CLONING AND CDNA SEQUENCE OF A BOVINE SUBMAXILLARY-GLAND MUCIN-LIKE PROTEIN CONTAINING 2 DISTINCT DOMAINS
CLONING AND CDNA SEQUENCE OF A BOVINE SUBMAXILLARY-GLAND MUCIN-LIKE PROTEIN CONTAINING 2 DISTINCT DOMAINS
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DOI：
10.1073/pnas.87.17.6798
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发表时间：
1990-09-01
影响因子：
11.1
通讯作者：
BHAVANANDAN, VP
中科院分区：
文献类型：
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作者：
BHARGAVA, AK;WOITACH, JT;BHAVANANDAN, VP
用抗脱细胞牛颌下腺粘蛋白的多克隆抗体筛选了牛颌下腺mRNA的lambda.gt11cDNA文库，获得了该粘蛋白核心蛋白的氨基酸序列。其中一个阳性克隆的插入片段为1.8kb，编码一个不完整的蛋白质。用1.8kb的cDNA对文库进行再筛选，获得了2.0kb的克隆。对全长2.0kb的cDNA进行核苷酸序列分析，发现有一个开放阅读框，编码一个563个氨基酸的蛋白质。克隆蛋白质的一个显著特征是氨基酸分布不对称，最显著的是羟基氨基酸和半胱氨酸。339个氨基酸残基的氨基末端结构域富含苏氨酸、丝氨酸和甘氨酸，而缺乏半胱氨酸、天冬氨酸、酪氨酸、苯丙氨酸和色氨酸。相比之下，224个残基的羧基末端结构域富含半胱氨酸、天冬氨酸、酪氨酸、赖氨酸和天冬氨酸，而苏氨酸、丝氨酸和甘氨酸相对较少。在蛋白质数据库中搜索与推导出的氨基酸序列的同源性，发现与几种蛋白质有统计学意义的匹配，包括猪的颌下无粘蛋白片段。富含半胱氨酸的结构域本身与任何注册的多肽序列在统计上都不是同源的。使用与粘蛋白样区和半胱氨酸富集区相对应的DNA探针进行RNA印迹分析，检测到几乎相同的转录本模式，表明特征克隆不是构建cDNA文库的产物。杂交结果还显示牛颌下腺中存在多分散的转录本，但在肝脏或脑RNA中没有检测到杂交信号。
A .lambda.gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.