The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation

Abstract

The cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that inhibits proliferation and enhances differentiation of human embryonal carcinoma cells. CREG binds to the cation-independent mannose 6-phosphate (M6P)/insulin-like growth factor II (IGF2) receptor (IGF2R) (M6P/IGF2R), and this receptor has been shown to be required for CREG-induced growth suppression. To better understand CREG function in cellular growth and differentiation, we solved the 3D crystal structure of this protein to 1.9-Å resolution. CREG forms a tight homodimeric complex, and CREG monomers display a β-barrel fold. The three potential glycosylation sites on CREG map to a confined patch opposite the dimer interface. Thus, dimerization of glycosylated CREG likely presents a bivalent ligand for the M6P/IGF2R. Closely related structural homologs of CREG are FMN-binding split-barrel fold proteins that bind flavin mononucleotide. Our structure shows that the putative flavin mononucleotide-binding pocket in CREG is sterically blocked by a loop and several key bulky residues. A mutant of CREG lacking a part of this loop maintained overall structure and dimerization, as well as M6P/IGF2R binding, but lost the growth suppression activity of WT CREG. Thus, analysis of a structure-based mutant of CREG revealed that binding to M6P/IGF2R, while necessary, is not sufficient for CREG-induced growth suppression. These findings indicate that CREG utilizes a known fold.

CREG1 enhances p16INK4a-induced cellular senescence

“…20 CREG1 functions as transcriptional repressor of E2F transcriptional activation capable of antagonizing the ability of E1A and ras to transform primary cells. 20,21 CREG1 enhances differentiation of embryonal carcinoma cells and its expression increases in differentiated cells. 22 Overexpression of CREG1 in human teratocarcinoma cells inhibits cell cycle progression and cell proliferation.…”

Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

“…Creg2 encodes for a secreted glycoprotein that enhances the differentiation of pluripotent stem cells. 38 One insertion was mapped in exon 3 of the Fmnl3 gene (formin-like 3) that encodes for a protein involved in the Rho-signaling pathway. 39 Additional sequences were located 9567 bp upstream of BC043301, encoding for a nucleid acid and metal ion binding protein, and 25 085 bp downstream of 2610301B20Rik.…”

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli

“…Eight-stranded antiparallel b-barrel is a very common protein fold. The stability of this structure is provided by a tightly packed hydrophobic core and extensive hydrogen bond network linking all b-strands (Cantini et al 2006;Sacher et al 2005).…”