Serum Mannose-binding Protein Research Articles

Particular Authoritative of N-acetylglucosamine to the Chicken Hepatic Lectin

Among Ca2+-dependent (C-type) creature lectins, the chicken hepatic lectin (CHL) is special in showing nearly total selectivity for N-acetylglucosamine over other monosaccharide ligands. The gem structures of the carbohydrate-recognition space (CRD) from serum mannose-binding protein (MBP) and of a complex between the CRD from liver MBP and the methyl glycoside of N-acetylglucosamine were utilized to show the official location in CHL. Substitution of parcels of CHL into the MBP system did not considerably increment selectivity. A bacterial expression framework for the CRD of CHL was created so that particular buildups anticipated to be close the 2-acetamido substituent of N-acetylglucosamine might be changed by site-directed mutagenesis. The comes about indicate that the ligand is bound to CHL within the same introduction because it ties to liver MBP. A tyrosine and a valine buildup that likely contact the the N-acetyl bunch have been recognized.

Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system.

The β1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the β1,2-gluco-oligosaccharides, with degrees of polymerization 2–13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the β1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CβG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by β1,2-glucans in mammalian systems.

Organization of the extracellular portion of the macrophage galactose receptor: A trimeric cluster of simple binding sites for N-acetylgalactosamine

The properties of the human macrophage galactose receptor have been investigated. Specificity for N-acetylgalactosamine (GalNAc) residues with exposed 3- and 4-hydroxyl groups explains virtually all of the results obtained from a recently expanded array of synthetic glycans and is consistent with a model for the structure of the binding site. This simple interaction is sufficient to explain the ability of the receptor to bind to tumor-cell glycans bearing Tn and sialyl-Tn antigens, but not to more elaborate O-linked glycans that predominate on normal cells. This specificity also allows for binding of parasite glycans and screening of an array of bacterial outer membrane oligosaccharides confirms that the receptor binds to a subset of these structures with appropriately exposed GalNAc residues. A key feature of the receptor is the clustering of binding sites in the extracellular portion of the protein, which retains the trimeric structure observed in the cell membrane. Chemical crosslinking, gel filtration, circular dichroism analysis and differential scanning calorimetry demonstrate that this trimeric structure of the receptor is stabilized by an α-helical coiled coil that extends from the surface of the membrane to the globular carbohydrate-recognition domains. The helical neck domains form independent trimerization domains. Taken together, these results indicate that the macrophage galactose receptor shares many of the features of serum mannose-binding protein, in which clusters of monosaccharide-binding sites serve as detectors for a simple epitope that is not common on endogenous cell surface glycans but that is abundant on the surfaces of tumor cells and certain pathogens.

Conformations, dynamics and interactions of di-, tri- and pentamannoside with mannose binding lectin: a molecular dynamics study

The binding of serum mannose-binding protein A (MBP-A) to high mannose N-linked glycoproteins, present on the surface of microorganism, activates the complement system. It is very important to explore the overall conformations of these ligands in the binding site of the MBP-A, which is very much dependent on the conformation of the manno-di-, tri- and the penta-saccharides that represent the component structures of these high-mannose type oligosaccharides. Herein, we report the possible conformations of α-(1→6)-linked dimannoside, benzyl-substituted trimannoside and core pentamannoside of the N-linked glycan in the binding site of MBP-A, with the help of molecular dynamics simulations. The results indicate that for all three ligands in addition to the non-reducing terminal mannose moiety the reducing moieties also interact with protein. Binding free energy calculations also indicate that the benzyl-substituted trisaccharide has higher affinity in comparison to the methyl substituted one. We have also found some conformers of the pentasaccharide, which have higher binding affinity than the monosaccharide.

Targeted glycoproteomic identification of cancer cell glycosylation

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewisx and Lewisy structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewisx antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Widely Divergent Biochemical Properties of the Complete Set of Mouse DC-SIGN-related Proteins

The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose-terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors.

Two Mechanisms for Mannose-binding Protein Modulation of the Activity of Its Associated Serine Proteases

Serum mannose-binding protein (MBP) neutralizes invading microorganisms by binding to cell surface carbohydrates and activating MBP-associated serine proteases-1, -2, and -3 (MASPs). MASP-2 subsequently cleaves complement components C2 and C4 to activate the complement cascade. To analyze the mechanisms of activation and substrate recognition by MASP-2, zymogen and activated forms have been produced, and MBP.MASP-2 complexes have been created. These preparations have been used to show that MBP modulates MASP-2 activity in two ways. First, MBP stimulates MASP-2 autoactivation by increasing the rate of autocatalysis when MBP.MASP-2 complexes bind to a glycan-coated surface. Second, MBP occludes accessory C4-binding sites on MASP-2 until activation occurs. Once these sites become exposed, MASP-2 binds to C4 while separate structural changes create a functional catalytic site able to cleave C4. Only activated MASP-2 binds to C2, suggesting that this substrate interacts only near the catalytic site and not at accessory sites. MASP-1 cleaves C2 almost as efficiently as MASP-2 does, but it does not cleave C4. Thus MASP-1 probably enhances complement activation triggered by MBP.MASP-2 complexes, but it cannot initiate activation itself.

