Hoffmann Anita, Funkner Andreas, Neumann Piotr, Juhnke Susanne, Walther Matthias, Schierhorn Angelika, Weininger Ulrich, Balbach Jochen, Reuter Gunter, Stubbs Milton T
Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle/Saale, Germany.
J Biol Chem. 2008 Nov 21;283(47):32598-609. doi: 10.1074/jbc.M801815200. Epub 2008 Sep 12.
The Drosophila Spätzle protein, involved in the embryonic development of the dorsal-ventral axis and in the adult immune response, is expressed as a proprotein and is activated by the serine proteinases Easter or Spätzle-processing enzyme. Proteolytic cleavage generates a 106-amino acid COOH-terminal fragment, C106, homologous to the mature form of nerve growth factor NGF, a cystine knot protein. Through alternative splicing, the Spätzle gene encodes for several isoforms that (with one exception, the "propeptide isoform") share C106 but differ in the prosequence. Three isoforms have been expressed recombinantly in Escherichia coli strains. The propeptide isoform could be expressed in soluble form and is unstructured according to CD and NMR measurements. Dimeric full-length Spätzle isoforms have been refolded from insoluble inclusion bodies and are able to rescue Spätzle-deficient embryos. Although the two full-length isoforms exhibit similar far-UV CD spectra, large differences in tryptophan fluorescence quenching by the respective pro-parts are observed. Both full-length isoforms exhibited highly cooperative folding transitions. Proteolytic digestion using trypsin resulted in C106, whose unfolding exhibits lower thermodynamic stability and cooperativity compared with the full-length proteins. The structure of C106 reveals a T-shaped dimer with significant differences to NGF and a deep internal cavity. Substantial beta-sheet formation is observed between the two monomers, whereas a long loop containing the single tryptophan residue is disordered in the crystals. Our results suggest that the propeptides stabilize the tertiary structure of the "mature" Spätzle cystine knot.
果蝇的斯佩兹尔蛋白参与背腹轴的胚胎发育和成虫免疫反应，它以前体蛋白形式表达，并被丝氨酸蛋白酶伊斯特或斯佩兹尔加工酶激活。蛋白水解切割产生一个106个氨基酸的羧基末端片段C106，它与神经生长因子NGF的成熟形式同源，NGF是一种胱氨酸结蛋白。通过可变剪接，斯佩兹尔基因编码几种异构体，这些异构体（有一种异构体除外，即“前肽异构体”）共享C106，但前序列不同。三种异构体已在大肠杆菌菌株中重组表达。前肽异构体可以以可溶形式表达，根据圆二色性（CD）和核磁共振（NMR）测量，它是无结构的。二聚体全长斯佩兹尔异构体已从不溶性包涵体中复性，并能够拯救斯佩兹尔缺陷型胚胎。尽管两种全长异构体表现出相似的远紫外CD光谱，但观察到各自前体部分对色氨酸荧光猝灭的差异很大。两种全长异构体都表现出高度协同的折叠转变。用胰蛋白酶进行蛋白水解消化产生C106，与全长蛋白相比，C106的去折叠表现出较低的热力学稳定性和协同性。C106的结构揭示了一个T形二聚体，与NGF有显著差异，且有一个深的内部腔。在两个单体之间观察到大量β-折叠的形成，而包含单个色氨酸残基的长环在晶体中是无序的。我们的结果表明，前肽稳定了“成熟”斯佩兹尔胱氨酸结的三级结构。
