Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.     

Isolation and characterization of a specific antibody population against human fibrinogen

A specific antibody population against human fibrinogen was isolated from a rabbit antiserum by affinity chromatography on fibrinogen-bound Sepharose gel. Using a sensitive competitive radioimmunoassay, the antibody population was found to recognize epitopes on native fibrinogen but crossreacted minimally with fibrinogen fragment D and an early plasmin-degraded fibrinogen A alpha-chain product, but not at all with fragment E or fibrinopeptides A and B. Fibrin monomers shared part of these epitopes. The antibody population crossreacted to a small extent with bovine, horse and baboon fibrinogens and not at all with fibrinogens from sheep, rat, pig, goat, guinea pig, dog and rabbit.     

Human fibrinogen specifically binds hyaluronic acid

Fibrin and hyaluronic acid (HA) are macromolecules whose concentrations are elevated at the same time in the extracellular space of damaged tissues. We have investigated whether HA can bind to fibrinogen using solid phase and soluble assays. Purified human fibrinogen specifically bound to HA-Sepharose to a greater extent (greater than 5-fold) than did alpha 1-acid glycoprotein, DNaseI, ovalbumin, haptoglobin, or lysozyme. Fibrinogen did not bind to ethanolamine-Sepharose, a control chromatographic support. Treatment of HA-Sepharose containing bound 125I-fibrinogen with ovine testicular hyaluronidase released 44% of the 125I radioactivity, indicating that fibrinogen was specifically bound to HA. Moreover, 125I-fibrinogen bound to HA-Sepharose could be displaced by free HA but not by either of the monosaccharide components of this polymer, glucuronic acid, or N-acetylglucosamine. Chondroitin sulfate and polygalacturonic acid competed only weakly for bound 125I-fibrinogen. Bound 125I-fibrinogen was also not released by high concentrations of NaCl (up to 4 M), indicating that the interaction is not simply ionic. The apparent affinity of fibrinogen for HA covaried with the molecular weight of the HA. Small HA oligosaccharides (Mr = 3900) were only 50% as effective as larger HA (Mr = 8 X 10(5)) in eluting bound 125I-fibrinogen from HA-Sepharose. The optimal oligosaccharide size for displacement of bound 125I-fibrinogen was greater than or equal to 200 monosaccharides. Additionally, the amount of 125I-fibrinogen bound to HA-Sepharose was directly related to the size of the HA-amine linked to the affinity support. The affinity constant for fibrinogen binding to 125I-HA (approximately 150 monosaccharides) is estimated to be at least 2 X 10(7) M-1. These results demonstrate for the first time a specific, reversible binding between HA and fibrinogen.     

Comparative studies on the gastric glycopeptide in eleven animal species.

1. Glycopeptides in the stomachs of eleven mammalian species, including human, rabbit, horse, cow, pig, goat, sheep, dog, cat, guinea pig and rat were assayed by determining the carbohydrate content of materials which remained after proteolysis. 2. The glycopeptide content was higher in the mucosa than in the muscular layer including serosa, especially in the porcine stomach and the fourth stomachs of the ruminants than in the stomachs of any other animals. 3. The glycopeptide, which was stained with both alcian blue and PAS, was absent or sparingly present in the mucosae of the human, rabbit, horse stomachs and in the mucosae of the first to third stomachs of the cow, goat and sheep, whereas in the mucosae of the pig, dog, cat, guinea pig and rat stomachs and in the mucosae of the fourth stomachs of the cow, goat and sheep, it was found in noticeable extents.     

