Structural studies of human glioma pathogenesis-related protein 1

Abstract

Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn2+ complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

Figure 1

GLIPR1 was expressed in P. pastoris as a glycosylated monomeric protein. (a) SDS–PAGE Western blot analysis of purified sGLIPR1 using anti-GLIPR1_75–95 peptide antibody under reducing (R) or nonreducing (NR) conditions. The positions of molecular-weight markers are indicated in kDa. (b) Coomassie-stained SDS–PAGE gel depicting sGLIPR1 deglycosylation by peptide N-glycosidase F (PNGase F) and endoglycosidase H (Endo H), which resulted in faster migration of the protein (right arrow) compared with nondigested control (left arrow). The band indicated with a star is Endo H.

Figure 2

Structural features of sGLIPR1 and primary sequence alignment of sGLIPR1 with other members of the GLIPR1 family and representative CAP-protein structures. This figure was generated with ESPript (Gouet et al., 2003 ▶). Also shown are the structural elements of GAPR-1. Secondary-structure elements are shown as follows: α-helices are shown as large squiggles labeled α, 3₁₀-helices are shown as small squiggles labeled η, β-strands are shown as arrows labeled β and β-­turns are labeled TT. Identical residues are highlighted in red and conserved residues are highlighted in yellow. The locations of the cysteine residues involved in sGLIPR1 disulfide bonds are numbered in green and the signature CRISP motifs are identified with red bars. The members of the GLIPR1 subfamily used are GLIPR1 (NP_006842), RTVP-1b (ABV21587.1), GLIPR1L1 (NP689992.1) and GLIPR1L2 (NP689649). RTVP-1b is a GLIPR1 splice variant with C-terminal divergence. The highest variability for this group is in the signal peptide and loop regions. The representative CAP structures are Na-ASP-2 (PDB entry 1u53), Ves v 5 (PDB entry 1qnx), GAPR-1 (PDB entry 1smb) and stecrisp (PDB entry 1rc9). The AEAEF leader sequences in Na-ASP-2 and Ves v 5 are from the expression vector. In these representative structures, the helices and strand regions of the core CAP domain are well conserved, with the highest variability in the loop regions of sGLIPR1.

Figure 3

Comparison of the sGLIPR1 structure with representative CAP structures. The top row shows a ribbon diagram of sGLIPR1 (PDB entry 3q2u), revealing a conserved core CAP domain similar to those observed in the representative CAP structures GAPR-1, stecrisp, Ves v 5 and Na-ASP-2. This core α–β–α sandwich is formed by the three core β-strands flanked by the labeled helices. Arrows indicate the locations of loop/turn regions in sGLIPR1 that are longer than in the other structures. The locations of disulfide bonds are shown in gray. The bottom row reveals that the surface-charge distributions differ for these representative CAP structures. These are colored from red for negatively charged regions to blue for positively charged regions. The same view is shown for all CAP structures.

Figure 4

Comparison of CAP-protein central cavities. The cavity of sGLIPR1 complexed with Zn²⁺ (magenta) forms similar interactions with Zn²⁺ as both snake-venom CRISP structures: natrin with Zn²⁺ (yellow) and pseudecin with Zn²⁺ (cyan). The uncomplexed structure of sGLIPR1 (orange) superposes well with that of the Zn²⁺ complex.

Figure 5

Putative model of functional full-length GLIPR1. This model is after removal of the signal peptide and translocation to the cell membrane. Left panel, front view of model of full-length GLIPR1 with conserved regions from all GLIPR1 protein-family members shown in magenta; the CRISP1 motif is shown in orange. The side chains of the putative CAP cavity residues are shown. The CRISP2 motif that is implicated in sperm–oocyte binding in other CAP proteins is shown in blue. The transmembrane helix (red) links the intracellular and extracellular portions of GLIPR1. Right panel, a rear view of the model rainbow-colored from blue (carboxy-terminus) to red (amino-terminus) reveals an alternative view of full-length GLIPR1 with Zn²⁺ in the CAP cavity.