Expression, Processing, and Glycosaminoglycan Binding Activity of the Recombinant Human 315-kDa Hyaluronic Acid Receptor for Endocytosis (HARE)

Abstract

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 ...

Probing Structural Selectivity of Synthetic Heparin Binding to Stabilin Protein Receptors

“…The Stab2/190-HARE cDNA encoding the C-terminal 1416 amino acids is cloned in pSecTag/FRT/V5-6ϫHIS-TOPO, which provides a secretion signal for the 190-HARE protein (8). Plasmids encoding the secreted ectodomain were generated by single primer deletion mutagenesis (7) in which the transmembrane and cytoplasmic domain encoding regions were deleted, and the resulting plasmids were then used to create stable cells that secreted properly folded and functional ectodomains in the medium. The ectodomains comprised amino acids M1-P2475 for Stab1, M1-T2458 for Stabilin-2/315-HARE, and S1136-V2453 for Stab2/190-HARE.…”

“…Receptors-Cell lines stably expressing Stab2 and empty vector were created as described previously (7,8). The cell line stably expressing human Stab1 cDNA was also constructed with identical methodology.…”

“…A subset of the 315-kDa receptor undergoes proteolytic cleavage to produce the smaller 190-kDa receptor. Both 315-kDa and 190-kDa Stab2 receptors bind to and participate in systemic clearance of heparin (7,8). The binding affinity of Stab2 to UFH is higher than LMWH, suggesting that interactions between Stab2 and heparin are dependent on the length of the polymer.…”

Anticancer Therapeutics: Targeting Macromolecules and Nanocarriers to Hyaluronan or CD44, a Hyaluronan Receptor

“…29 HARE binds chondroitin sulfate and HA; CD44 binds only to HA. 39 In rats with hepatic metastases of colon adenocarcinoma cells, pretreatment by intravenous injection of either chondroitin sulfate or HA resulted in higher tumor accumulation of a subsequent intravenous HA dose. 67 In perfused liver, pre-blocking with an anti-HARE antibody or immediate pre-dosing with free HA resulted in decreased HA clearance of the second dose.…”

Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation

“…CD44, a type 1 transmembrane receptor expressed on the surface of smooth muscle cells and platelets. CD44 exists in many different isoforms that all have HA binding properties [10] and regulate several signaling pathways including Src family kinases, Rho family GTPases, extracellular signal-regulated kinases and mitogen activated protein kinase (MAPK) [11,12] and thereby mediates both cell adhesion and cell growth [13,14].…”