Carboxyl-terminal residues of mammalian fibrinogen and fibrin

The carboxyl-terminal residues of mammalian fibrinogens of six different species and the chain peptides, alpha(A), beta(B) and gamma, isolated from these fibrinogens were determined by hydrazinolysis, digestion with carboxypeptidases and selective tritium labelling. The C-terminal ends of bovine fibrinogen and fibrin were identified as proline and valine, in the molar ratio of approximately 1:2. Proline was identified as the C-terminus of the alpha(A)-chain, and C-terminal valine was found on both the beta(B)- and gamma-chains. On hydrazinolysis after selective tritium labelling of fibrinogen, radioactive C-terminal valine was also identified. The same C-terminal ends as those of bovine fibrinogen were found on the corresponding chain peptides isolated from sheep fibrinogen. The C-terminal residues of all the chain peptides of human and horse fibrinogens, however, were valine. In hog and dog fibrinogens, proline was identified at the C-termini of the alpha(A)-chains, and C-terminal valine and isoleucine were found on the beta(B)- and gamma-chains, respectively. Thus, the C-terminal amino acid residues of the fibrinogens of all mammalian species tested were very similar. It should be noted that hydrophobic amino acids, like isoleucine, valine and proline, are mainly located in the C-terminal ends of all three chain peptides in the fibrinogen molecule.     

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

The endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells is Ca(+2)-independent and distinct from a Ca(+2)-dependent hyaluronan binding activity.

Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I-hyaluronan (HA) and internalize it using a coated pit pathway [Biochem J, 257:875-884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I-HA binding to intact LECs at 4 degrees C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I-HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca(+2)-independent HA binding activity increased approximately 743%, while the Ca(+2)-dependent binding activity was enhanced only approximately 46%. Therefore, the Ca(+2)-dependent HA binding activity appears not to be intracellular, whereas the Ca(+2)-independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I-HA at 37 degrees C in 10 mM EGTA or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I-HA in the presence of 10 mM Ca+2, the amount of cell-associated radioactivity increased approximately 20-50-fold. However, this additional cell-associated 125I-HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10 mM EGTA at 4 degrees C. Therefore, the Ca(+2)-dependent cell-associated 125I-HA had accumulated on the cell surface and had not been internalized. From these studies we conclude that LECs have at least two types of specific HA binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells

125I-Hyaluronic acid (HA) uniquely modified only at the reducing end (Raja, R.H., LeBoeuf, R. D., Stone, G.W., and Weigel, P.H. (1984) Anal. Biochem. 139, 168-177) binds specifically to rat liver endothelial cells in suspension or in culture. About 67-85% of the HA binding sites in isolated cells in suspension and 50% in cultured cells were intracellular, since they were exposed after permeabilizing cells with digitonin. Specific 125I-HA binding at 4 degrees C varied from 60 to 80% for intact cells and from 70 to 90% for permeabilized cells. Freshly isolated permeabilized cells bound about 500,000 HA molecules/cell at saturation. Within 5 h of culture, however, total HA binding decreased to 250,000 molecules/cells and then remained constant for at least 36 h. Surface HA receptor activity was essentially the same on cultured cells or cells in suspension (approximately 10(5)/cell). Cultured cells had 1.8 x 10(5) fewer intracellular receptors/cell. The affinities of surface and intracellular receptors of cells in culture and in suspension were essentially the same. The average Kd, determined by equilibrium binding studies, was 5.8 +/- 2.8 x 10(-8) M (n = 12). Dissociation of bound 125I-HA from permeable cultured cells was rapid (t1/2 = 30.9 min;kappa off = 3.7 x 10(-4) s-1). A variety of carbohydrates had essentially identical effects on 125I-HA binding to surface or total cellular receptors in cells in culture or in suspension. Chondroitin sulfate and heparin competed almost as effectively as unlabeled HA for 125I-HA binding at 4 degrees C. Other saccharides including polygalacturonic acid, dextran, glucuronic acid, and N-acetylglucosamine competed poorly or not at all. We conclude that (i) the 125I-HA binding sites within liver endothelial cells are HA receptors, identical in affinity and specificity to those on the cell surface; (ii) the distribution of cellular HA receptors is similar to other receptor systems with about 50-80% being intracellular; (iii) the liver endothelial cell HA receptor recognizes several glycosaminoglycans; and (iv) the liver endothelial receptor is different in function and characteristics than the fibroblast HA receptor.     

