Akita H, Fukae M, Shimoda S, Aoba T
Forsyth Dental Center, Boston, MA 02115.
Arch Oral Biol. 1992 Nov;37(11):953-62. doi: 10.1016/0003-9969(92)90067-i.
The present study was undertaken to investigate glycosylation of porcine enamel proteins secreted in the secretory stage of amelogenesis and to gain insight into functional roles of glycosylated proteins in enamel mineralization. Enamel proteins, isolated from various zones of the secretory enamel, were separated by SDS-PAGE and then transferred on to a nitrocellulose membrane. The transblotted proteins were visualized with either antibodies against porcine amelogenins or various biotin-conjugated lectins. The lectins used were Con-A, GS-II, STA, WGA, s-WGA, GS-I, MPA, VVA, PNA, RCA-I, DBA, SJA, UEA-I, Lotus-A and LPA. The results of the immuno- and lectin blottings revealed that most of the lectins did not bind to porcine amelogenins, while a large number of non-amelogenins having various molecular masses were stained strongly with the conjugated WGA, Con A and MPA lectins. On the basis of the binding specificity with the lectins, porcine non-amelogenins were classified into two groups: WGA (and Con A)-binding moieties at 60-90 kDa (WGA-HMW); and MPA-binding moieties at 13-17 kDa (MPA-LMW). These two groups of non-amelogenins differed distinctly in terms of their localization and stability in the secretory tissue and their adsorption properties onto hydroxyapatite. The WGA-HMW were concentrated in the outer region adjacent to the ameloblasts and disappeared (due to degradation) in the underlying inner secretory enamel. In contrast, the MPA-LMW were found in all zones of the secretory enamel and their quantity remained relatively constant. Histochemical studies using FITC-conjugated WGA and MPA showed that the fluorescence-labelling of WGA was localized in the core region of prism rods, while the fluorescence-labelling of MPA was locally limited at the rim of prism rods or at the prism sheath. In separate adsorption studies, it was found that the WGA-HMW, as well as the intact amelogenins, displayed a high adsorption affinity on to apatite crystals, whereas the MPA-LMW showed only marginal adsorption on to apatitic surfaces. The overall results indicate that part of the heterogeneity found in porcine enamel proteins can be ascribed to variations of carbohydrate moieties attached to non-amelogenins.(ABSTRACT TRUNCATED AT 400 WORDS)
本研究旨在调查在釉质形成分泌期分泌的猪釉质蛋白的糖基化情况，并深入了解糖基化蛋白在釉质矿化中的功能作用。从分泌期釉质的不同区域分离出的釉质蛋白，通过SDS-PAGE进行分离，然后转移到硝酸纤维素膜上。用抗猪釉原蛋白的抗体或各种生物素偶联凝集素对转印后的蛋白进行可视化分析。所使用的凝集素有伴刀豆球蛋白A（Con-A）、大豆凝集素（GS-II）、荆豆凝集素（STA）、小麦胚凝集素（WGA）、琥珀酰化小麦胚凝集素（s-WGA）、麦芽凝集素（GS-I）、曼陀罗凝集素（MPA）、荆豆凝集素（VVA）、花生凝集素（PNA）、蓖麻凝集素I（RCA-I）、双花扁豆凝集素（DBA）、雪花莲凝集素（SJA）、欧洲荆豆凝集素（UEA-I）、百脉根凝集素（Lotus-A）和扁豆凝集素（LPA）。免疫印迹和凝集素印迹结果显示，大多数凝集素不与猪釉原蛋白结合，而大量具有不同分子量的非釉原蛋白被偶联的WGA、Con A和MPA凝集素强烈染色。根据与凝集素的结合特异性，猪非釉原蛋白分为两组：60 - 90 kDa的WGA（和Con A）结合部分（WGA-高分子量部分）；以及13 - 17 kDa的MPA结合部分（MPA-低分子量部分）。这两组非釉原蛋白在分泌组织中的定位和稳定性以及它们在羟基磷灰石上的吸附特性方面有明显差异。WGA-高分子量部分集中在成釉细胞相邻的外层区域，并在下方的内层分泌釉质中消失（由于降解）。相反，MPA-低分子量部分在分泌期釉质的所有区域都有发现，其数量相对保持恒定。使用异硫氰酸荧光素（FITC）偶联的WGA和MPA进行的组织化学研究表明，WGA的荧光标记位于棱柱体的核心区域，而MPA的荧光标记局部局限于棱柱体边缘或棱柱鞘处。在单独的吸附研究中发现，WGA-高分子量部分以及完整的釉原蛋白对磷灰石晶体表现出高吸附亲和力，而MPA-低分子量部分在磷灰石表面仅表现出微弱吸附。总体结果表明，猪釉质蛋白中发现的部分异质性可归因于附着在非釉原蛋白上的碳水化合物部分的变化。（摘要截选至400字）
