title: The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers.
abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood-brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.
authors.affiliation: Bio-engineering Institute of Jinan University, Jinan University, Guangzhou, Guangdong, PR China. rongjie_yu1123@163.com
authors.forename: Rongjie
authors.initials: R
authors.lastname: Yu
authors.forename: Zhixing
authors.initials: Z
authors.lastname: Zeng
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Guo
authors.forename: Huahua
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Ding
authors.forename: Jiansu
authors.initials: J
authors.lastname: Chen
mesh_terms: D000284:Administration, Oral; D000818:Animals; D015824:Blood-Air Barrier / Q000502:physiology*; D001812:Blood-Brain Barrier / Q000378:metabolism*; D001814:Blood-Testis Barrier / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015696:Gene Products, tat / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D006801:Humans; D007274:Injections, Intraperitoneal; D008168:Lung / Q000378:metabolism; D008297:Male; D051379:Mice; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics; D013737:Testis / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D015696:Gene Products, tat; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011993:Recombinant Fusion Proteins