Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.
Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.
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DOI：
10.1073/pnas.90.15.7144
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发表时间：
1993-08
影响因子：
11.1
通讯作者：
M. Verma;E. Davidson
中科院分区：
文献类型：
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作者：
M. Verma;E. Davidson
迄今为止，只有一个哺乳动物粘蛋白的cDNA，MUC 1，已被报道的完整序列，虽然一些粘蛋白的蛋白质已被部分表征。在这里，我们报告了犬气管粘蛋白cDNA的核苷酸序列，含有两个潜在的翻译起始密码子，一个翻译终止密码子和一个poly（A）尾。从犬气管上皮细胞制备的λ gt 11 cDNA文库中筛选与多克隆抗脱辅基-犬气管粘蛋白抗体的目的是获得推导的粘蛋白核心蛋白的氨基酸序列。纯化含有各种长度的重叠插入片段的抗体阳性克隆并用于核苷酸测序。根据测序数据，合成寡核苷酸引物，并确定cDNA的两端（5'和3'）。完整序列为3.7 kb，包括一个具有1118个氨基酸编码能力的开放阅读框、两个翻译起始ATG密码子（与Kozak共有序列相关）、一个聚腺苷酸化位点和一个聚腺苷酸片段。该蛋白质富含苏氨酸、脯氨酸、丝氨酸、甘氨酸和丙氨酸，而缺乏酪氨酸、苯丙氨酸和色氨酸。虽然推导的犬气管粘蛋白序列中不存在氨基酸的串联重复序列，但TPTPTP和TTTTPV基序分别出现了13次和19次。C-末端区域含有富含Cys的结构域（尽管在蛋白质的中间也存在一些Cys残基），如已经报道的牛颌下粘蛋白、猪颌下粘蛋白、大鼠肠粘蛋白、人肠粘蛋白和青蛙皮肤粘蛋白。这表明，一个广泛的组粘蛋白含有这样一个富含Cys的结构域，其功能意义尚待了解。犬气管粘蛋白中存在三个潜在的N-糖基化位点，其氨基酸序列与人气管和肠粘蛋白具有同源性。当通过威斯康星州大学遗传学计算机组程序包分析以确定预测的二级结构时，N-末端结构域显示出更大的灵活性（可能是由于该区域中Pro残基的数量高）。使用犬粘蛋白cDNA作为探针的成绩单的评价表明与总RNA的多分散性的消息。
To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.