Megumi Yokoyama

Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance.

Background: Type I collagen is the most abundant organic component in bone, providing form and stability. Results: Lysyl hydroxylase 3-mediated glucosylation occurs at specific sites in collagen, including cross-linking sites, and suppression of this modification results in defective collagen and mineralization. Conclusion: The data indicate the critical importance of this modification in bone physiology. Significance: Alterations of this collagen modification may cause bone defects. Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.

Lysyl Hydroxylase 3 Glucosylates Galactosylhydroxylysine Residues in Type I Collagen in Osteoblast Culture

Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.

Sialolipoma of the palate: a rare case and review of the literature

Sialolipoma is a new variant of salivary gland lipoma, which was first described in 2001. We report a rare case of sialolipoma of the palate, and review another 10 cases affecting the minor salivary gland and 13 affecting the major salivary gland, together with details of the clinical and histopathological findings.

Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation

The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

Secretory proteins without a transport signal are retained in secretory granules during maturation in rat parotid acinar cells

OBJECTIVE The acinar cells of the parotid gland are filled with numerous secretory granules (SGs), which accumulate the digestion enzyme amylase. SGs mature accompanied with membrane remodelling such as fusion and budding of small vesicles. However, little is understood about the mechanism of the condensation of SG contents during maturation. In this study, we examined whether secretory proteins need a specific signal to be retained in SGs. DESIGN To induce internalization of the luminal membrane after exocytosis, we injected the β-adrenergic agonist isoproterenol into rats. Acinar cells were then incubated with Lucifer Yellow (LY) dye as a tracer for 3h for uptake into immature secretory granules (ISGs). To observe whether LY was retained in SGs after maturation, we continued incubating the cultured acinar cells for 2 days. RESULTS The localization of LY into ISGs was confirmed by the following four methods: (1) co-localization of the fluorescence of LY and amylase by confocal laser microscopy, (2) detection of the fluorescence from purified ISGs, (3) secretion of the fluorescence together with amylase upon stimulation, and (4) observation of the intracellular localization of LY by electron microscopy. Moreover, we observed co-localization of some of the SGs with the fluorescence of LY after cell culture. CONCLUSIONS Although the fusion and budding of small vesicles may contribute to the process of granule maturation, LY remained in the SGs even after maturation. These results suggest that secretory proteins that have no transport signal are not excluded from SGs, and they are retained in SGs during granule maturation in exocrine parotid glands.

Maintenance of claudin-3 expression and the barrier functions of intercellular junctions in parotid acinar cells via the inhibition of Src signaling

OBJECTIVES Salivary acinar and duct cells show different expression patterns of claudins, which may reflect their different functions. To study the role of claudins in saliva secretion, we examined alterations in the expression patterns of cell adhesion molecules in parotid glands of γ-irradiated rats and analyzed the influence of those changes on intercellular barrier function using primary cultures of parotid acinar cells. DESIGN Rats were γ-irradiated with doses of 5, 15 or 20Gy, and expression levels of cell adhesion molecules were examined by immunoblotting analysis. Acinar cells were isolated from parotid glands and were cultured in the absence or presence of the Src kinase inhibitor PP1. Changes in protein and mRNA expression patterns were determined by immunoblotting and by RT-PCR analyses, respectively. Intercellular barrier function was examined by measuring transepithelial electrical resistance and the paracellular flux of FITC-dextran. RESULTS In irradiated parotid glands, the expression of claudin-4 was enhanced at 15Gy or higher, levels that induce the hyposecretion of saliva, although that increase was transient. At 30days after irradiation, expression levels of cell adhesion molecules were decreased. In primary cultures, the expression of claudin-4 was also increased transiently but the expression of claudin-3 and E-cadherin was decreased. The barrier function of tight junctions was disrupted although the localization of occludin was maintained. The Src kinase inhibitor PP1 suppressed those changes in gene expression and retained the intercellular barrier function. CONCLUSIONS These results suggest that the inhibition of Src signaling maintains the barrier functions of intercellular junctions in salivary glands, which can be lost due to tissue injury.