The Crystallographic And Functional Research On Human GRP & The Preliminary X-ray Characterization Of Human Galectin-8

(I) Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates among the vast array expressed in animal tissues. Most animal lectins can be classified into four distinct families: C-type lectins (including the selectins); P-type lectins; pentraxins; and galectins. Galectins are a family of lectins that bindβ-galactosides by means of a carbohydrate recognition domain (CRD) that has many conserved sequence elements. In addition to galectins expressed in vertebrates (fish, birds, amphibians, and mammals), galectins have also been found in invertebrates (worms and insects) and even in protists (sponge and fungus).The galectins can be classified into three groups: the proto-type which contains one carbohydrate recognition domain (CRD), the chimera-type which has a proline/glycine-rich repetitive sequence connected to a CRD, and the tandem-repeat-type which contains two homologous CRDs in tandem separated by a short linker. Proto-type galectins are non-covalent homodimers composed of two identical CRDs except galectin-5 which exists as a monomer. The only member of the chimera-type is galectin-3 which is predominantly found in mammals. The tandem-repeat-type includes galectin-4, -6, -8, -9, and -12. In the past years, the x-ray crystal structures of a few galectins such as gal-1, 2, 3, 7, and 10 have been reported and they are all similar and show jelly-roll topologies typical of legume lectins. Their CRDs are all composed of 11 or 12-strand antiparallelβ-sandwich. Some of them have short 3₁₀ helices. The general architectures of the carbohydrate-binding site in galectins of known three dimension structures are very similar. The structure of human Gal-1 -β-galactoside complex reveals that the amino acids His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71 and Arg73 are directly involved in interactions with the bound disaccharide.GRP (previously known as HSPC159) is a novel human galectin-related protein whose gene was originally deduced by partial sequence alignment and confirmed by a full-length sequence for an mRNA isolated from CD34+ hematopoietic stem cells. The human GRP gene (locus #29094) is located on chromosome 2p13 (NT-031752) and is composed of five exons with exon/intron junctions located in positions generally conserved across the galectin family. GRP sequence is evolutionarily ancient and highly conserved as very similar cDNA sequences have been found in human, mouse, chicken, frog and fish. GRP shares consensus amino acids at 51 of the 64 most highly conserved residues in other galectins. On the other hand, its sequence deviates significantly at five of the seven most critical residues for carbohydrate-binding.In this work, we expressed and purified the C-terminal fragment of human GRP (GRP-C; residues 38-172) containing the CRD. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG400 and 2M ammonium sulfate in 100mM Tris-HCl buffer pH7.5. Diffraction data with resolution limit of about 1.9 A were collected at the beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a=123.07, b=96.67, c=61.56 A,β=118.72°. The estimated Matthews coefficient was 2.6A³/Da, corresponding to 51.8% solvent content. Then, we determined the crystal structure of carbohydrate-recognition-domain of hGRP at 1.9A resolution. In this structure, hGRP-C adopts a fold of 10-strand anti-parallelβ-sandwich similar to that known for other galectin structures. However, the architectures of carbohydrate-binding site between hGRP-C and other known structural galectins are completely different, which suggests a novel mode in which GRP carries out its biological function in vivo.(II) Galectin-8 belongs to the family of tandem-repeat type galectins. It consists as several isoforms, each made of two domains of -140 amino-acids, both having a carbohydrate recognition domain (CRD). These domains are joined by a 'link peptide' of variable length. The human galectin-8 gene covers 33 kbp of genomic DNA. It is localized on chromosome 1 (1q42.11) and contains 11 exons. The gene produces by alternative splicing 14 different transcripts, altogether encoding 6 proteins. Galectin-8, like other galectins, is a secreted protein. Upon secretion galectin-8 acts as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of a selective subset of cell surface integrin receptors. Complex formation between galectin-8 and integrins involves sugar-protein interactions and triggers integrin-mediated signaling cascades such as Tyr phosphorylation of FAK and paxillin. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Such a mechanism allows local signals emitted by secreted galectin-8 to specify territories available for cell adhesion and migration. Due to its dual effects on the adhesive properties of cells and its association with fibronectin, galectin-8 might be considered as a novel type of a matricellular protein. Galectin-8 levels of expression positively correlate with certain human neoplasms, prostate cancer being the best example studied thus far. The overexpressed lectin might give these neoplasms some growth and metastasis related advantages due to its ability to modulate cell adhesion and cellular growth. Hence, galectin-8 may modulate cell-matrix interactions and regulate cellular functions in a variety of physiological and pathological conditions.In this work, we have expressed and purified the C-terminal CRD of human galectin-8 (CCRD). The CCRD protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 27.5% PEG8000 and 0.2M ammonium phosphate in 100mM Na Cacodylate buffer pH6.5 with additive 5% Ethylene Glycol. Diffraction data with resolution limit of 3.0 A were collected in house at 100 K. The crystals belong to the orthorhombic space group P222, with unit-cell parameters a=54.15, b=73.13, c=179.42 A. The estimated Matthews coefficient was 2.84A³/Da, corresponding to 56.71% solvent content.