Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking
Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking
复制标题
DOI：
10.1111/j.1742-4658.2005.04891.x
复制
发表时间：
2005-09-01
期刊：
影响因子：
5.4
通讯作者：
Kawabata, S
中科院分区：
文献类型：
--
作者：
Iijima, M;Hashimoto, T;Kawabata, S
节肢动物表皮细胞作为抵御病原体入侵的第一道屏障发挥着重要作用。我们广泛地确定了鲎表皮蛋白的序列。提取的蛋白质的腹侧的角质层的一部分，通过几丁质亲和层析纯化，并通过二维SDS/PAGE分离。出现在凝胶上的蛋白质被指定为高分子量几丁质结合蛋白，然后根据它们的近似等电点和主要氨基酸组成将这些蛋白质分组。被指定为碱性G、碱性Y和酸性S基团的组的成员包含在节肢动物表皮蛋白中发现的所谓的Rebers和Riddiford共识。命名为酸性DE 25和DE 29的蛋白质各自含有富含Cys的结构域，其序列与昆虫围食基质蛋白和几丁质酶的序列相似。相比之下，基本QH 4和QH 10不包含已知几丁质结合蛋白中发现的共有序列。或者，低分子量几丁质结合级分制备的尺寸排阻色谱法，和15个低分子量几丁质结合蛋白，命名为P1至P15，被分离出来。除P9和P15外，发现它们都与已知的抗微生物肽相同。P9由Kunitz型胰凝乳蛋白酶抑制剂序列组成，P15含有在胰岛素样生长因子结合蛋白中发现的富含Cys的基序。有趣的是，我们观察到几乎所有高分子量几丁质结合蛋白的转氨酶依赖性聚合，这一发现表明，转氨酶依赖性交联在节肢动物角质层中的宿主防御中起着重要作用，类似于在哺乳动物表皮角质层细胞包膜中观察到的。
Arthropod cuticles play an important role as the first barrier against invading pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin-affinity chromatography, and separated by two-dimensional SDS/PAGE. Proteins appearing on the gel were designated high molecular mass chitin-binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. Members of groups designated basic G, basic Y, and acidic S groups contained a so-called Rebers and Riddiford consensus found in arthropod cuticular proteins. Proteins designated acidic DE25 and DE29 each contained a Cys-rich domain with sequences similar to those of insect peritrophic matrix proteins and chitinases. In contrast, basic QH4 and QH10 contained no consensus sequences found in known chitin-binding proteins. Alternatively, a low molecular mass chitin-binding fraction was prepared by size exclusion chromatography, and 15 low molecular mass chitin-binding proteins, named P1 through P15, were isolated. With the exception of P9 and P15, all were found to be identical to known antimicrobial peptides. P9 consisted of a Kunitz-type chymotrypsin inhibitor sequence, and P15 contained a Cys-rich motif found in insulin-like growth factor-binding proteins. Interestingly, we observed transglutaminase-dependent polymerization of nearly all high molecular mass chitin-binding proteins, a finding suggests that transglutaminase-dependent cross-linking plays an important role in host defense in the arthropod cuticle, analogous to that observed in the epidermal cornified cell envelope in mammals.