Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

Ray, Darryl A.; Oka, Janet A.; Weigel, Paul H.

doi:10.1021/bi00368a039

Cited by 5 publications

(3 citation statements)

References 40 publications

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“…(iii) The two complete trimeric sugar binding sites can be simultaneously occupied by small glycopeptide ligands, but large ligands bound to one domain sterically preclude binding to the adjacent one (Hardy et al, 1985). (iv) In the presence of some nonionic detergents there is a doubling of the valency of the affinity-purified receptor even when immobilized on Sepharose (Ray et al, 1986). Presumably, the hexamer is distorted by the detergent to allow the second binding domain to also bind ligand, (v) The mole ratio of RHL 1, 2, and 3 in affinity-purified receptor preparations will be 4:1:1.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Herzig¹,

Weigel²

1989

Biochemistry

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We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.

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Section: Resultsmentioning

confidence: 99%

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Herzig¹,

Weigel²

1989

Biochemistry

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“…Experiments to determine whether surface vs internal receptors are preferentially affected by mannitol have thus far been inconclusive. One possibility is that at low concentrations mannitol can act like a weak nonionic detergent and stimulate the binding of ligand by increasing the valency of receptor [27]. Although Stowell et al [28] and Sarkar et al [29] have mapped the binding site of the mammalian hepatocyte lectin by using a variety of saccharides, glycoproteins , and neoglycoproteins, to our knowledge no one has examined alditols, such as mannitol, for inhibitory capability.…”

Section: Discussionmentioning

confidence: 99%

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Oka

Weigel

1988

J of Cellular Biochemistry

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Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.

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“…If this is true, a large ligand, such as ASOR, may be bound to the hepatocyte membrane receptors primarily by the 52-kDa (and the 60-kDa) bands. Recently, Ray et al (1986) showed that certain detergents, such as Triton X-100, increased the number of 125I-ASOR bound to permeabilized-fixed hepatocytes more than 2-fold. This increased binding capacity can be explained as the result of the Triton-induced, increased availability of the Gal combining site(s) of the 43-kDa subunit.…”

Section: Discussionmentioning

confidence: 99%

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

Lee¹,

Lee²

1987

Biochemistry

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The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which [43 kilodalton (kDa)] exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an 125I-labeled high-affinity reagent [a triantennary glycopeptide containing an aryl azide group on galactosyl residues; Lee, R. T., & Lee, Y. C. (1986) Biochemistry 25, 6835-6841], the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. We postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work [Hardy, M. R., Townsend, R. R., Parkhurst, S. M., & Lee, Y. C. (1985) Biochemistry 24, 22-28].

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Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

Cited by 5 publications

References 40 publications

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

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