Structure dissection of human progranulin identifies well‐folded granulin/epithelin modules with unique functional activities

Abstract

Progranulin is a secreted protein with important functions in several physiological and pathological processes, such as embryonic development, host defense, and wound repair. Autosomal dominant mutations in the progranulin gene cause frontotemporal dementia, while overexpression of progranulin promotes the invasive progression of a range of tumors, including those of the breast and the brain. Structurally, progranulin consists of seven-and-a-half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides. We have expressed all seven human GEMs using recombinant DNA in Escherichia coli. High-resolution NMR showed that only the three GEMs, hGrnA, hGrnC, and hGrnF, contain relatively well-defined three-dimensional structures in solution, while others are mainly mixtures of poorly structured disulfide isomers. The three-dimensional structures of hGrnA, hGrnC, and hGrnF contain a stable stack of two b-hairpins in their N-terminal subdomains, but showed a more flexible C-terminal subdomain. Interestingly, of the well-structured GEMs, hGrnA demonstrated potent growth inhibition of a breast cancer cell line, while hGrnF was stimulatory. Poorly folded peptides were either weakly inhibitory or without activity. The functionally active and structurally well-characterized human hGrnA offers a unique opportunity for detailed structure-function studies of these important GEM proteins as novel members of mammalian growth factors.Keywords: granulin/epithelin module; progranulin; NMR; structure; functional activity Abbreviations: PGRN, progranulin, also called the granulin/epithelin precursor or GEP; GEM, granulin/epithelin-like module; hGrnX, human granulin X, where X is A, B, C, D, E, F, G, or P; cGrn1, carp granulin-1; S2H, stack of two b-hairpins; S4H, stack of four b-hairpins; CDAP, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate; TCEP, tris(2-carboxyethyl)phosphine; itz, 2-iminothiazolidine-4-carboxyl, resulting from the aqueous ammonia cleavage of a peptide on the Nterminal side of the S-cyanylated cysteines; hGrnA-itz-a-b-red, a fully reduced fragment of hGrnA obtained after CN-induced cleavage before cysteines in positions a and (b + 1); hGrnA-(-2)-a-red, a fully reduced N-terminal fragment of hGrnA obtained after CN-induced cleavage before cysteine in position (a + 1); m/z, mass to charge ratio; TFA, trifluoroacetic acid.Article and publication are at

The granulin gene family: from cancer to dementia

“…Two-dimensional nuclear magnetic resonance analysis of purified carp GRN-1 revealed that the peptide backbone adopts a unique conformation of a parallel stack of beta-hairpins in the form of a left-handed helix. (3) Recombinant mammalian GRN modules show a similar, but less rigid structure (4) (Fig. 1B).…”

“…B: The three-dimensional structure of human GRN-A determined by twodimensional nuclear magnetic resonance. (4) The two images are identical but turned approximately 908 in the vertical plane. The disulfide bridges (yellow) form an axial ''rod'' through the center of the molecule.…”

Serum Progranulin Concentrations May Be Associated With Macrophage Infiltration Into Omental Adipose Tissue

“…Progranulin is a secreted protein with important functions in several processes, including immune response and embryonic development (6). Progranulin, also known as granulin, acrogranin, proepithelin, and PC cell-derived growth factor, is a 593-amino acid glycoprotein, the mRNA of which is expressed in many epithelial cells both in vitro and in vivo.…”

Cloning, expression and cytotoxicity of granulin A, a novel polypeptide contained in human progranulin

“…All members of the GRN family contain 12 cysteine residues, arranged in highly conserved positions, and the cysteine residues form intramolecular disulfide bridges, resulting in characteristic tightly packed structures (1). The GRN family includes GRN A, B, C, D, E, F and G (2).…”

“…The polypeptide contains 12 cysteine residues, and peptides bearing this domain are found in a wide range of organisms, from eubacteria to mammals (3,4). Studies have shown that GRN A plays a critical role in epithelial homeostasis, tumorigenesis, and in reproductive, immunological, and neuronal functions (5,6).…”