Characterization and purification of the hyaluronan-receptor on liver endothelial cells

doi:10.1016/0167-4838(91)90085-E

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1078, Issue 1, 30 May 1991, Pages 12-18

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(91)90085-E Get rights and content

Abstract

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with ¹²⁵I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6–6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDA protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of ³H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.

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A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer
2016, Journal of Controlled Release
Citation Excerpt :
While not relevant for OC treatment, for the sake of completeness the biodistribution of the bioconjugate given i.v. was also investigated. As previously demonstrated [38], i.v.-injected bioconjugate disclosed a marked accumulation in the liver, mainly due to the presence of the HARE receptors in this district [47], and was not toxic. Because of this peculiar accumulation of the i.v.-administered HA-IFNα2a, the bioconjugate can be envisaged also as a safe and targeted treatment for liver diseases, especially HBV or HCV infections.
While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.
A double-strand decoy DNA oligomer for NF-κB inhibits TNFα-induced ICAM-1 expression in sinusoidal endothelial cells
2002, Biochemical and Biophysical Research Communications
Citation Excerpt :
The polycation PLL and negatively charged DNA form a stable and condensed nanoassociate surrounded by highly hydrated HA glycocalyx. The PLL-g-HA/DNA complex is specifically taken up by SECs through an HA receptor mediated pathway since SECs possess receptors that recognize and internalize HA [21,22]. As expected, the PLL-g-HA/NF-κB31 facilitated its entry into LSE cells (Fig. 2).
Altered gene expression of liver sinusoidal endothelial cells (SECs) is associated with impaired immune response. Here we report that the decoy technique effectively suppresses TNFα-induced ICAM-1 expression in SEC. An NF-κB decoy (NF-κB31: 5^′-TG $GGGACTTTCC$ AGTTTCT $GGAAAGTCCC$ CA-3), which contains a consensus sequence for NF-κB, was complexed to PLL-g-HA [hyaluronate-grafted poly(l-lysine) copolymer] that permits transfer of exogenous DNA selectively to the SEC. The PLL-g-HA/NF-κB31 complex was added to the culture media of LSE cells, a human SEC-derived cell line. Then, cells were stimulated with TNFα (5 ng/mL). PLL-g-HA/NF-κB31, but not control oligodeoxynucleotides having a reverse or scrambled sequence, inhibited the intranuclear localization of NF-κB induced by TNFα, with almost complete inhibition at 2.5 μg/mL as DNA. NF-κB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells. The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions.
The hyaluronan receptor for endocytosis (HARE) is not CD44 or CD54 (ICAM-1)
2002, Biochemical and Biophysical Research Communications
Citation Excerpt :
Protein content was determined by the method of Bradford [21] using BSA as standard. Forsberg and Gustafson [22] initially identified a 100-kDa protein as the putative LEC HA receptor. Subsequently, McCourt et al. [16] attempted to purify the endocytic HA receptor without the use of a quantitative assay to measure specific HA-binding activity, such as a dot-blot [20,23] or ligand-blot [24] assay.
Mammalian liver contains an endocytic, recycling receptor that mediates the clearance of hyaluronan (HA) and chondroitin sulfate from the circulation. McCourt et al. [J. Biol. Chem. 269 (1994) 30081] previously reported that this endocytic liver HA receptor was ICAM-1. In contrast, we purified this HA receptor for endocytosis (HARE) from rat liver sinusoidal endothelial cells (LECs) and obtained two novel large proteins [Zhou et al., J. Biol. Chem. 274 (1999) 33831]. The goal of the present study was to clarify this inconsistency and determine whether CD44, which is also an HA receptor, or ICAM-1 (CD54) is identical to, or is part of, HARE. Although isolated liver LECs contain HARE, CD44, and ICAM-1, confocal fluorescence microscopy showed that the two latter proteins have cellular distributions that are distinct from and essentially nonoverlapping with HARE. HA accumulation by cultured LECs was inhibited >98% by an antibody against HARE and unaffected by antibodies to ICAM-1 or CD44, indicating that virtually all specific HA uptake is mediated by HARE and not by ICAM-1 or CD44. Finally, no reactivity was observed against purified HARE in an ELISA-based assay using CD44 or ICAM-1 antibodies. The results confirm that the mammalian endocytic HA receptor is HARE and is not ICAM-1 or CD44.
Identification of the hyaluronan receptor for endocytosis (HARE)
2000, Journal of Biological Chemistry
Citation Excerpt :
ICAM-1 is a cell surface adhesion molecule that binds HA (14). ICAM-1 was incorrectly identified as the LEC endocytic HA receptor (16). This report, although later explained as an artifact (17), has not been formally withdrawn, and subsequent reports (52, 53) based on these erroneous findings may still confuse the field.
Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, α, β, and γ at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831–33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit ¹²⁵I-HA binding and endocytosis by LECs at 37 °C. All of these results confirm that the mAbs recognize thebone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HAreceptor for endocytosis).
Purification and subunit characterization of the rat liver endocytic hyaluronan receptor
1999, Journal of Biological Chemistry
The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and ∼300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451–20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a ∼185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at ∼260, 230, and 97 kDa. These proteins designated, respectively, the α, β, and γ subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species containN-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.
An ultrasensitive, nonisotopic immunoassay for hyaluronan using the streptavidin-biotin system
1998, Clinica Chimica Acta
A time-resolved fluoroimmunoassay for measuring hyaluronan concentrations in plasma and several biological fluids is described. The solid-phase immunoassay is based on the competition between aggregation of hyaluronan with the cartilage proteoglycan monomer, followed by binding of a monoclonal antibody to keratan sulfate of the proteoglycan and a biotinylated anti-mouse IgG. Fluorescence can be measured by a time-resolved fluorometer after binding of Eu³⁺-labelled streptavidin to the biotinylated IgG. The assay is precise and correlates well (r=0.986) with the only established radioimmunoassay known. The results show that it is essential to perform a blank run without addition of proteoglycan, as endogenous proteoglycan disturbs the measurement and causes underestimation of plasma hyaluronan. The distinguishing feature of this assay is its extreme sensitivity (<0.24 μg/l of plasma). The mean analytical recovery after serial dilutions and addition was 100.3 and 101.3%, the within-assay and between-assay coefficients of variation were 3.67% and 7.02%, respectively.

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Characterization and purification of the hyaluronan-receptor on liver endothelial cells

Abstract

J. Biol. Chem.

Biochim. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Dev. Biol.

Exp. Cell Res.

Methods Enzymol.

Analyt. Biochem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Anal. Biochem.

Anal. Biochem.