Mapping the Protein Domain Structures of the Respiratory Mucins: A Mucin Proteome Coverage Study
Mapping the Protein Domain Structures of the Respiratory Mucins: A Mucin Proteome Coverage Study
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DOI：
10.1021/pr300058z
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Kesimer, Mehmet
中科院分区：
文献类型：
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作者：
Cao, Rui;Wang, T. Tiffany;Kesimer, Mehmet
粘蛋白基因编码了人类基因组中表达量最大的蛋白家族。这些蛋白质被o -连接的低聚糖高度取代，极大地限制了进入肽骨架的途径。大多数粘蛋白的n端、o糖基化和c端区域的基因组组织已经确定，并可在序列数据库中获得。然而，对于它们暴露的蛋白质区域在翻译和分泌后的命运知之甚少，迄今为止，与基因组研究相辅相成的详细蛋白质组学研究相当有限。使用从培养的人气道上皮细胞分泌物中分离的粘蛋白，胰蛋白酶消化和质谱，我们研究了负责气道上皮维持和保护的粘蛋白的蛋白质组覆盖范围。除去严重糖基化的粘蛋白结构域，凝胶形成的粘蛋白MUC5B和MUC5AC的N端区域覆盖率高达85%，c端区域覆盖率高达60%，这表明在c端有更多的N-和稀疏的o -糖基化区域以及可能的其他修饰。所有可能的肽从富含半胱氨酸的区域中断重糖基化粘蛋白结构域被确定。有趣的是，从MUC5B和MUC5AC的10个不同结构域中鉴定出43个切割位点，其中在肽的n端具有非色氨酸切割位点，表明可能暴露于蛋白水解和/或“自发切割”。其中一些非色氨酸裂解可能对分泌前后分子的适当成熟很重要。从MUC16中鉴定出的大部分肽来自SEA区域。令人惊讶的是，三个肽被清楚地从其重度糖基化区域识别出来。MUC4覆盖范围达到25%，覆盖了该分子的7个不同结构域。MUC1细胞质结构域的所有肽以及该区域的三个非色氨酸切割都被检测到。仅从MUC20中鉴定出一个肽，这使我们成功地构建了针对该分子的抗血清。综上所述，本报告代表了我们目前在剖析粘蛋白大分子复杂性方面的努力。鉴定可进行蛋白水解的区域有助于设计有效的抗体，并指出可能用于粘蛋白相互作用的区域，鉴定裂解位点将有助于了解其在正常和疾病环境下的分泌前和分泌后过程。
Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.