Structural Flexibility of Multifunctional HABP1 May Be Important for Regulating Its Binding to Different Ligands

Abstract

Hyaluronan-binding protein 1 (HABP1)/p32/gC1qR was characterized as a highly acidic and oligomeric protein, which binds to different ligands like hyaluronan, C1q, and mannosylated albumin. It exists as trimer in high ionic and reducing conditions as shown by crystal structure. In the present study, we have examined the structural changes of HABP1 under a wide range of ionic environments. HABP1 exhibits structural plasticity, which is influenced by the ionic environment under in vitro conditions near physiological pH. At low ionic strength HABP1 exists in a highly expanded and loosely held trimeric structure, similar to that of the molten globule-like state, whereas the presence of salt stabilizes the trimeric structure in a more compact fashion. It is likely that the combination of the high net charge asymmetrically distributed along the faces of the molecule and the relatively low intrinsic hydrophobicity of HABP1 result in its expanded structure at neutral pH. Thus, the addition of counter ions in the molecular environment minimizes the intramolecular electrostatic repulsion in HABP1 leading to its stable and compact conformations, which reflect in its differential binding toward different ligands. Whereas the binding of HABP1 toward HA is enhanced on increasing the ionic strength, no significant effect was observed with the two other ligands, C1q and mannosylated albumin. Thus, although HA interacts only with compact HABP1, C1q and mannosylated albumin can bind to loosely held oligomeric HABP1 as well. In other words, structural changes in HABP1 mediated by changes in the ionic environment are responsible for recognizing different ligands.The ubiquitous glycosaminoglycan hyaluronan is an unbranched polysaccharide composed entirely of a repeating disaccharide, D-glucuronic acid (␤133) N-acetyl-D-glucosamine (␤134), which, unlike other glycosaminoglycans, is neither attached to a protein core nor O-or N-sulfated. It has diverse biological roles in vertebrates; these include acting as a vital structural component of connective tissues; the formation of loose hydrated matrices that allow cells to divide and migrate during development, immune cell adhesion, and activation; and in intracellular signaling (1-5). This wide range of activities in fact results from its interactions with a large number of proteins termed as hyaladherin that exhibit significant differences in their tissue expression, cellular localization, specificity, affinity, and regulation (2).Hyaluronan-binding protein 1 (HABP1) 1 one of the members of hyaladherin family, is originally purified as a novel receptor of hyaluronan by our group (6 -8). We established its role in cell adhesion and tumor invasion (8 -9) and sperm maturation and motility (10, 11). The cDNA encoding hyaluronan-binding protein 1 has been cloned by immunoscreening the human skin fibroblast expression library, sequenced, and overexpressed (7). Sequence analysis confirmed that although HABP1 is synthesized as pro-protein form of 282 amino acids, the first 73 NH 2 -terminal resi...

Overexpression of Hyaluronan-binding Protein 1 (HABP1/p32/gC1qR) in HepG2 Cells Leads to Increased Hyaluronan Synthesis and Cell Proliferation by Up-regulation of Cyclin D1 in AKT-dependent Pathway

“…The crystal structure of HABP1 shows it to be a trimer having a doughnut shaped quaternary structure with an asymmetric charge distribution along its surface that attributes to its functional diversity (9). HABP1 also exhibits structural flexibility influenced by the ionic environment, which plays an important role in its binding toward different ligands (10). HABP1 has been detected in a number of cellular compartments including the mitochondria, nucleus, and cytoplasm and cell surface where it is shown to interact with many different cellular proteins (11).…”

pH and Cation-induced Thermodynamic Stability of Human Hyaluronan Binding Protein 1 Regulates Its Hyaluronan Affinity

“…For all practical purposes the concentration of a known aliquot was determined in 20 mM phosphate buffer, pH 6.5, containing 6 M guanidine-HCl by measuring the absorbance at 280 nm at 25°C on a Cary 100 Bio UV visible double beam spectrophotometer (Varian Inc., Mulgrave, Australia) interfaced with a Peltier thermal controller. The molar extinction coefficient of denatured HABP1 was calculated and found to be 22,190 …”

“…The protein was purified by a 65-90% ammonium sulfate fractionation, followed by ion exchange chromatography on a Resource-Q (6 ml) column (Amersham Biosciences), interfaced with a Pharmacia FPLC TM system (Amersham Biosciences) using a linear gradient of 0 -1 M NaCl in 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 5% glycerol, and 0.2% 2-mercaptoehanol, pH 7.5, followed by hyaluronan-Sepharose affinity column chromatography as reported earlier (19,22). Affinity purified HABP1 was further purified by size exclusion chromatography as described earlier (19).…”

“…Recently, we have reported the structural flexibility of HABP1 under a wide range of ionic environments. At low ionic strength HABP1 exists in highly expanded and loosely held trimeric structures, whereas, the presence of salt induces compact trimeric structure (22). Thus the possibility of minimizing the intramolecular repulsion by counter ions leading to stable conformation needs to be examined.…”

“…excluded volume effects (21). Previously, we have shown that HABP1 exists in an expanded molten globule-like state at low ionic strength around alkaline pH because of charge-charge repulsion emanating from the presence of multiple negatively charged carboxyl groups, and that the addition of low concentrations of cations leads to a substantial compaction of HABP1 (22). The effect of cation is attributed to their minimizing the charge repulsion by binding to the negatively charged groups, which in turn diminish the repulsive interaction (23).…”