Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface.

Hyaluronic acid binding protein (HABP) has been purified to homogeneity from normal adult rat kidney by hyaluronate Sepharose affinity chromatography, and its apparent molecular mass was found to be 68 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of HABP under reducing as well as nonreducing conditions revealed a single protein band of 34 kDa, thus indicating that kidney HABP is a homodimer and lacks interchain disulfide bond. Its glycoprotein nature was demonstrated by Con-A binding analysis. The pI value of kidney HABP was 6, indicating its acidic nature. Polyclonal antibodies were raised against it, and the monospecificity of the antibodies towards HABP was confirmed by Western blot analysis of tissue extracts. Immunoblot analysis has elucidated the occurrence of this glycoprotein in various tissues. Moreover, HABP present in these tissues are shown to be structurally and immunologically identical. However, this glycoprotein is antigenically distinct from other well characterized extracellular proteins, e.g., fibronectin, laminin and collagen type IV. With the help of enzyme-linked immunosorbent assay (ELISA) and iodinated [125I]HABP, it has been shown that kidney HABP binds specifically to hyaluronic acid (HA) amongst all the glycosaminoglycans (GAGs), however, HABP can interact with other matrix proteins, e.g., laminin, fibronectin, and collagen type IV. The apparent dissociation constants of HABP for HA, laminin, fibronectin, and collagen type IV were approximately in the range of 10(-9) M, and kinetic analysis showed that these binding interactions were complex and of positive cooperative nature. Indirect immunofluorescence staining demonstrated its localization on human fetus lung fibroblast cell surface. Detection of 34 kDa HABP in the serum-free supernatant culture medium of fibroblasts was further evident by immunoblot analysis, thus confirming the secretory nature of HABP and its occurrence in the extracellular matrix.     

Increased Hyaluronan Levels in HABP1/p32/gC1qR Overexpressing HepG2 Cells Inhibit Autophagic Vacuolation Regulating Tumor Potency

Tumor growth and development is influenced by its microenvironment. A major extracellular matrix molecule involved in cancer progression is hyaluronan (HA). Hyaluronan and expression of a number of hyaladherin family proteins are dramatically increased in many cancer malignancies. One such hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) has been considered to be a biomarker for tumor progression. Interestingly, overexpression of HABP1 in fibroblast has been shown to increase autophagy via generation of excess reactive oxygen species (ROS) and depletion of HA leading to apoptosis. Cancerous cells are often found to exhibit decreased rate of proteolysis/autophagy in comparison to their normal counterparts. To determine if HABP1 levels alter tumorigenicity of cancerous cells, HepR21, the stable transfectant overexpressing HABP1 in HepG2 cell line was derived. HepR21 has been shown to have increased proliferation rate than HepG2, intracellular HA cable formation and enhanced tumor potency without any significant alteration of intracellular ROS. In this paper we have observed that HepR21 cells containing higher endogenous HA levels, have downregulated expression of the autophagic marker, MAP-LC3, consistent with unaltered levels of endogenous ROS. In fact, HepR21 cells seem to have significant resistance to exogenous ROS stimuli and glutathione depletion. HepR21 cells were also found to be more resilient to nutrient starvation in comparison to its parent cell line. Decline in intracellular HA levels and HA cables in HepR21 cells upon treatment with HAS inhibitor (4-MU), induced a surge in ROS levels leading to increased expression of MAP-LC3 and tumor suppressors Beclin 1 and PTEN. This suggests the importance of HABP1 induced HA cable formation in enhancing tumor potency by maintaining the oxidant levels and subsequent autophagic vacuolation.     

Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients

A practical fluorescence-based assay method for determination of hyaluronan (HA) was developed. Plates were coated with hyaluronan-binding proteins (HABP) obtained from bovine cartilage and successively incubated with samples containing standard solutions of hyaluronan or serum from normal and cyrrhotic patients, biotin-conjugated HABP, and europium-labeled streptavidin. After release of europium from streptavidin with enhancement solution the final fluorescence is measured in a fluorometer. The method is specific for HA even in the presence of substantial amounts of other glycosaminoglycans (chondroitin, dermatan sulfate, and heparan sulfate, and heparin) or proteins. It is possible to quantify HA between 0.2 and 500.0 microg/L. Analyses of HA concentration in 545 normal subjects and 40 cirrhotic patients gave average values of 14.5 and 542.0 microg/L, respectively. It was also shown that older subjects (> or =51 years old) have more HA (28.0 microg/L) than younger subjects (12.0 to 14.0 microg/L). This new sandwich technique has shown high precision and sensitivity similar to those of a recently described fluorescence-based assay method, being able to measure HA in amounts as small as 0.2 microg/L. In addition, this noncompetitive assay avoids preincubation, consumes less time (<5 h) than the previous competitive fluorescence-based assay (>72 h), and avoids the use of radioactive materials.     

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

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.     

pH and cation-induced thermodynamic stability of human hyaluronan binding protein 1 regulates its hyaluronan affinity

Hyaluronan-binding protein 1 (HABP1) is a trimeric protein with high negative charges distributed asymmetrically along the faces of the molecule. Recently, we have reported that HABP1 exhibits a high degree of structural flexibility, which can be perturbed by ions under in vitro conditions near physiological pH (Jha, B. K., Salunke, D. M., and Datta, K. (2003) J. Biol. Chem. 278, 27464-27472). Here, we report the effect of ionic strength and pH on thermodynamic stability of HABP1. Trimeric HABP1 was shown to unfold reversibly upon dissociation ruling out the possibility of existence of folded monomer. An increase in ionic concentration (0.05-1 M) or decrease in pH (pH 8.0-pH 5.0) induced an unusually high thermodynamic stability of HABP1 as reflected in the gradual increase in transition midpoint temperature, enthalpy of transition, and conformational entropy. Our studies suggest that the presence of counter ions in the molecular environment of HABP1 leads to dramatic reduction of the intramolecular electrostatic repulsion either by de-ionizing the charged amino acid residues or by direct binding leading to a more stable conformation. A regulation on cellular HA-HABP1 interaction by changes in pH and ionic strength may exist, because the more stable conformation attained at higher ionic strength or at acidic pH showed maximum affinity toward HA as probed either in solid phase binding assay on HA-immobilized plates or an in-solution binding assay using intrinsic fluorescence of HABP1.     

Autophagic Vacuolation Induced by Excess ROS Generation in HABP1/p32/gC1qR Overexpressing Fibroblasts and Its Reversal by Polymeric Hyaluronan

The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca⁺⁺ efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07.     

Pep-1 as a novel probe for the in situ detection of hyaluronan.

Hyaluronan (HA) is expressed by most tissues, including skin. Localization of HA in the skin is assessed by histology with HA-binding protein (HABP) serving as the probe. Reports have suggested that HA expression in skin is altered in a number of diseases. However, interlaboratory variations in HABP staining profiles, even in normal skin, suggest a need to standardize methods and/or identify new probes. We report the staining patterns of a HA-binding peptide (termed "Pep-1") in human and mouse skin. After acetone fixation, Pep-1 stained HA in the intercellular spaces of the epidermis, whereas staining in the dermis was weak and diffuse in both human and mouse skin. HABP staining of the epidermis and dermis were comparable in human skin but failed to stain the vital epidermis of mouse skin. In human skin, Pep-1 stained the basal, spinous, and granular layers, whereas HABP failed to stain the basal layer. Precipitation of HA in situ resulted in dermal staining but weak staining in the epidermis for HABP and Pep-1. Our results may suggest that Pep-1 is sensitive to HA conformation. Furthermore, Pep-1 may represent a new probe to study HA expression in the skin.     

