Mechanism of pH-dependent N-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor

Efficient release of ligands from the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of the hepatic asialoglycoprotein receptor at endosomal pH requires a small set of conserved amino acids that includes a critical histidine residue. When these residues are incorporated at corresponding positions in an homologous galactose-binding derivative of serum mannose-binding protein, the pH dependence of ligand binding becomes more like that of the receptor. The modified CRD displays 40-fold preferential binding to N-acetylgalactosamine compared with galactose, making it a good functional mimic of the asialoglycoprotein receptor. In the crystal structure of the modified CRD bound to N-acetylgalactosamine, the histidine (His(202)) contacts the 2-acetamido methyl group and also participates in a network of interactions involving Asp(212), Arg(216), and Tyr(218) that positions a water molecule in a hydrogen bond with the sugar amide group. These interactions appear to produce the preference for N-acetylgalactosamine over galactose and are also likely to influence the pK(a) of His(202). Protonation of His(202) would disrupt its interaction with an asparagine that serves as a ligand for Ca(2+) and sugar. The structure of the modified CRD without sugar displays several different conformations that may represent structures of intermediates in the release of Ca(2+) and sugar ligands caused by protonation of His(202).     

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

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 receptor-ligand 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.     

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.     

Minimal requirements for the binding of selectin ligands to a C-type carbohydrate-recognition domain.

The C-type carbohydrate-recognition domains of E-selectin and rat serum mannose-binding protein have similar structures. Selectin/mannose-binding protein chimeras created by transfer of key sequences from E-selectin into mannose-binding protein have previously been shown to bind the selectin ligand sialyl-Lewis(X) through a Ca(2+)-dependent subsite, common to many C-type lectins, and an accessory site containing positively charged amino acid residues. Further characterization of these chimeras as well as analysis of novel constructs containing additional regions of E-selectin demonstrate that selectin-like interaction with sialyl-Lewis(X) can be faithfully reproduced even though structural evidence indicates that the mechanisms of binding to E-selectin and the chimeras are different. Selectin-like binding to the nonfucosylated sulfatide and sulfoglucuronyl glycolipids can also be reproduced with selectin/mannose-binding protein chimeras that contain the two subsites involved in sialyl-Lewis(X) binding. These results indicate that binding of structurally distinct anionic glycans to C-type carbohydrate-recognition domains can be mediated by the Ca(2+)-dependent subsite in combination with a positively charged region that forms an ionic strength-sensitive subsite.     

Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor

The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes.Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 A resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not alpha-helical, while in tetranectin they form an alpha-helical coiled-coil.     

Histidine pK(a) values for the N-lobe of human transferrin: effect of substitution of binding site Asp by Ser (D63S)

The pK(a) values have been determined for eight of the nine histidine residues and the amino terminus of the N-lobe of human apo-transferrin (hTF/2N), and for seven of the nine histidine residues and the amino terminus of the protein Asp63Ser hTF/2N containing a mutation of the Fe(3+)-ligand Asp63 to Ser63. Calculations suggested that substitution of aspartate by serine would result in decreases of the pK(a) values of most of the histidine residues in the protein. This was found to be the case experimentally, and allowed assignment of the varepsilonCH resonance of His249. For the wild-type protein, the His residue with a pK(a) of 7.40 was assigned as His249, whereas for the mutant, no observable His residue had a pK(a) value higher than 6.9. The protonated form of His249 appears to be stabilised by interactions with Asp63, and the high pK(a) value may be critical for ensuring the release of iron at endosomal pH (5.5). The mutation lowered the apparent binding constant of hTF/2N for the synergistic anion oxalate from log K 4.0 to log K 3.3. (1)H NMR spectral changes induced by Ga(3+) binding to the mutant are compared to those observed for the wild-type protein.     

Calcium coordination and pH dependence of the calcium affinity of ligand-binding repeat CR7 from the LRP. Comparison with related domains from the LRP and the LDL receptor.

We have determined the X-ray crystal structure to 1.8 A resolution of the Ca(2+) complex of complement-like repeat 7 (CR7) from the low-density lipoprotein receptor-related protein (LRP) and characterized its calcium binding properties at pH 7.4 and 5. CR7 occurs in a region of the LRP that binds to the receptor-associated protein, RAP, and other protein ligands in a Ca(2+)-dependent manner. The calcium coordination is identical to that found in LB5 and consists of carboxyls from three conserved aspartates and one conserved glutamate, and the backbone carbonyls of a tryptophan and another aspartate. The overall fold of CR7 is similar to those of CR3 and CR8 from the LRP and LB5 from the LDL receptor, though the low degree of sequence homology of residues not involved in calcium coordination or in disulfide formation results in a distinct pattern of surface residues for each domain, including CR7. The thermodynamic parameters for Ca(2+) binding at both extracellular and endosomal pHs were determined by isothermal titration calorimetry for CR7 and for related complement-like repeats CR3, CR8, and LB5. Although the drop in pH resulted in a reduction in calcium affinity in each case, the changes were very variable in magnitude, being as low as a 2-fold reduction for CR3. This suggests that a pH-dependent change in calcium affinity alone cannot be responsible for the release of bound protein ligands from the LRP at the pH prevailing in the endosome, which in turn requires one or more other pH-dependent effects for regulating protein ligand release.     

