Trimeric coiled-coil domain of human pulmonary surfactant protein D enhances zinc-binding ability and biologic activity of soluble TRAIL.
Unlike other TNF ligand family members, the homotrimeric tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contains a buried zinc atom coordinated by three Cys230 residues from each subunit. The bound zinc ion is essential for maintaining the structure stability and bioactivity of TRAIL. To improve characteristics of TRAIL by modification to enhance its zinc-binding ability, we constructed a new variant of TRAIL in which the extracellular region of the ligand was N-terminally fused with a trimeric coiled-coil domain derived from human pulmonary surfactant-associated protein D (ST), and compared its characteristics with that of native TRAIL. Circular dichroism (CD) studies and metal assays showed that two versions of TRAIL folded as predicted into secondary and tertiary structures, and contained stoichiometric Zn(2+) through optimizing bacterial expression and purification. The addition of the human trimeric coiled-coil domain, however, significantly increased the antitumor activity of soluble TRAIL in vitro and in vivo. The accelerated thermal stability studies demonstrated that human serum album (HSA) promoted the aggregation and degradation of native TRAIL, but not ST, and the addition of ZnSO(4) to the solution of native TRAIL with HSA partially inhibited its aggregation, suggesting ST is more difficult to lose its bound zinc ion than native TRAIL. The issue was further confirmed by dialysis assess. This is the first example of modified TRAIL with enhanced zinc-binding ability.
Wu
Xuechang
X
Institution of Microbiology, College of Life Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, PR China. mblab2008@hotmail.com
Li
Ping
P
Qian
Chaodong
C
Li
Ou
O
Zhou
Yinyan
Y
D000818:Animals; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009154:Mutation; D009368:Neoplasm Transplantation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D037663:Pulmonary Surfactant-Associated Protein D / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D012709:Serum Albumin / Q000737:chemistry / Q000494:pharmacology; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014183:Transplantation, Heterologous; D015032:Zinc / Q000378:metabolism*; D019287:Zinc Sulfate / Q000494:pharmacology
D037663:Pulmonary Surfactant-Associated Protein D; D012709:Serum Albumin; D053221:TNF-Related Apoptosis-Inducing Ligand; D019287:Zinc Sulfate; D015032:Zinc; D003545:Cysteine