Janet A. Weigel

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

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 (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of ∼3–4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound 125I-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201–36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade 125I-HA. Competition studies with live cells indicate that 190-HARE and 315-HARE bind HA with higher apparent affinity (Kd ∼10–20 nm) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.

The Human Hyaluronan Receptor for Endocytosis (HARE/Stabilin-2) Is a Systemic Clearance Receptor for Heparin

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The Kd for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and 23.4 ± 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized 125I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of ∼12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations.

Characterization of the Recombinant Rat 175-kDa Hyaluronan Receptor for Endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339–349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained ∼25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass ∼ 133 kDa) to the 175-kDa HARE at 4 °C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 °C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 °C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.

The Cytoplasmic Domain of the Hyaluronan Receptor for Endocytosis (HARE) Contains Multiple Endocytic Motifs Targeting Coated Pit-mediated Internalization

The hyaluronic acid (HA) receptor for endocytosis (HARE) is the primary scavenger receptor for HA and chondroitin sulfates in mammals. The two human isoforms of HARE (full-length 315-kDa and a 190-kDa proteolytic cleavage product), which are type I single-pass membrane proteins, are highly expressed in sinusoidal endothelial cells of lymph nodes, liver, and spleen. Their identical HARE cytoplasmic domains contain four candidate AP-2/clathrin-mediated endocytic signaling motifs as follows: YSYFRI2485, FQHF2495, NPLY2519, and DPF2534 (315-HARE numbering). Stably transfected cells expressing 190-HARE(ΔYSYFRI), 190-HARE(ΔFQHF), or 190-HARE(ΔNPLY) (lacking Motifs 1, 2, or 3) had decreased 125I-HA endocytosis rates of ∼49, ∼39, and ∼56%, respectively (relative to wild type). In contrast, 190-HARE(ΔDPF) cells (lacking Motif 4) showed no change in HA endocytic rate. Deletions of motifs 1 and 2 or of 1, 2, and 4 decreased the rate of HA endocytosis by only ∼41%. Endocytosis was ∼95% decreased in mutants lacking all four motifs. Cells expressing a 190-HARE(Y2519A) mutant of the NPLY motif retained 85–90% of wild type endocytosis, whereas this mutation in the triple motif deletant decreased endocytosis to ∼7% of wild type. Tyr in NPLY2519 is thus important for endocytosis. All HARE mutants showed similar HA binding and degradation of the internalized HA, indicating that altering endocytic motifs did not affect ectodomain binding of HA or targeting of internalized HA to lysosomes. We conclude that, although NPLY may be the most important motif, it functions together with two other endocytic motifs; thus three signal sequences (YSYFRI, FQHF, and NPLY) provide redundancy to mediate coated pit targeting and endocytosis of HARE.

The hyaluronan receptor for endocytosis (HARE) is not CD44 or CD54 (ICAM-1).

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.

Nonpalmitoylated Human Asialoglycoprotein Receptors Recycle Constitutively but Are Defective in Coated Pit-mediated Endocytosis, Dissociation, and Delivery of Ligand to Lysosomes

The hepatic asialoglycoprotein receptor (ASGP-R) internalizes desialylated glycoproteins via the clathrin-coated pit pathway and mediates their delivery to lysosomes for degradation. The human ASGP-R contains two subunits, H1 and H2. Cytoplasmic residues Cys36 in H1, as well as Cys54 and Cys58 in H2 are palmitoylated (Zeng, F.-Y., and Weigel, P. H. (1996) J. Biol. Chem. 271, 32454). In order to study the function(s) of ASGP-R palmitoylation, we mutated these Cys residues to Ser and generated stably transfected SK-Hep-1 cell lines expressing either wild-type or nonpalmitoylated ASGP-Rs. Compared with wild-type ASGP-Rs, palmitoylation-defective ASGP-Rs showed normal ligand binding, intracellular distribution and trafficking patterns, and pH-induced dissociation profiles in vitro. However, continuous ASOR uptake, and the uptake of prebound cell surface ASOR were slower in cells expressing palmitoylation-defective ASGP-Rs than in cells expressing wild-type ASGP-Rs. Unlike native ASGP-Rs in hepatocytes or hepatoma cells, which mediate endocytosis via the clathrin-coated pit pathway and are almost completely inhibited by hypertonic medium, only ∼40% of the ASOR uptake in SK-Hep-1 cells expressing wild-type ASGP-Rs was inhibited by hyperosmolarity. This result suggests the existence of an alternate nonclathrin-mediated internalization pathway, such as transcytosis, for the entry of ASGP-R·ASOR complexes into these cells. In contrast, ASOR uptake mediated by cells expressing palmitoylation-defective ASGP-Rs showed only a marginal difference under hypertonic conditions, indicating that most of the nonpalmitoylated ASGP-Rs were not internalized and processed normally through the clathrin-coated pit pathway. Furthermore, cells expressing wild-type ASGP-Rs were able to degrade the internalized ASOR, whereas ASOR dissociation was impaired and degradation was barely detectable in cells expressing nonpalmitoylated ASGP-Rs. We conclude that palmitoylation of the ASGP-R is required for its efficient endocytosis of ligand by the clathrin-dependent endocytic pathway and, in particular, for the proper dissociation and delivery of ligand to lysosomes.

