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, ␣, ␤, and ␥ 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 125 I-HA binding and endocytosis by LECs at 37°C. All of these results confirm that the mAbs recognize the bone 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 (HA receptor for endocytosis). HA1 is an important and often abundant extracellular matrix component of all tissues, in particular cartilage, skin, and vitreous humor (1). HA plays a key role in development, morphogenesis, and differentiation, in cell adhesion and proliferation, and in inflammation and wound healing (1-4). In humans the total body turnover of HA is several grams per day (1). Although local turnover of HA occurs in avascular tissues, particularly cartilage (5, 6), two major clearance systems are responsible for HA degradation and removal in the body (4). The first is the lymphatic system, which accounts for ϳ85% of the HA turnover, and the second is in the liver, which accounts for the other ϳ15% of the total body HA turnover. Throughout the body, HA is continuously synthesized and degraded in almost all tissues. At the same time, chondroitin sulfate and other glycosaminoglycans are also released from the cleavage of proteoglycans, especially aggregating proteoglycans associated with HA. Large native HA molecules (ϳ10 7 Da) are partially degraded to large fragments (ϳ10 6 Da) that are released from the matrix and enter the lymphatic system, flowing to lymph nodes.The lymph nodes completely degrade the majority of HA (ϳ85%) by unknown mechanisms. Neither the responsible cell type, the receptor involved, nor the location in lymph nodes at which HA uptake and degradation occurs has been determined. The remaining HA (ϳ15%) that escapes degradation in the lymph nodes ultimately flows into the blood at the thoracic duct. Since HA is an exceptionally viscous polysaccharide in solution, it would be deleterious for the blood concentration of HA, even at relatively low molecular weight, to increase. Clearance of this circulating HA and the other glycosaminoglycan degradation fragments is presumably important for normal health (1, 4). Elevate...

The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms ( The glycosaminoglycan (GAG)2 hyaluronic acid (HA) is a protein-free polymer of disaccharide units containing glucuronic acid and N-acetylglucosamine (1, 2). HA is involved in many physiological processes (3), such as wound healing, development, and metastasis of some cancers (4 -8). The typical molecular mass of the polysaccharide ranges from just a few thousand Da (tens of sugars) that are thought to be important in cellular signaling (6) to several million Da (tens of thousands of sugars). These larger forms of HA are present throughout the body and are particularly concentrated within the bursa of major joints, such as the knee, where they help to provide shock absorbance in cartilage or lubrication in synovial fluid (9, 10), and the eye, where HA maintains structural integrity of the vitreous humor (11). The adult human body contains ϳ15 g of HA, of which about 5 g are turned over daily (12). Partially degraded HA perfuses from extracellular matrices (ECMs) and enters the lymphatic and vascular circulation systems, where it is catabolized to shorter fragments. This active maintenance of HA turnover must be efficient in order to maintain homeostatic conditions for total body HA.All of the other GAGs, including the chondroitin sulfates (CSs), heparan sulfate (HS), and keratan sulfate, are linked to core proteins (as proteoglycans) that help to form ECMs, such as the basement membranes of tissues, or structural components of organs, such as the vitreous humor. There are over 30 known core proteins that are essential for a diverse array of functions, such as neural development, growth factor signaling, and pathogen recognition (13). These core proteins are found as prevalent components of tissue ECMs or as specialized components needed for the development of microenvironments that interface a specialized tissue cell type with the ECM. Both the proteoglycans and their attached GAG chains may combinatorially interact with ligands and contribute to modulation of the functional aspects of a particular microenvironment (e.g. CS interacting with apolipoprotein E for uptake of ␤-very low density lipoprotein in hippocampal neurons) (14). Although numerous studies have focused on how the inhibition of some CS proteoglycans enhances neural development, especially in injured spinal cord models, there is very little information on how CS and HS are catabolized. The current model is that extracellular chondroitinases, heparinases, and proteases initially break down these GAGs and proteoglycans, and their final digestion can then take place intracellularly at the local tissue * This research was supported by NIGMS, National Institutes of Health, Grant GM69961. The costs of publication of this article were defrayed in part by the payment of page charges. This article ...

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Heparin (Hep)2 is the most anionic proteoglycan, due to the extensive sulfation of its glycosaminoglycan (GAG) chains, and contains many different sulfated disaccharide isomers of N-acetylgalactosamine and glucuronic acid or iduronic acid. Hep binds to many different soluble, matrix, and cell surface proteins and receptors, and has many functions, including its roles as an anti-coagulant and as a co-receptor for some growth factors (1, 2). Hep is a highly prescribed drug in surgical patients and those at risk for thromobosis. The genes and metabolic pathway for Hep biosynthesis in mast cells are understood reasonably well, and many of the biological and clinical activities of Hep have been well studied for several decades (3-6). In contrast, we know less about the catabolism of Hep and how total body homeostasis of this multifunctional proteoglycan is maintained. In particular, the mechanisms controlling systemic turnover of Hep, whether as endogenous proteoglycan or free chain drugs, are not known.Although receptors for Hep have been characterized on a variety of cell types (e.g. macrophages, vascular smooth muscle cells), none of these mediate substantial clearance of Hep (7-9). A few reports implicated a role for Kupffer cells in Hep clearance, but were not followed up (10, 11). The possible contribution of liver sinusoidal endothelial cells to Hep uptake in these primary cell preparations was not examined. Because Hep is a widely prescribed drug, it is even more important to understand the factors that control its clearance and, therefore, pharmokinetics. Animal studies of Hep clearance, except by renal function, have been difficult to perform, because Hep binds to so many soluble, cell surface or matrix proteins and become widely distributed after injection.Unfractioned Hep (3000 -30000 Da), low molecular mass Hep (300 -8000 Da), and the pentasaccharide, Fondaparinux, are the three classes of Hep drugs used to treat venous thrombosis, acute myocardial infarction, trauma, obesity, and coronary and peripheral vascular procedures; all situations wherein patients need immediate platelet anti-coagulation therapy (12). Following an intravenous bolus, unfractioned Hep has a halflife of ϳ1 h and is cleared from the circulation by the liver and kidney (13). Low molecular mass Hep and the pentasaccharide, subcutaneously injected, have half-lives of ϳ3-6 and ϳ17 h, respectively (14). Larger more structurally diverse Hep is more readily cleared than the lower molecular weight fragments. Although clinical handbooks declare that Hep is metabolized and cleared by the reticuloendothelial system, the mechanisms for Hep clearance are not known.The primary scavenger receptor for systemic turnover of HA and most types of chondroitin sulfate, but not HS, is HARE/ Stab-2, which mediates most of the total body HA turnover per day (15)(16)(17). HARE is found primarily in the sinusoidal endothelial cells of the lymph nodes, liver, and spleen (18 -21), and * This work was supported, in whole or in part, by National Institutes of Hea...

We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and approximately 300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5'-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig kappa-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of (125)I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the approximately 300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.