A hyaluronate binding protein transiently codistributes with p21 in cultured cell lines

Hyaluronate (HA) affects the migratory and adhesive properties of cells. HABP, one of the sites which bind HA, localizes in the ruffling lamellae of normal migrating fibroblasts. Similarly, p21 in K-ras oncogene-transformed cells appears enhanced in membrane ruffles. To investigate the possibility that p21 and HABP are functionally linked, their subcellular distribution in two K-ras-transformed lines was examined by double label immunofluorescence and correlated with motility. In both lines, the majority of cells were p21^k-ras and HABP positive at 24 h after subculture. However, immunofluorescence for HABP both decreased and relocated, from ruffles and cell processes to cell bodies, with time whereas the intensity and distribution of staining for p21 remained constant. In doubly positive cells, HABP and p21 colocalized in the ruffles at 24 h, but not at 72 h after subculture. The times after subculture at which changes in the immunofluorescent pattern of HABP occurred differed with cell type and correlated with their migratory rate. Thus, the migratory rate of KNRK cells, which was less than in the K-C3H-10T1/2 cells, correlated with both an earlier decrease in HABP and an earlier loss of codistribution between HABP and p21 compared to K-C3H-10T1/2 cells. Further evidence of a functional link between HABP and p21^k-ras was suggested by the ability of hyaluronic acid, which induces ruffling in K-C3H-10T1/2 cells, to promote the coassociation of p21^k-ras and HABP. These results demonstrate a transient codistribution of p21 and HABP, in ruffles, that is possibly related to migratory activity and/or cell-surface changes following subculture.     

Evidence for clustered mannose as a new ligand for hyaluronan- binding protein (HABP1) from human fibroblasts

We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment.     

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 +/- 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.     

Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.     

Multifunctional activities of human fibroblast 34-kDa hyaluronic acid-binding protein

We have already reported that human fibroblast 34-kDa hyaluronic acid-binding protein (HABP) is identical with P32, the protein co-purified with splicing factor SF-2 [Deb and Datta (1996) J. Biol. Chem. 271, 2206–2212]. Data search further revealed that it has 92% sequence homology with a murine protein YL2 which interacts with HIV1 Rev. In this paper we have successfully demonstrated that HIV1 Rev binds with labeled 34-kDa HABP which can be competed with excess unlabeled HABP, suggesting this protein can be a cellular factor promoting HIV1 Rev to function. Interestingly, the multifunctional nature of HABP has been elucidated as it has 100% homology with another protein gClq, the complement protein. The distinct non-overlapping binding motifs for HA and gClq have been identified in the same protein, suggesting that either the protein can function independently or its activity is regulated by ligand binding, wherein its binding to one of the ligands may modulate the receptor activity of the other ligand.     

Possible role of the 34-kilodalton hyaluronic acid-binding protein in visceral Leishmaniasis.

Leishmania donovani, the causative organism of human visceral leishmaniasis, invades host macrophages through its interaction with the cell surface molecules of target cells. The presence of a cell surface protein (Mr 34 kDa) having specific affinity toward hyaluronan (HA), a major extracellular matrix component, has been previously reported in macrophage cell lines. In order to identify the possible role of this HA-binding protein (HABP) in leishmaniasis, initially we demonstrated its overexpression in spleen, liver, macrophages, and serum of hamsters infected with L. donovani. We further observed higher levels of HABP in the macrophage cell line J774.G8 upon infection with L. donovani. Finally, we observed a significant increase in the level of HABP in the serum of patients with kala-azar. In order to understand its functional role in leishmaniasis, we report here a significant inhibition of cellular phosphorylation of HABP in hamster macrophages infected with L. donovani. Interestingly, the 34-kDa HABP was shown to bind with 2 proteins of promastigotes as well as amastigotes of L. donovani (with molecular masses of 55 kDa and 30 kDa respectively), suggesting a possible role for HABP in adhesion during the interaction of promastigotes and macrophages.     

Structural flexibility of multifunctional HABP1 may be important for regulating its binding to different ligands

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.     

A method for the quantitation of hyaluronan (hyaluronic acid) in biological fluids using a labeled avidin-biotin technique.

An improved method for the detection and quantitation of hyaluronan (hyaluronic acid) (HA) in biological fluids is described. The principle on which the method is based is that HA binds strongly to a biotinylated HA-binding protein (B-HABP) which was prepared from cartilage proteoglycans. HA was immobilized on polyvinyl chloride plates which had been precoated with poly-L-lysine. The unknown sample or HA standards together with excess B-HABP are then added. The B-HABP that binds to the immobilized HA is then incubated with the enzyme-conjugated avidin (e.g., alkaline phosphatase), and the color which develops on addition of enzyme substrate (e.g., p-nitrophenyl phosphate) is determined by light absorption using a microtitration plate reader. The assay is not only convenient and reliable but is capable of measuring HA in solution at the picogram level. The assay was used to determine HA levels in human sera and synovial fluid taken from volunteers and patients with rheumatoid arthritis and osteoarthritis.     

