Recombinant production and structural studies of the human Lypd6 and Lypd6b proteins

Autor: Alexander S. Paramonov, Ekaterina N. Lyukmanova, E. V. Loktyushov, D. A. Dolgikh, Andrey V Tsarev, D.S. Kulbatskii, Mikhail P. Kirpichnikov, Zakhar O. Shenkarev
Rok vydání: 2017
Předmět:
Zdroj: Russian Journal of Bioorganic Chemistry. 43:644-652
ISSN: 1608-330X
1068-1620
DOI: 10.1134/s1068162017060127
Popis: Lypd6 and Lypd6b are human three-finger proteins expressed in various tissues and sharing a high degree of sequence homology (~54%). Unlike other members of the Ly6/uPAR family, both proteins additionally bear extended N- and С-terminal sequences flanking the LU-domain. The role of these sequences is unclear. It is known that Lypd6 can increase the amplitude of nicotine-induced calcium currents in mouse trigeminal ganglion neurons. The Danio rerio fish Lypd6 is involved in the regulation of the Wnt/β-cathenin signaling pathway and the knockdown of the lypd6 expression leads to the impairment of embryonic development. The Lypd6b expression in X. laevis oocytes increased the sensitivity of nicotinic acetylcholine receptors to acetylcholine and increased their desensitization rate. Molecular mechanisms of Lypd6 and Lypd6b action as well as their spatial structures remain unknown. In this work, the genes encoding water-soluble variants of human Lypd6 and Lypd6b lacking N-terminal sequences (rLypd6 and rLypd6b) and Lypd6 bearing the N-terminal sequence (N-rLypd6) were obtained and expressed. The proteins were expressed in cytoplasmic inclusion bodies followed by solubilization under denaturing conditions and renaturation. The renaturation conditions were screened to optimize recombinant protein yields. The analysis of NMR spectra of recombinant N-rLypd6, rLypd6, and rLypd6b demonstrated that N-rLypd6 may not have a structured form. The production of milligram quantities of isotope-labeled rLypd6 and rLypd6b analogues allowed characterization of the secondary structure of these proteins and study of their intramolecular dynamics. It was found that rLypd6 and rLypd6b have structural elements typical for the Ly6/uPAR family, although with some unique features, particularly, an additional disulfide bond in the third loop and helical regions in the first and third loops.
Databáze: OpenAIRE
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