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

Role of conserved and nonconserved residues in the Ca(2+)-dependent carbohydrate-recognition domain of a rat mannose-binding protein. Analysis by random cassette mutagenesis.

The carbohydrate-recognition domain of rat serum mannose-binding protein A has been subjected to random cassette mutagenesis. Mutant domains, expressed in bacteria, were initially screened for binding to invertase-coated nitrocellulose and then analyzed further for Ca2+ affinity, saccharide binding, resistance to proteolysis, and oligomerization. The results are consistent with previous evolutionary and structural studies. Six out of seven completely inactive mutants have changes in residues directly involved in ligating Ca2+. Most changes in conserved residues which form part of the hydrophobic core characteristic of Ca(2+)-dependent (C-type) animal lectins result in decreased affinity for Ca2+, even though these residues are distant from the Ca2+ sites. Changes can be made in large portions of the surface without affecting saccharide binding. The results indicate that the precise arrangement of the regular portion of the domain containing the hydrophobic core is necessary for formation of a stable Ca(2+)-ligated structure under physiological conditions. The data also suggest that the saccharide-binding site is likely to be in close proximity to the bound Ca2+.     

Scavenger receptor C-type lectin binds to the leukocyte cell surface glycan Lewis(x) by a novel mechanism

The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.     

Physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat

A portion of rat mannose-binding protein A (MBP-A), a Ca(2+)-dependent animal lectin, has been overproduced in a bacterial expression system, biochemically characterized, and crystallized. A fragment corresponding to the COOH-terminal 115 residues of native MBP-A, produced by subtilisin digestion of the bacterially expressed protein, contains the carbohydrate-recognition domain (CRD). Gel filtration, chemical cross-linking, and crystallographic self-rotation function analyses indicate that the subtilisin fragment is a dimer, although the complete bacterially expressed fragment, containing the neck and CRD of MBP-A, is a trimer. Crystals of the minimal CRD, obtained only as a complex with a Man6GlcNAc2Asn glycopeptide, diffract to Bragg spacings of at least 1.7 A. Several trivalent lanthanide ions (Ln3+) can substitute for Ca2+, as assessed by their ability to support carbohydrate binding and to protect the CRD from proteolysis in a manner similar to that observed for Ca2+. These assays indicate that Ln2+ binds about 30 times more tightly than Ca2+ to the CRD, and that two Ca2+ or Ln3+ bind to each monomer, a result confirmed by determination of the Ho3+ positions in a Ho(3+)-containing crystal of the CRD. Crystals grown in the presence of Ln3+ belong to different space groups from those obtained with Ca2+ and are therefore not useable for traditional crystallographic phase determination methods, but are well-suited for high resolution structure determination by multiwavelength anomalous dispersion phasing.     

Twists and turns of the cation-dependent mannose 6-phosphate receptor. Ligand-bound versus ligand-free receptor.

Mannose 6-phosphate receptors (MPRs) participate in the biogenesis of lysosomes in higher eukaryotes by transporting soluble acid hydrolases from the trans-Golgi network to late endosomal compartments. The receptors release their ligands into the acidic environment of the late endosome and then return to the trans-Golgi network to repeat the process. However, the mechanism that facilitates ligand binding and dissociation upon changes in pH is not known. We report the crystal structure of the extracytoplasmic domain of the homodimeric cation-dependent MPR in a ligand-free form at pH 6.5. A comparison of the ligand-bound and ligand-free structures reveals a significant change in quaternary structure as well as a reorganization of the binding pocket, with the most prominent change being the relocation of a loop (residues Glu(134)-Cys(141)). The movements involved in the bound-to-free transition of the cation-dependent MPR are reminiscent of those of the oxy-to-deoxy hemoglobin transition. These results allow us to propose a mechanism by which the receptor regulates its ligand binding upon changes in pH; the pK(a) of Glu(133) appears to be responsible for ligand release in the acidic environment of the late endosomal compartment, and the pK(a) values of the sugar phosphate and His(105) are accountable for its inability to bind ligand at the cell surface where the pH is about 7.4.     

Molecular studies of pH-dependent ligand interactions with the low-density lipoprotein receptor

The release of ligand from the low-density lipoprotein receptor (LDLR) has been postulated to involve a "histidine switch"-induced intramolecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescently tagged variant apolipoprotein E N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp-null apoE3-NT. In binding experiments with wild-type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562, and His586 in sLDLR in pH-dependent ligand binding and discharge, site-directed mutagenesis studies were performed. Compared to WT sLDLR, triple His --> Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and a decreased level of ligand release as a function of low pH. When these His residues were substituted for Lys, the positively charged side chain of which does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, the evidence suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased when the pH was reduced from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys/His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization.     

