Abstract
Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.
Highlights
After Meyer and Palmer [1] discovered hyaluronan (HA),1 it was found to be a component of essentially all vertebrate extracellular matrices (ECMs)
Cell 13, 2853–2868) of this cell surface receptor were achieved after we developed monoclonal antibodies against hyaluronan receptor for endocytosis (HARE)
The results indicate that monoclonal antibodies (mAbs)-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance
Summary
Materials and Media—125I-HA was prepared as described [33] using a hexylamine derivative of HA (oligosaccharides of Mr ϳ70,000). Washed livers were pre-perfused for 15 min with recirculation with a recirculation medium The perfusate was switched to 60 ml of fresh recirculation medium containing 125I-HA (0.25 g/ml in all experiments) and other additions as noted, and the liver was allowed to take up HA for up to 60 min at 35 °C. The tissue sections on the slides were digested for 15 min at 37 °C in pre-warmed 0.1 N HCl containing 0.32 mg/ml pepsin followed by a 2-min water wash and a 2-min PBS wash. After a 1 min PBS wash, the slides were treated with biotinylated horse antimouse IgG (1:200) for 30 min at room temperature. Western blotting procedures were performed essentially as described by Burnette [39] with minor modifications [21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
