Identification of the Ca(2+)-independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linking reagent.

J Yannariello-Brown,S.J Frost,P.H Weigel

doi:10.1016/s0021-9258(19)88723-9

Abstract

The Ca(2+)-independent endocytic hyaluronan (HA) receptor in rat liver sinusoidal endothelial cells (LECs) was identified using a novel cross-linking derivative of HA. The heterobifunctional, photoactivatable, reducible reagent sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) was coupled to the terminal amino group of uniquely modified HA-amine oligosaccharides (M(r) approximately 60,000) and subsequently iodinated. 125I-ASD-HA bound to cultured LECs with similar specificity and affinity as a previously characterized 125I-HA-amine/Bolton-Hunter adduct. Permeabilized LECs were incubated with 125I-ASD-HA with 10 mM EGTA and photolysed with UV light. Detergent extracts were reduced to release the HA oligosaccharides and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Two polypeptides were consistently and equally labeled at M(r) = 175,000 and 166,000. Photoaffinity labeling of these two proteins was virtually identical in cultured LECs or membranes and was competed greater than 90% with a 100-fold excess of HA. As with the previously characterized bona fide LEC HA receptor, cross-linking was also competed by chondroitin sulfate and heparin, but less efficiently by chondroitin and not with galacturonan. We conclude that the Ca(2+)-independent LEC HA receptor is composed of at least two polypeptides of M(r) approximately 175,000 and 166,000 and may exist as a heterodimer of M(r) approximately 340,000. We also conclude that the LEC HA receptor is distinct from the CD44 family of HA-binding proteins.

Highlights

HA oligosaccharides and radiolabeled proteins were not dependent on the extracellular presence of divalent catanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresisandautoradiography
The receptor ias thermally fold excessof HA.As with thepreviously characterized labile integral membrane protein and is sensitive rtoeducing bona fide LEC HA receptor,cross-linkingwasalso agents, suggesting that disulfide bonds are critical for maincompeted by chondroitin sulfate and heparin, butless taining a biologically active conformation
The possibility exists that the 86, 66, and 55-kDa bands are distinct HA-binding proteins unrelated to the LEC en

Summary

HO-CH2-CHpSH

The broad, heavily labeled band at 55 kDa, which was competed >80%, was much more intense relative to the 175/166-kDa doublet in the isolated membranes than in the permeable LECs (compare to Fig. 2). A minor increase in the intensity of the 175/165-kDa bands was detected after digitonin treatment (compare lanes 1 and 3 in Fig. 3), suggesting that the vesicles are already permeable or that the binding domain of HA receptors are preferentially localized to theouter vesicle face. The cross-linking of the photoaffinity derivative to the 175- and 166-kDa polypeptides showed the same pattern of polysaccharide competition (Fig. 4) previously observed for the HA receptor in cultured LECs using 12'I-BH-HA [2, 5]. CS, chondroitin sulfate; dCS, desulfated chondroitin sulfate; Hep, heparin; pGAL, Dolvgalacturonic acid (ealacturonan)

Method

Findings

DISCUSSION