Wu Y, Adam R, Williams S A, Bianco A E
Liverpool School of Tropical Medicine, UK.
Mol Biochem Parasitol. 1996 Jan;75(2):207-19. doi: 10.1016/0166-6851(95)02529-4.
State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.
通过对蜱中丝状线虫棘唇旋尾丝虫感染性幼虫进行代谢标记，鉴定出了220 kDa和75 kDa的种特异性产物。合成对温度敏感，在27℃时发生，而在37℃时不发生。这些产物在离开媒介后的感染后发育过程中3 - 6天分泌，但随后未检测到后续表达。通过二维电泳纯化了pI为6.2的较小蛋白质，并推导了其N端氨基酸序列。这初步确定该蛋白质为几丁质酶，通过糖基几丁质底物凝胶电泳进行生化确认。聚合酶链反应用于从棘唇旋尾丝虫感染性幼虫的cDNA文库中扩增产物。核苷酸序列编码一个20个氨基酸的推定信号肽和一个504个残基的成熟蛋白（Mr 56 kDa），与马来布鲁线虫微丝蚴中描述的MF1几丁质酶具有69%的同一性（考虑保守取代时为81%的相似性）。N - 连接糖基化可能解释了预测的多肽与天然寄生虫产物（75 kDa）之间Mr差异的部分或全部原因。基于棘唇旋尾丝虫序列的引物用于从盘尾丝虫感染性幼虫的cDNA文库中扩增相关序列。盘尾丝虫的cDNA编码一个20个氨基酸的信号肽，随后是477个残基，Mr为54 kDa，与棘唇旋尾丝虫几丁质酶具有67%的同一性（考虑保守取代时为80%的相似性），与马来布鲁线虫MF1分子具有69%的同一性。有人提出，这些丝状线虫感染阶段表达的几丁质酶可能在感染后发育过程中的蜕皮中起作用。
