Abstract
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 approximately 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 approximately 133 kDa) to the 175-kDa HARE at 4 degrees 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 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees 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.
Highlights
In a typical 70-kg adult, the total HA1 content is ϳ15 g, and about one-third of this turns over every day [1]
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
The HA/CS clearance and degradation in liver and lymph nodes is mediated by the same endocytic receptor, which is expressed in the sinusoidal endothelial cells of these tissues [13, 14]
Summary
Materials and Buffers—Na125I was from Amersham Pharmacia Corp. 125I-HA was prepared as described previously [29] using a hexylamine derivative of HA oligosaccharides (number-average molecular mass ϭ 133,000 based on gel permeation chromatography coupled to multiangle light scattering analysis), modified only at the reducing ends. For the ligand blot assay, the nitrocellulose membrane was treated first with TBS containing 0.1% Tween 20 at 4 °C for 2 h, or TBST (Tris-buffered saline containing 0.05% Tween 20) overnight, and incubated with 1–2 g/ml 125I-HA in 150 mM NaCl, 10 mM HEPES, pH 7.4, and 5 mM EDTA without, or with, a 100- to 150-fold excess of nonlabeled HA (as competitor) to assess total and nonspecific binding, respectively [31]. The membrane was incubated with anti-rat HARE mAbs (e.g. 1 g/ml IgG) at 22 °C for 1 h, washed three times for 5 min each with TBST, and incubated with goat anti-mouse IgG-alkaline phosphatase conjugate (1:1500 dilution) for 1 h at room temperature. N-terminal amino acid sequence analysis was performed by the University of Oklahoma Health Sciences Center Molecular Biology Resource facility
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