Species variation in the binding of hGH to hepatic membranes

The binding of 125I-labeled human growth hormone (hGH) to liver membranes from several different species was studied to determine the lactogenic or somatotropic hormone nature of the receptors. Liver membranes from several species of the class of Mammalia bound significant quantities of 125I-hGH. Goat, sheep, rat, mouse, and rabbit liver membranes exhibited the highest binding with cow, pig, human, and hamster liver membranes exhibiting severalfold less binding. The binding of the dog and cat liver membranes exhibited relatively high nonspecific binding. Fish and chicken liver membranes did not bind appreciable quantities of 125I-hGH. In all species except for dog and cat in which 125I-hGH bound to the membranes, hGH was the most effective competitor for binding. The mean ID50 for hGH and all membranes was 2.4 X 10(-9) M. Human liver membranes exhibited the smallest ID50, 4.9 X 10(-10) M. In sheep liver membranes, bovine growth hormone (bGH) was equipotent to hGH in competing for 125I-hGH binding. bGH also demonstrated significant competition for 125I-hGH binding in pig and cow membranes. Ovine prolactin (oPrl) exhibited significant competition for 125I-hGH only in rodent membranes. The ID50 for oPrl was 3- to 10-fold greater than for hGH in the rat, hamster, and mouse liver membranes. The ID50 for oPrl in the sheep liver membranes was 13-fold greater than that of hGH. We conclude the following: (1) There appears to be a species specificity of hGH binding that may be phylogenetically significant and may result from variations in the structure of the hormone or the receptor. (2) The competitive binding properties of hGH are fairly consistent within phylogenetic orders. (3) The simple designation of lactogenic or somatotropic for hormones and receptors is insufficient to characterize the binding properties of this group of hormones.     

Comparison of mammalian VIP bioactivities in dispersed acini from guinea pig pancreas

Mammalian VIP is identical in pig, cow, human, rat, dog and goat but differs in the guinea pig (GP) in positions 5, 9, 19, and 26. We now demonstrate that GP, goat, rat and synthetic mammalian VIP are indistinguishable in their inhibition of binding of 125I-labelled synthetic VIP to dispersed acini from GP pancreas and that GP, pig, dog, goat and synthetic VIP are also similar in their efficacy and potency in stimulating amylase release from these acini. Thus in spite of the differences in amino acid sequence, GP VIP appears to have full biologic potency in its action on dispersed acini from GP pancreas.     

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 approximately 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 approximately 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.     

Common senescent cell-specific antibody epitopes on fibronectin in species and cells of varied origin.

The phenomenon of in vitro cellular senescence has been demonstrated in cultured cells derived from humans and various other species. We have previously shown that monoclonal antibodies SEN-1, SEN-2, and SEN-3 react to epitopes on fibronectin that are exposed when human diploid fibroblasts become senescent. We here present results demonstrating that exposure of these epitopes is specific to senescence for a variety of human cells: epidermal keratinocytes, mammary epithelial cells, as well as fibroblasts. Fibronectin from 11 additional species was also analyzed by Western immunoblot for ability to bind the SEN antibodies. SEN-1 bound only human and gorilla fibronectin, whereas SEN-2 and SEN-3 bound fibronectin from those two species as well as the horse, cow, sheep, goat, dog, and chick. None of the antibodies reacted with fibronectin from the rabbit, rat, or mouse. These data indicated a correlation between the ability of the SEN antibodies to bind fibronectin from a particular species and the ability of cells from that species to exhibit a stable senescent phenotype in vitro. Therefore, exposure of this region of fibronectin may be important in the establishment and maintenance of cellular senescence. In addition, the ability of the SEN antibodies to react with fibronectin from a variety of senescent cells emphasizes their usefulness as markers for cellular senescence.     

The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma

Background

Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings

Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions

Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

     

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A

The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.     

Interaction of heterologous fibrinogens with human platelet receptors.

The interaction of ADP-stimulated human platelets with human 125I-fibrinogen as well as with pig and bovine fibrinogens was analysed. It was found that the fibrinogens studied were bound to the same platelet receptors but the affinity of animal preparations was about half the value observed for human fibrinogen (in a homologous system).     

