Ho Samuel B, Luu Ying, Shekels Laurie L, Batra Surinder K, Kandarian Brandon, Evans David B, Zaworski Phillip G, Wolfe Cindy L, Heinrikson Robert L
Department of Medicine, University of California-San Diego, San Diego, CA, USA.
Biochim Biophys Acta. 2010 Jul;1800(7):629-38. doi: 10.1016/j.bbagen.2010.03.010. Epub 2010 Mar 20.
The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and have cytoprotective properties. Their extracellular regions contain two EGF-like Cys-rich domains (CRD1 and CRD2) connected by an intervening linker segment with SEA module (L), and may function to stimulate intestinal cell restitution. The purpose of this study was to determine the effect of size, recombinant host source, and external tags on mucin CRD1-L-CRD2 protein activity.
Four recombinant Muc3-CRD proteins and three MUC17-CRD proteins were generated using Escherichiacoli or baculovirus-insect cell systems and tested in colonic cell cultures for activity related to cell migration and apoptosis.
N-terminal glutathione-S-transferase (GST) or C-terminal His(8) tags had no effect on either the cell migration or anti-apoptosis activity of Muc3-CRD1-L-CRD2. His-tagged Muc3-CRD1-L-CRD2 proteins with truncated linker regions, or the linker region alone, did not demonstrate biologic activity. The human recombinant MUC17-CRD1-L-CRD2-His(8) was shown to have anti-apoptotic and pro-migratory activity, but did not stimulate cell proliferation. This protein showed similar in vitro biologic activity, whether produced in E. coli or a baculovirus-insect cell system.
Recombinant mucin proteins containing a bivalent display of Cys-rich domains accelerate colon cell migration and inhibit apoptosis, require a full-length intervening Linker-SEA segment for optimal biologic activity, and are functional when synthesized in either E. coli and insect cell systems.
These results indicate that an Escherichiacoli-derived full-length His(8)-tagged human MUC17 CRD1-L-CRD2 recombinant protein is a biologically active candidate for further development as a therapeutic agent.
膜结合黏蛋白MUC17（人类）和Muc3（小鼠）在肠上皮细胞的顶端表面高度表达，并具有细胞保护特性。它们的细胞外区域包含两个由带有SEA模块（L）的中间连接段相连的表皮生长因子样富含半胱氨酸结构域（CRD1和CRD2），可能具有刺激肠细胞修复的功能。本研究的目的是确定大小、重组宿主来源和外部标签对黏蛋白CRD1-L-CRD2蛋白活性的影响。
使用大肠杆菌或杆状病毒-昆虫细胞系统生成了四种重组Muc3-CRD蛋白和三种MUC17-CRD蛋白，并在结肠细胞培养物中测试其与细胞迁移和凋亡相关的活性。
N端谷胱甘肽-S-转移酶（GST）或C端His(8)标签对Muc3-CRD1-L-CRD2的细胞迁移或抗凋亡活性均无影响。带有截短连接区域的His标签的Muc3-CRD1-L-CRD2蛋白或单独的连接区域均未表现出生物学活性。人重组MUC17-CRD1-L-CRD2-His(8)具有抗凋亡和促迁移活性，但不刺激细胞增殖。无论在大肠杆菌还是杆状病毒-昆虫细胞系统中产生，该蛋白都显示出相似的体外生物学活性。
含有二价富半胱氨酸结构域展示的重组黏蛋白可加速结肠细胞迁移并抑制凋亡，需要全长的中间连接-SEA段以实现最佳生物学活性，并且在大肠杆菌和昆虫细胞系统中合成时均具有功能。
这些结果表明，源自大肠杆菌的全长His(8)标签的人MUC17 CRD1-L-CRD2重组蛋白是作为治疗剂进一步开发的具有生物学活性的候选物。
