Alonso Edurne, Gómez Laura, Madrid Juan Francisco, Sáez Francisco José
Departamento de Biología Celular e Histología, Facultad de Ciencia y Tecnología, Universidad del País Vasco/Euskal Herriko Unibertsitatea, B Sarriena s/n, E 48940 Leioa (Vizcaya), Spain.
Microsc Res Tech. 2006 Jul;69(7):595-9. doi: 10.1002/jemt.20318.
The presence of mannose (Man) in the glycoconjugates of primordial germ cells (PGCs) of Xenopus embryos was elucidated by lectin histochemistry with Concanavalin A (Con A) and snowdrop (Galanthus nivalis) bulb lectin (GNA), in combination with deglycosylative pretreatments: beta-elimination, which removes O-linked oligosaccharides, and incubation with Peptide N glycosidase F (PNGase F), which removes N-linked glycan chains. In addition, histochemistry with Con A, which binds to Man and glucose (Glc), was also performed after glucose-oxidase incubation, which converts Glc into gluconic acid, and GNA was carried out after acid hydrolysis, which removes terminal sialic acid (NeuAc) moieties. PGCs were analyzed during their migration over the mesentery until the genital ridge, and after colonization of this gonad anlage. The results showed that for both lectins: (1) the PGCs and other surrounding tissue showed a similar binding pattern, and (2) the staining in the PGCs was similar in the developmental stages studied. Labeling with Con A was due to Man, and not to Glc, as shown after incubation with glucose-oxidase, and it was assumed that Man was in N-linked oligosaccharides. However, GNA labeling was mainly due to O-linked oligosaccharides, because the pretreatment of beta-elimination turned cells negative. Moreover, acid hydrolysis pretreatment gave rise to a stronger GNA-staining, suggesting that either Man was also in subterminal position to NeuAc or some Man-containing glycans were unmasked after removal of NeuAc from other oligosaccharide chains.
通过伴刀豆球蛋白A（Con A）和雪花莲（Galanthus nivalis）球茎凝集素（GNA）的凝集素组织化学，结合去糖基预处理（β-消除，去除O-连接寡糖；与肽N-糖苷酶F（PNGase F）孵育，去除N-连接聚糖链），阐明了非洲爪蟾胚胎原始生殖细胞（PGCs）糖缀合物中甘露糖（Man）的存在情况。此外，在葡萄糖氧化酶孵育（将葡萄糖转化为葡萄糖酸）后，进行了与结合Man和葡萄糖（Glc）的Con A的组织化学实验，在酸水解（去除末端唾液酸（NeuAc）部分）后进行了GNA实验。对PGCs在其穿过肠系膜迁移至生殖嵴的过程中以及在该性腺原基定植后进行了分析。结果表明，对于这两种凝集素：（1）PGCs和其他周围组织呈现相似的结合模式，（2）在所研究的发育阶段，PGCs中的染色相似。如葡萄糖氧化酶孵育后所示，Con A标记是由于Man而非Glc，并且推测Man存在于N-连接寡糖中。然而，GNA标记主要归因于O-连接寡糖，因为β-消除预处理使细胞呈阴性。此外，酸水解预处理导致更强的GNA染色，这表明要么Man也处于NeuAc的次末端位置，要么在从其他寡糖链去除NeuAc后，一些含Man的聚糖被暴露出来。