Hyaluronan binding protein 1 (HABP1)/C1QBP/p32 is an endogenous substrate for MAP kinase and is translocated to the nucleus upon mitogenic stimulation

The role of hyaluronan binding protein 1 (HABP1) in cell signaling was investigated and in vitro kinase assay demonstrated that it is a substrate for MAP kinase. Phosphorylation of endogenous HABP1 was also observed following treatment of J774 cells with PMA. HABP1 was coimmunoprecipitated with activated ERK, confirming their physical interaction in the cellular context. Upon PMA stimulation of normal rat fibroblast (F111) and transformed (HeLa) cells, the HABP1 level in the cytoplasm gradually decreased with a parallel increase in the nucleus. In HeLa cells, within 6 h of PMA treatment, HABP1 was completely translocated to the nucleus, which was prevented by PD98059, a selective inhibitor of ERK. We also observed that the nuclear translocation of HABP1 is concurrent with that of ERK, suggesting that ERK activation is a requirement for the translocation of HABP1. It is thus established for the first time that HABP1 is a substrate for ERK and an integral part of the MAP kinase cascade.     

Localization of hyaluronate and hyaluronate-binding protein on motile and non-motile fibroblasts

The distribution of a hyaluronate-binding (HABP) and rhodamine B-isothiocyanate (RITC)-labeled hyaluronate (HA) were studied on both actively motile and stationary chick heart fibroblasts to assess the relationship of these molecules to each other, to other extracellular matrix molecules, to membrane protrusions and to adhesion sites. RITC-HA and HABP, detected by indirect immunofluorescence, were concentrated in the perinuclear region, the leading lamella and retraction processes of actively motile cells, although RITC-HA also occurred diffusely over the rest of the cell body. Double immunofluorescence confirmed that HA and HABP co-localized in the former three regions, suggesting that, at these locations, the HABP may act as a cell surface-binding site for HA. With increasing culture confluency and consequent slowing of fibroblast motility, the localization of both polymers changed to a uniform and diffuse distribution over the cell body and processes. On actively motile cells, RITC-HA and HABP did not co-distribute with fibronectin, heparan sulfate proteoglycan or laminin. Areas coated with RITC-HA and HABP often contained specialized adhesion sites as determined by interference reflection microscopy (IRM) but neither polymer appeared to particularly localize to adhesion sites. However, the occurrence of RITC-HA and HABP in the leading lamellae of motile cells consistently coincided with ruffling activity. These results are discussed with respect to a possible instructive role of HA in cell motility.     

Quantitative histochemical study of hyaluronic acid binding protein and the activity of uridine diphosphoglucose dehydrogenase in the synovium of patients with rheumatoid arthritis

OBJECTIVE: To examine hyaluronic acid (HA) dynamics in synovia with rheumatoid arthritis (RA), relying on a new quantitative technique introduced into histochemistry. STUDY DESIGN: Synovial lesions from 28 patients were classified into four histologic stages of RA according to the degree of inflammation. The distribution of HA was histochemically investigated with a hyaluronic acid binding protein (HABP) and that of HA-producing cells enzyme histochemically with the expression of uridine diphosphoglucose dehydrogenase (UDPGD) activity in the synovium of RA patients. The results were quantified using an image processor for analytical pathology. The positive area of HABP reaction, the number of UDPGD-positive cells and the color density of the enzyme-histochemistry of UDPGD activity were measured with the IPAP system. RESULTS: HA was shown to be diffusely distributed in the synovia, particularly densely in the superficial layer, and the distribution overlapped with that of UDPGD activation. HA distribution and UDPGD activity varied with the severity of synovial inflammation, and the positive area was the most extensive in the early stage, while it completely disappeared in the fibrotic stage. CONCLUSION: We assume that for HA, not only does the production decrease, but the range of distribution contracts with time.