non-cross-linked, hydroxylated, and glycosylated residues in mice skin type I collagen.</p

Summary of nanoindentation modulus (KPa) by atomic force microscopy (AFM)-based nanoindentation in mice skin.

Summary of nanoindentation modulus (KPa) by atomic force microscopy (AFM)-based nanoindentation in mice skin.</p