The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Abstract

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 A resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded 8-sheets on the inside and six a-helices on the outside. There is a 5-A-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor.Each subunit has a new class of a + 13 sandwich structure consisting of two P-a-P3 motifs. These P-a-38 motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.Glycosylation-inhibiting factor (GIF), is a 13-kDa cytokine involved in the formation of IgE-suppressive factor (1). GIF inhibits N-glycosylation of IgE-binding factors, and the unglycosylated IgE-binding factor then selectively suppresses IgE synthesis. Furthermore, GIF facilitates generation of antigenspecific suppressor T cells (2), and appears to be a subunit of antigen-specific suppressor T-cell factors (1). Indeed, the major cell source of GIF activity is antigen-specific suppressor T cells (3). After molecular cloning of GIF, however, it was found that various cell line cells contained 0.6-kb mRNA, which hybridized with GIF cDNA (4), and secreted the 13-kDa protein that reacted with polyclonal antibodies against recombinant GIF (5). Nevertheless, only the peptide from suppressor T cell had GIF bioactivity. More recent experiments provided evidence that the bioactive GIF and inactive GIF have an identical amino acid sequence and suggested that the bioactivity of GIF is generated by posttranslational modifications of the inactive protein (5, 6). Since neither N-glycosylation nor phosphorylation is involved in the generation of bioactive GIF, we speculated that the heterogeneity of GIF is a conformational transition of the protein (5). The present experiments were undertaken to determine the three-dimensional structure of recombinant human GIF in order to get an insight into the structural basis of its bioactivity. It was found that the crystal structure of GIF is a novel structure with a trimeric barrel with three 6-stranded P-sheets. ¶ MATERIALS AND METHODSCrystallization of GIF. The recombinant human GIF was expressed in Escherichia coli and was purified as described (6 Table 2.

Insight into the biology of Macrophage Migration Inhibitory Factor (MIF) revealed by the cloning of its cell surface receptor

“…It wasn't until 1989 that a discrete gene sequence was determined to be associated with MIF [4] and in 1993, pure recombinant protein became available for biologic and structure-function studies [5]. The threedimensional crystal structure of MIF was solved by three laboratories in 1996, thus revealing a new protein fold and structural superfamily with MIF as its defining member [6][7][8]. These studies also provided insights into the likely native form of the protein -a homotrimer with a MW of 37.5 kDa (Figure 1).…”

Structure activity studies of the cytokine macrophage migration inhibitory factor (MIF) reveal a critical role for its car☐y terminus

“…These studies showed that MIF is an important cytokine that is critically involved in the regulation of the host immune and stress responses. With respect to the molecular properties, the recent elucidation of the three-dimensional structure of MIF [12,[14][15][16][17] has added further to our understanding of the role of MIF. However, because a receptor for MIF has not yet been identified and because the identity of a catalytic center that is responsible for the enzymatic activity of MIF is unclear, the molecular mechanism of MIF action continues to be unknown.…”

“…In particular, it is not clear if the C-terminus is composed of a 13-strand configuration, or if it is structurally undefined. For example, no structure was found for the Cterminus of rat MIF [12], whereas for human MIF, none, one, or two short C-terminal [3-strand elements have been proposed [14][15][16][17] (Fig. 1B).…”

“…The three-dimensional structure of MIF was elucidated recently by X-ray crystallography and NMR methods and MIF was found to be a trimer of identical subunits [12,[14][15][16][17]. Each monomer contains two antiparallel c~-helices that pack against a four-stranded [3-sheet.…”

GIF Inhibits Th Effector Generation by Acting on Antigen-Presenting B Cells

“…The molecular structure of this cytokine was elucidated (6,7). It was recently found that T cell-secreted GIF that can inhibit IgE Ab formation is cysteinylated at Cys-60 (8,9).…”