Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan‐binding protein 1 (HABP1)/p32/gC1qR

Abstract

Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human ®broblast exists in dierent oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modi®cation of Cys186. The gradual structural transition caused by cysteine-mediated disul®de linkage is evident as the¯uorescence intensity increases with increasing Hg 2+ concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the anity of the hexamer for dierent ligands. The hexamer shows enhanced anity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function.Keywords: clustered mannose; hyaluronan; hyaluronanbinding protein 1 (HABP1); oligomerization; p32.Hyaluronan-binding protein 1 (HABP1), a 68-kDa protein, was originally puri®ed as a novel receptor of hyaluronan, an important component of the extracellular matrix [1]. Subsequently, we characterized the protein and con®rmed its localization on the cell surface [2] and its role in cell adhesion and tumour invasion [3], sperm maturation, and motility [4,5]. The role of this protein in hyaluronan-mediated cellular signalling is well documented, as hyaluronan binding to lymphocyte and hyaluronanmediated lymphocyte aggregation were inhibited by pretreatment of the cells with antibodies to HABP1 [6]. This is further strengthened by the observation of enhanced phosphorylation of HABP1 and increased formation of inositol trisphosphate and phospholipase C-c in hyaluronan-supplemented cells, which have been shown to be inhibited by pretreatment with antibodies to HABP1 [7]. As a continuation of this study, the cDNA encoding HABP from human skin ®broblast has been cloned and sequenced [8]. The presence of the hyaluronanbinding motif was con®rmed and the overexpressed protein subsequently shown to bind hyaluronan. The gene encoding this protein has been assigned to human chromosome 17p12-p13 and has been named HABP1 [9]. A computer search of the sequence encoding HABP1 revealed identity with p32, a protein copuri®ed with splicing factor SF2 [10], and with the receptor for globular head of complement subcomponent C1q (gC1qR) [11], and substantial homology (92%) wi...

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

“…This interpretation is strengthened by the fact that iodide, a polar fluorescence quencher, quenches fluorescence emission intensity at much faster rate in the buffer of low ionic strength than that of high ionic strength. Solvent accessibility of cysteine also seems to be affected by the ionic strength of the environment around the molecule, which is consistent with the earlier observations (18) where trimeric HABP1 is shown to dimerize through the only cysteine present in each molecule under oxidative conditions.…”

“…Recently we have reported that HABP1, a multifunctional protein, exists in different oligomeric forms under various in vitro conditions in solution, although the molecule is predominantly trimeric in anaerobic conditions at 150 mM salt concentration (18). HABP1, having an HA-binding motif (Lys 119 -Lys 128 ), is reported to bind with HA with a K d of 1.1 ϫ 10 Ϫ9 , with C1q through the first 24 residues on the NH 2 terminus with a nanomolar dissociation constant (13), and with clustered mannose (29).…”

“…Among these structural arrangements, the HAbinding motif in each trimeric assembly is solvent-exposed. In our previous report (18), we have shown that HABP1 exists predominantly as a non-covalently linked trimer near physiological conditions and as a hexamer (dimer of trimers) in the oxidative environment. The isoelectric point (pI) of HABP1 is 4.10, and there are more than average polar amino acid residues (43%) present in the molecule.…”

“…to Met 74 substitution) was overexpressed in Escherichia coli as described earlier (7). HABP1 was purified by a 65-90% ammonium sulfate cut, followed by ion exchange chromatography on MonoQ TM HR 10/10 column (Amersham Biosciences), interfaced with an 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 with the modification of an earlier report (18). Affinity-purified HABP1 was further purified by size exclusion chromatography in 10 mM phosphate-buffered saline containing 150 mM NaCl and 0.2% 2-mercaptoethanol.…”

Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex

“…In the next step, the interaction between p32 and LBR was compared with a panel of other potential interaction pairs. As shown previously, p32 is able to interact strongly with the cytomegaloviral protein kinase pUL97 (Marschall et al, 2005) and with itself Jha et al, 2002;Marschall et al, 2005). A point mutant of p32 (L243H) showed a loss of interaction with pUL97 and with the LBR but not with p32 (Fig.…”