Lectin labelling of glycans and glycosyl residues in extracellular matrices. The example of the Crustacean exoskeleton
- 作者：Compè ; re ; Philippe
- 刊名：Biology of the Cell
- 出版年：1998
- 期刊代码：11_02484900
- 类别：imm
- 出版时间：June, 1998
- 卷：90
- 期：3
- 页码：289
- 文件大小：140 K
摘要
In mineralised extracellular matrices such as many invertebrate exoskeletons, the location of carbohydrates by use of lectins raises some problems due to their involvement in structural macromolecules and their possible loss, alteration or masking during tissue processing (fixation, decalcification and resin embedding). The distribution of glycans and glycosyl residues was investigated in the calcified cuticle of the Atlantic shore crab Carcinus maenas L. (Crustacca, Decapoda). The labelling is based on the binding of biotinylated lectins that are secondarily recognised by a gold(10 nm)-albumin-streptavidin complex. It is visualised either in transmission electron microscopy or in light microscopy after silver enhancement (Compère, Ph. (1996) Chitin in life Sciences, J. André Publ., 66-87). The results allowed to analyse different factors influencing the lectin labelling of carbohydrates involved in the molecular architecture of the cuticle layers, i.e. the thin outermost epicuticle and the procuticle that mainly consists of a chitin-protein fibrous network impregnated by CaCO3. 1) The accessibility of sugar residues for lectin labelling seems to depend on the chemical form of the target carbohydrate and its association with other compounds. In the procuticle, the sugar oligomers (a-Man, Gal(b-1,3)GalNAc) of interfibrillar glycoproteins are freely recognised by Con-A and Jac on frozen sections as well as on resin sections. In contrast, the glycosyl residues (a-Man, GalNAc, Gal(b-1,3)GalNAc, NcuNAc) enclosed in the compact proteinaceous matrix of the inner epicuticle bind lectins (Con A, SBA, Jac, MAA II and SNA) only when exposed at the surface of resin sections. A similar effect was also observed in the case of long polymer glycans such as the chitin crystallites forming the core of the procuticular chitin-protein microfibres (Compère, op. cit.). 2) The nature of the embedding resin has a major influence and must be accorded to the kind of target residues to be recognised. Indeed, Con A and Jac intensely bind one of the procuticle layers on acrylic resin sections but not on epoxide sections. However, acrylic resins, in contrast to epoxide resins, does not allow a good recognition of some residues such as sialic acids by MAA II and SNA. 3) The fixation is an important prerequisite for retention and preservation of glycosaminoglycans. For this purpose, additives were tested in the fixative and decalcification media. The results show that cationic dyes, especially Ruthenium red, enhances the labelling by Con A, Jac, MAA II and SNA.