Affinity chromatography with monolithic capillary columns: I. Polymethacrylate monoliths with immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography

Monolithic capillary columns with surface-immobilized mannan have been introduced for affinity-based micro-column separations by nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylate– co-ethylene dimethacrylate) and poly(glycidyl methacrylate– co-ethylene dimethacrylate– co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. While neutral monoliths with immobilized mannan were only useful for affinity nano-LC, the cationic monoliths with surface-bound mannan were useful in both affinity nano-LC and affinity CEC. The cationic monoliths allowed a relatively high electro-osmotic flow (EOF) when mannan was immobilized to the epoxy monolith via a positively charged spacer arm, triethylenetetramine. The neutral monoliths exhibited lower permeability under pressure-driven flow (PDF) than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized mannan exhibited strong affinity toward mannose-binding proteins (MBP) such as the plant lectins concanavalin A and Lens culinaris agglutinin and a mammalian lectin (e.g. rabbit serum mannose-binding protein). Due to the strong binding affinity, the monoliths with surface bound mannan allowed the injection of large volume of rabbit serum and to isolate in a single run the mannose-binding protein in an amount sufficient to run with it sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), thus demonstrating their capability in “nano-proteomics”.

Affinity chromatography with monolithic capillary columnsI. Polymethacrylate monoliths with immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography

Monolithic capillary columns with surface-immobilized mannan have been introduced for affinity-based micro-column separations by nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylate–co-ethylene dimethacrylate) and poly(glycidyl methacrylate–co-ethylene dimethacrylate–co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. While neutral monoliths with immobilized mannan were only useful for affinity nano-LC, the cationic monoliths with surface-bound mannan were useful in both affinity nano-LC and affinity CEC. The cationic monoliths allowed a relatively high electro-osmotic flow (EOF) when mannan was immobilized to the epoxy monolith via a positively charged spacer arm, triethylenetetramine. The neutral monoliths exhibited lower permeability under pressure-driven flow (PDF) than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized mannan exhibited strong affinity toward mannose-binding proteins (MBP) such as the plant lectins concanavalin A and Lens culinaris agglutinin and a mammalian lectin (e.g. rabbit serum mannose-binding protein). Due to the strong binding affinity, the monoliths with surface bound mannan allowed the injection of large volume of rabbit serum and to isolate in a single run the mannose-binding protein in an amount sufficient to run with it sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), thus demonstrating their capability in “nano-proteomics”.

Localization of the Serine Protease-binding Sites in the Collagen-like Domain of Mannose-binding Protein: INDIRECT EFFECTS OF NATURALLY OCCURRING MUTATIONS ON PROTEASE BINDING AND ACTIVATION

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.

Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2.

Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca(2+)-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity.

Orientation of Bound Ligands in Mannose-binding Proteins: IMPLICATIONS FOR MULTIVALENT LIGAND RECOGNITION

Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca(2+) coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or alpha1-6-linked mannose bind to MBP-A in one orientation, whereas alpha1-2- or alpha1-3-linked mannose binds in an orientation rotated 180 degrees around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His(189) to its MBP-C equivalent, valine, causes Man alpha 1-3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands.

Dominant effects of mutations in the collagenous domain of mannose-binding protein.

Individuals heterozygous for mutant alleles encoding serum mannose-binding protein (MBP, also known as mannose-binding lectin) show increased susceptibility to infections caused by a wide range of pathogenic microorganisms. To investigate the molecular defects associated with heterozygosity, wild-type rat serum MBP polypeptides (MBP-A: 56% identical in sequence to human MBP) and rat MBP polypeptides containing mutations associated with human immunodeficiency have been coexpressed using a well-characterized mammalian expression system. The resulting proteins are secreted almost exclusively as heterooligomers that are defective in activating the complement cascade. Functional defects are caused by structural changes to the N-terminal collagenous and cysteine-rich domains of MBP, disrupting interactions with associated serine proteases. The dominant effects of the mutations demonstrate how the presence of a single mutant allele gives rise to the molecular defects that lead to the disease phenotype in heterozygous individuals.

Structural and Functional Aspects of Complement Activation by Mannose-binding Protein

Serum mannose-binding protein (MBP) is the first component of the lectin pathway of the complement cascade. It binds to sugars on the surface of pathogenic microorganisms and triggers complement fixation by activating an associated serine protease, designated MBP-associated serine protease-2 (MASP-2). Recent studies have provided insight into the interactions between MBP and MASP-2 that trigger complement activation. MBP/MASP complexes share many features with the C1 complex of the classical pathway. The relatively simple MBP/MASP complexes serve as useful models for understanding activation of the classical pathway of the complement cascade.

