NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

Abstract

Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca 2+ binding sites (sites 1, 2 and 3), with Ca 2+ at site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptorligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in the early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca 2+ and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca 2+ binding sites, site 3 is low affinity, and that Ca 2+ ions bind to sites 1 and 2 cooperatively. The pH and Ca 2+ concentration dependences of Ca 2+ binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca 2+ makes it possible for ASGP-R to be more sensitive to Ca 2+ concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca 2+ and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.

Computational and Experimental Prediction of Human C-Type Lectin Receptor Druggability

“…We argue that a druggable pocket located in close proximity of the long loop renders it a potential binding site for an inhibitor. The loop exhibits considerable movement in the absence of calcium as observed for other CRDs (65, 67, 83, 84) and adjacent sites have been proposed to communicate with the primary carbohydrate recognition site (22, 23). Four categories of sites were defined out of which only two, namely categories (i) and (ii), are either directly or indirectly associated with calcium ion binding.…”

“…We assumed the existence of allosteric sites originating from the flexibility of the long loop and cooperativity between the adjacent sites as previously described for other CTLRs (22, 23, 65, 67, 83, 84). In this context, it should be noted that accounting for conformational dynamics is recognized as a particular challenge for the development of improved algorithms (34).…”

“…Canonical carbohydrate recognition is mediated by a calcium ion and although there are four Ca 2+ binding sites, only the second site (Ca 2+ -2) is described to be involved in coordinating glycans (21). While Ca 2+ -4 has not been associated with carbohydrate binding, positive cooperative effects are observed between the other sites (22, 23). Not all potential Ca 2+ sites are occupied in every CTLD, which reflects the fine-tuned physiological role of this interaction.…”

“…Not all potential Ca 2+ sites are occupied in every CTLD, which reflects the fine-tuned physiological role of this interaction. For endocytic CTLRs the pH sensitivity of the heptad-repeat neck formation and Ca 2+ coordination as well as active Ca 2+ export from the endosome are major contributors to endosomal ligand release (23, 24). Some CTLRs bind carbohydrates in a Ca 2+ -independent, non-canonical binding site with Dectin-1 being the prime example (25).…”

Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy

“…When ASGPRs bind its targeted ligands, the complexes can be internalized by clathrin-mediated endocytosis, and the ligands are released from ASGPRs to allow receptors to recycle to the cell membrane. The key to keeping the concentration of receptors on cell surfaces is the fast cycling of the internalized receptors 3. Owing to the specific binding of galactose to ASGPR, galactose moieties can be used as targeted ligands for drug delivery and internalized by ASGPR-mediated endocytosis 4, 5.…”