The existence of galactosylceramide I3-sulfate in serums of various mammals and its anticoagulant activity.

Human, porcine, goat, sheep, bovine, horse, canine, rat, mouse, guinea pig, and chicken serums were investigated for the existence of sulfatide. Among the ten mammal serums, seven were found to be sulfatide positive, and the amounts of sulfatide were determined to be: 16.29 nmol/ml serum (porcine), 9.39 (bovine), 12.71 (goat), 7.75 (horse), 1.21 (sheep), 0.64 (human), and 0.16 (dog). The existence of sulfatide in the serums of human, goat, sheep, cow, horse, and dog is here reported for the first time. It is suggested that sulfatide is widely distributed in the serums of various mammals except for rodents and that it takes part in the anticoagulant systems. The fatty acids of those sulfatides comprised mainly non-hydroxy fatty acids and a significant amount (18-53% of the total fatty acid) of hydroxy fatty acids with chain lengths of C16, C22, C23, and C24. The long chain bases comprised sphingenine, sphinganine, and 4-D-hydroxysphinganine. Experiments to elucidate the mechanism of the anticoagulant activity of sulfatide revealed that it was specific to sulfatide and that the galactose-bound sulfate group is essential for this activity. The activity of clusters of sulfatide molecules was much more pronounced than that of single molecules.     

Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in 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). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains approximately 25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous (125)I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was approximately 8.5 min. The average K(d) for the binding of HA to the 190-kDa hHARE at 4 degrees C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 degrees C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 degrees C, none of the glycosaminoglycans tested, except HA, competed for (125)I-HA binding by 190hHARE cells at 4 degrees C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit (125)I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.     

Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling.

Hyaluronic acid (HA) is cleared from the blood by liver endothelial cells through receptor-mediated endocytosis [Eriksson, Fraser, Laurent, Pertoft & Smedsrod (1983) Exp. Cell Res. 144, 223-238]. We have measured the capacity of cultured rat liver endothelial cells to endocytose and degrade 125I-HA (Mr approximately 44,000) at 37 degrees C. Endocytosis was linear for 3 h and then reached a plateau. The rate of endocytosis was concentration-dependent and reached a maximum of 250 molecules/s per cell. Endocytosis of 125I-HA was inhibited more than 92% by a 150-fold excess of non-radiolabelled HA. HA, chondroitin sulphate and heparin effectively competed for endocytosis of 125I-HA, whereas glucuronic acid, N-acetylglucosamine, DNA, RNA, polygalacturonic acid and dextran did not compete. In the absence of cycloheximide, endothelial cells processed 13 times more 125I-HA in 6 h than their total (cell-surface and intracellular) specific HA-binding capacity. This result was not due to degradation and rapid replacement of receptors, because, even in the presence of cycloheximide, these cells processed 6 times more HA than their total receptor content in 6 h. Also, in the presence of cycloheximide, no decrease in 125I-HA-binding capacity was seen in cells processing or not processing HA for 6 h, indicating that receptors are not degraded after the endocytosis of HA. During endocytosis of HA at 37 degrees C, at least 65% of the intracellular HA receptors became occupied with HA within 30 min. This indicates that the intracellular HA receptors (75% of the total) function during continuous endocytosis. Hyperosmolarity inhibits endocytosis and receptor recycling in the asialoglycoprotein and low-density-lipoprotein receptor systems by disrupting the coated-pit pathway [Heuser & Anderson (1987) J. Cell Biol. 105, 230a; Oka & Weigel (1988) J. Cell. Biochem. 36, 169-183]. Hyperosmolarity inhibited 125I-HA endocytosis in liver endothelial cells by more than 90%, suggesting use of a coated-pit pathway by this HA receptor. We conclude that liver endothelial cell HA receptors are recycled during the continuous endocytosis and processing of HA.     

A blocking antibody to the hyaluronan receptor for endocytosis (HARE) inhibits hyaluronan clearance by perfused liver

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.     

Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.