Collagen-binding matrix proteins from elastomeric extraorganismic byssal fibers
Collagen-binding matrix proteins from elastomeric extraorganismic byssal fibers
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DOI：
10.1021/bm0255903
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发表时间：
2002-11-01
影响因子：
6.2
通讯作者：
Waite, JH
中科院分区：
文献类型：
--
作者：
Sun, CJ;Lucas, JM;Waite, JH
海洋贻贝的深海丝代表了一种特殊的胞外物质。线由纤维状嵌合胶原组成，如嵌入微纤维基质中的preCol-P（具有胶原、弹性蛋白样和富含组氨酸的结构域）。我们在这里报告的提取，纯化和表征的水溶性近端线程基质蛋白1（PTMP 1），这是优先位于每个深海线程的近端部分，并在近端到远端的方向减少。PTMP I具有约50 kDa的质量，如通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱法测定的。甘氨酸是最常见的残基，为12.2 mol %，其次是天冬酰胺/天冬氨酸和谷氨酰胺/谷氨酸，分别为11.4和9.9 mol %。用生物素标记的伴刀豆球蛋白A和中性糖分析通过蛋白质印迹法检测糖基化。根据N端序列设计简并引物，并通过Lys-C内肽酶酶切获得一个内肽，利用PCR技术从紫贻贝足cDNA文库中扩增得到该蛋白的完整cDNA序列。发现两个变异体仅在N-末端有微小的序列差异。cDNA推导的蛋白质序列显示两个对称的内部重复序列，它们共同占蛋白质的85%以上。PTMP 1与血管性血友病因子和整合素α（1）A结构域的序列和表位相似性表明其具有胶原结合能力。基于酶联免疫吸附测定（ELISA）的PTMP 1与固定化I型胶原结合的测量显示出高亲和力（表观K-D = 0.25 μ M），但结合表现出对金属的不依赖性。利用M. edulis中，我们还发现了一个类似PTMP 1的cDNA在一个相关的物种，M。galloprovincialis，与M. edulis变异体，同源性为99%。相应的cDNA序列的同源性分别为94%和96%。
The byssal threads of marine mussels represent a peculiar case of extraorganismic extracellular material. The threads consist of fibrous chimeric collagens such as preCol-P (with collagenous, elastin-like and histidine-rich domains) embedded in a microfibrillar matrix. We report here on the extraction, purification, and characterization of water-soluble proximal thread matrix protein 1 (PTMP1), which is preferentially located in the proximal portion of each byssal thread and decreases in a proximal to distal direction. PTMP I has a mass of about 50 kDa as determined by matrix-assisted laser desorption-ionization with time-of-flight (MALDI-TOF) mass spectrometry. Glycine is the most common residue at 12.2 mol %, followed by asparagine/aspartic acid and glutamine/glutamic acid at 11.4 and 9.9 mol %, respectively. Glycosylation has been detected by Western blotting with biotinylated concanavalin A and neutral sugar analysis. With degenerate primers designed from the N-terminal sequence and an additional internal peptide derived by Lys-C endopeptidase digestion, a complete cDNA sequence for this protein was obtained by polymerase chain reaction (PCR) amplification of a Mytilus edulis foot cDNA library. Two variants with minor sequence differences limited to the N-terminus were found. The cDNA-deduced protein sequence reveals two symmetric internal repeats that together account for >85% of the protein. Sequence and epitope similarity of PTMP1 to the A domains of von Willebrand factor and integrin alpha(1)I suggest a capacity for collagen binding. Enzyme-linked immunosorbent assay (ELISA)-based measurement of PTMP1 binding to immobilized type I collagen shows high affinity (apparent K-D = 0.25 muM), but the binding exhibits no dependence on metals. Using primers designed from M. edulis, we also found a PTMP1-like cDNA in a related species, M. galloprovincialis, with a deduced protein sequence having 97% identity with one M. edulis variant and 99% identity with the other. The corresponding cDNA sequences have 94% and 96% identity, respectively.