Study of Hyaluronan-Binding Proteins and Receptors Using Iodinated Hyaluronan Derivatives

This chapter detailed methodology for the purification of high molecular weight HA, as well as procedures to fragment the HA to prepare large oligosaccharides in the range of 40,000-80,000 Da. The aforementioned procedures used to prepare HA-alkylamine and HA-Bolton-Hunter adducts, as well as 125I-labeled HA, have been very reproducible, and the latter preparations are of adequate length to retain high-affinity interactions and specific binding, e.g., to human fibrinogen and HARE. For example, we were able to isolate, characterize, and clone the rat HARE using 125I-labeled HA initially with the dot blot assay to monitor solubilization and partial purification, and later with the ligand blot assay, to identify the protein after SDS-PAGE. The ligand blot assay enabled us to determine that HARE is actually present as two discrete isoreceptors of different molecular masses. These techniques should provide a means to analyze purification strategies and to characterize additional HA receptors and binding proteins involved in a variety of physiologic processes.

Systemic blockade of the hyaluronan receptor for endocytosis prevents lymph node metastasis of prostate cancer.

Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA receptor for endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis. In our study, we tested the hypothesis that HARE can act as an endothelial receptor for metastatic tumor cells with pericellular HA. In an orthotopic mouse model of prostate cancer, we delivered a monoclonal antibody against HARE that specifically blocks HA binding and internalization. This treatment fully blocked the formation of metastatic tumors in lymph nodes. No effects on primary tumor growth were observed and the antibody did not induce toxic outcomes in any other tissue. Our results implicate HARE for the first time in potentiation of tumor metastasis and suggest a novel mechanism by which tumor cell‐associated HA could promote tissue‐specific dissemination. “Published 2012 Wiley Periodicals, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America.”

Hyaluronan and its receptors in mucoepidermoid carcinoma

Hyaluronan (HA) is a prominent extracellular matrix component undergoing continuous production and degradation. Increased HA levels have been described in a variety of tumors. The objective of this study was to examine the staining patterns of HA and two of its associated receptors (CD44 and HARE) in relation to the metastatic potential of mucoepidermoid carcinoma (MC). Immunohistochemical staining of preserved surgical specimens was used.

PURIFICATION AND CHARACTERIZATION OF THE HYALURONAN RECEPTOR FOR ENDOCYTOSIS (HARE)

ABSTRACT Liver sinusoidal endothelial cells (LECs) express two HARE proteins of 175 kDa and ∼300 kDa that endocytically clear hyaluronan (HA) from the circulation. We have characterized eight monoclonal antibodies (mAbs) raised against the partially purified 175 kDa HARE. Seven mAbs recognize the 175 kDa HARE after nonreducing SDS-PAGE and in all cases also crossreact with the ∼300 kDa HARE. Two of the mAbs inhibit 125I-HA binding and endocytosis by LECs at 37°C. We purified these two HAREs and showed that the 175 kDa HARE is a single protein, whereas the ∼300 kDa species contains three subunits at 260, 230 and 97 kDa (Zhou, et al. J. Biol. Chem. 274, 33831–33834, 1999). Two mAbs recognized both the two nonreduced HARE species and three of the four proteins present after reduction. The 97 kDa subunit was not recognized by any mAbs in Western blots. Based on their cross-reactivity with the mAbs against the 175 kDa HARE, the 175, 230 and 260 kDa proteins are related to each other. Immunocytochemistry using these mAbs shows high protein expression levels in rat liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Little or no HARE expression was observed in muscle, heart, lung, kidney, brain, skin, eye, pancreas, thymus, testis, adipose, salivary gland, adrenal, thyroid, larynx, tongue, stomach or intestine. We propose the name HARE (HA Receptor for Endocytosis) because this receptor mediates the very rapid endocytosis of HA and its tissue distribution is not unique to liver.