Kneipp L F, Andrade A F, de Souza W, Angluster J, Alviano C S, Travassos L R
Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil.
Exp Parasitol. 1998 Jun;89(2):195-204. doi: 10.1006/expr.1998.4290.
The expression of chitin as a structural component of Trichomonas vaginalis and Tritrichomonas foetus was demonstrated by using enzymatic hydrolysis by recombinant (rec-) chitinase, chemical analysis, lectin, fluorescent Calcofluor and antibody binding, glycosidases of known specificity, high-performance liquid chromatography (HPLC), and flow cytometry. Chitinous structures were characterized by their insolubility in hot alkali and by releasing glucosamine on hydrolysis with 6 N HCl. N,N'-Diacetylchitobiose and N,N,'N''-triacetylchitotriose were identified by HPLC as enzymatic hydrolysis products of the alkali-resistant polysaccharide. The location of chitin on the surface of T. vaginalis and T. foetus was inferred from the decreased reactivity with whole parasites of ligands such as Lycopersicon esculentum (TOL) and Solanum tuberosum lectins, fluorescent Calcofluor, and anti-chitin antibody, after cell treatment with rec-chitinase. Binding of [125I]TOL showed that, in T. vaginalis and T. foetus, the numbers of lectin receptors per cell were 4.2 x 10(5) and 3.0 x 10(5), respectively. Binding of the lectin to the trichomonad surface was markedly decreased by treatment with rec-chitinase. TOL interaction with the parasites was not affected by N-acetyl-beta-D-glucosaminidase treatment, showing that the lectin receptors consisted of beta-linked GlcNAc polymers and not of terminal beta-linked GlcNAc residues.
通过使用重组几丁质酶进行酶促水解、化学分析、凝集素、荧光钙黄素和抗体结合、已知特异性的糖苷酶、高效液相色谱（HPLC）和流式细胞术，证实了几丁质作为阴道毛滴虫和胎儿三毛滴虫结构成分的表达。几丁质结构的特征在于它们在热碱中不溶，以及在用6 N HCl水解时释放出氨基葡萄糖。通过HPLC鉴定出N，N'-二乙酰壳二糖和N，N'，N''-三乙酰壳三糖是耐碱多糖的酶促水解产物。在用重组几丁质酶处理细胞后，根据与配体如番茄凝集素（TOL）和马铃薯凝集素、荧光钙黄素和抗几丁质抗体的全寄生虫反应性降低，推断出几丁质在阴道毛滴虫和胎儿三毛滴虫表面的位置。[125I]TOL的结合表明，在阴道毛滴虫和胎儿三毛滴虫中，每个细胞的凝集素受体数量分别为4.2×10（5）和3.0×10（5）。用重组几丁质酶处理后，凝集素与滴虫表面的结合明显减少。TOL与寄生虫的相互作用不受N-乙酰-β-D-氨基葡萄糖苷酶处理的影响，表明凝集素受体由β连接的GlcNAc聚合物组成，而不是由末端β连接的GlcNAc残基组成。