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.

Stoichiometry of Complexes between Mannose-binding Protein and Its Associated Serine Proteases: DEFINING FUNCTIONAL UNITS FOR COMPLEMENT ACTIVATION

Serum mannose-binding protein (MBP) initiates the lectin branch of the complement cascade by binding to sugars on the surfaces of microorganisms and activating two MBP-associated serine proteases (MASP-1 and MASP-2). Rat serum MBP consists of oligomers containing up to four copies of a subunit that is composed of three identical polypeptide chains. Biophysical analysis of intact and truncated MASPs indicates that each MASP is a homodimer that is stabilized through interactions involving an N-terminal CUB domain. The binding sites for MBP are formed from the three N-terminal MASP domains, in which two CUB modules interact with MBP. Each MASP dimer contains binding sites for two MBP subunits. Both sites must be occupied by subunits from a single MBP oligomer to form a stable complex. Thus, the smallest functional unit for complement activation consists of MBP dimers bound to MASP-1 or MASP-2 homodimers. Trimers and tetramers of MBP form complexes containing up to two MASPs. The results reveal how MASP-1 and MASP-2 can function independently to activate the complement cascade.

Mechanism of pH-dependentN-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).

Impaired secretion of rat mannose-binding protein resulting from mutations in the collagen-like domain.

Serum mannose-binding protein (MBP) or mannose-binding lectin initiates the lectin branch of the innate immune response by binding to the surface of potentially pathogenic microorganisms and initiating complement fixation through an N-terminal collagen-like domain. Mutations in this region of human MBP are associated with immunodeficiency resulting from a reduction in the ability of the mutant MBPs to fix complement as well as from reduced serum concentrations. Inefficient secretion of the mutant proteins, which is one possible cause of the reduced serum levels, has been investigated using a mammalian expression system in which each of the naturally occurring human mutations has been recreated in rat serum MBP. The mutations Gly25-->Asp and Gly28-->Glu disrupt the disulfide-bonding arrangement of the protein and cause at least a 5-fold increase in the half-time of secretion of MBP compared with wild-type rat serum MBP. A similar phenotype, including a 3-fold increase in the half-time of secretion, disruption of the disulfide bonding arrangement, and inefficient complement fixation, is observed when nearby glucosylgalactosyl hydroxylysine residues at positions 27 and 30 are replaced with arginine residues. The results suggest that defective secretion resulting from structural changes in the collagen-like domain is likely to be a contributory factor for MBP immunodeficiency.

Surfactant protein A binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity.

Collectins are a family of calcium-dependent collagenous lectins that appear to be important in innate host defense. We investigated the ability of three human collectins, namely, lung surfactant proteins A (SP-A) and D (SP-D) and the serum mannose-binding protein (MBP), to bind to the surface glycoproteins of respiratory syncytial virus (RSV). SP-A was shown to bind to the F (fusion) glycoprotein but not to the viral G (attachment) glycoprotein, and binding was completely abrogated in the presence of EDTA. Neither SP-D nor MBP bound to either glycoprotein. SP-A also neutralized RSV in a calcium dependent fashion. These results support a role for SP-A in the defense of infants against infection with RSV and indicate a possible mechanism for its protective activity.

Identification of Amino Acid Residues That Determine pH Dependence of Ligand Binding to the Asialoglycoprotein Receptor during Endocytosis

The rat hepatic asialoglycoprotein receptor mediates clearance of galactose- and N-acetylgalactosamine-terminated glycoproteins by endocytosis, binding ligands through a C-type, Ca(2+)-dependent carbohydrate-recognition domain (CRD) at extracellular pH and releasing them at lower pH in endosomes. At physiological Ca(2+) concentrations, the midpoint for ligand release from the CRD of the major subunit of the receptor is pH 7.1. In contrast, the midpoint is pH 5.0 for a galactose-binding derivative of the homologous C-type CRD of serum mannose-binding protein, which would thus not efficiently release ligand at an endosomal pH of 5.4. Site-directed mutagenesis of the CRD from the major subunit of the asialoglycoprotein receptor has been used to identify residues that are essential for efficient release of ligand at endosomal pH. The effects of changes to residues His(256), Asp(266), and Arg(270) singly and in combination indicate that these residues reduce the affinity of the CRD for Ca(2+), so that ligands are released at physiological Ca(2+) concentrations. The proximity of these three residues to the ligand-binding site at Ca(2+) site 2 of the domain suggests that they form a pH-sensitive switch for Ca(2+) and ligand binding. Introduction of histidine and aspartic acid residues into the mannose-binding protein CRD at positions equivalent to His(256) and Asp(266) raises the pH for half-maximal binding of ligand to 6.1. The results, as well as sequence comparisons with other C-type CRDs, confirm the importance of these residues in conferring appropriate pH dependence in this family of domains.