Selective binding of the scavenger receptor C-type lectin to Lewisx trisaccharide and related glycan ligands

The scavenger receptor C-type lectin (SRCL) is an endothelial receptor that is similar in organization to type A scavenger receptors for modified low density lipoproteins but contains a C-type carbohydrate-recognition domain (CRD). Fragments of the receptor consisting of the entire extracellular domain and the CRD have been expressed and characterized. The extracellular domain is a trimer held together by collagen-like and coiled-coil domains adjacent to the CRD. The amino acid sequence of the CRD is very similar to the CRD of the asialoglycoprotein receptor and other galactose-specific receptors, but SRCL binds selectively to asialo-orosomucoid rather than generally to asialoglycoproteins. Screening of a glycan array and further quantitative binding studies indicate that this selectivity results from high affinity binding to glycans bearing the Lewis(x) trisaccharide. Thus, SRCL shares with the dendritic cell receptor DC-SIGN the ability to bind the Lewis(x) epitope. However, it does so in a fundamentally different way, making a primary binding interaction with the galactose moiety of the glycan rather than the fucose residue. SRCL shares with the asialoglycoprotein receptor the ability to mediate endocytosis and degradation of glycoprotein ligands. These studies suggest that SRCL might be involved in selective clearance of specific desialylated glycoproteins from circulation and/or interaction of cells bearing Lewis(x)-type structures with the vascular endothelium.     

Focusing of the electrostatic potential at EF-hands of calbindin D(9k): titration of acidic residues

Biological functions for a large class of calmodulin-related proteins, such as target protein activation and Ca(2+) buffering, are based on fine-tuned binding and release of Ca(2+) ions by pairs of coupled EF-hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK(a) values for all aspartic and glutamic acid residues in apo calbindin D(9k), a representative of calmodulin-related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca(2+) ligands with high pK(a) under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca(2+) ligand of each site, and that the potential is significantly more negative at the N-terminal binding site.     

The three-dimensional structure of codakine and related marine C-type lectins

Codakine is a new Ca(2+)-dependent mannose-binding C-type lectin (MBL) isolated from the gill tissue of the tropical clam, Codakia orbicularis. Bioinformatic analyses with the BLAST program have revealed similarities with marine lectins involved in immunity whose three-dimensional (3D) structures were unknown up until recently. In this article, we present bioinformatic analyses of marine lectins that are homologous to codakine, in particular lectins from the sea worm Laxus oneistus, named mermaid. These lectins are involved in the symbiotic association with sulphur-oxidizing bacteria which are closely related to the C. orbicularis gill symbiont. Using homology modelling, folding that is characteristic of C-type lectins was observed in all the marine Ca(2+)-dependent lectins studied, with conservation of random coiled structures of the carbohydrate recognition domain (CRD) and Ca(2+)-binding sites. Like codakine, the marine lectins analysed contain a signal peptide commonly found in secreted and transmembrane proteins. The majority of the predictive 3D models established from the lectins exhibit a common feature, namely the involvement in invertebrate and vertebrate immunity (dendritic cell receptor, macrophage receptor, etc.). These bioinformatic analyses and the literature data support the hypothesis that codakine, like the L. oneistus mermaids, is probably involved in the cellular mediation of symbiosis and defence against pathogenic microorganisms.     

Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin

The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.     

Crystal structure of human CD69: a C-type lectin-like activation marker of hematopoietic cells

CD69 is a widely expressed type II transmembrane glycoprotein related to the C-type animal lectins that exhibits regulated expression on a variety of cells of the hematopoietic lineage, including neutrophils, monocytes, T cells, B cells, natural killer (NK) cells, and platelets. Activation of T lymphocytes results in the induced expression of CD69 at the cell surface. In addition, cross-linking of CD69 by specific antibodies leads to the activation of cells bearing this receptor and to the induction of effector functions. However, the physiological ligand of CD69 is unknown. We report here the X-ray crystal structure of the extracellular C-type lectin-like domain (CTLD) of human CD69 at 2.27 A resolution. Recombinant CD69 was expressed in bacterial inclusion bodies and folded in vitro. The protein, which exists as a disulfide-linked homodimer on the cell surface, crystallizes as a symmetrical dimer, similar to those formed by the related NK cell receptors Ly49A and CD94. The structure reveals conservation of the C-type lectin-like fold, including preservation of the two alpha-helical regions found in Ly49A and mannose-binding protein (MBP). However, only one of the nine residues coordinated to Ca(2+) in MBP is conserved in CD69 and no bound Ca(2+) is evident in the crystal structure. Surprisingly, electron density suggestive of a puckered six-membered ring was discovered at a site structurally analogous to the ligand-binding sites of MBP and Ly49A. This sugar-like density may represent, or mimic, part of the natural ligand recognized by CD69.