Lectin-Carbohydrate Interactions: Fine Specificity Difference Between Two Mannose-Binding Proteins

Two types of rat mannose-binding proteins (MBPs), MBP-A (serum type) and MBP-C (liver type), have similar binding specificity for monosaccharide and similar binding site construct according to the X-ray structure, but exhibit different affinity toward natural oligosaccharides and glycoproteins. To understand the basis for this phenomenon, we used cloned fragment of MBP-A and -C (entire carbohydrate-recognition domain and a short connecting piece) that exists as stable trimers in various binding studies. Binding of a number of mannose-containing di- and tri-saccharides and high-mannose type oligosaccharides indicated that MBP-C has an extended binding area of weak interaction with the second and the third mannose residues, whereas MBP-A recognizes just a single mannose residue. In addition, MBP-C has a weak secondary binding site some 25 Å away from the primary site. These findings explain the higher affinity of MBP-C for natural high-mannose type oligosaccharides as compared to MBP-A. A huge affinity differential manifested by natural glycoproteins (e.g., inhibitory potency of thyroglobulin is ~200 fold higher for MBP-C than for MBP-A in a solid-phase assay) may be due to steric hindrance experienced by MBP-A in the competition assay, and suggests different arrangement of subunit in the MBP trimers.     

Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications

Mannose-binding proteins (MBPs), members of the collectin family,have been implicated as lectin opsonins for various virusesand bacteria. Two distinct but related MBPs, MBP-A and MBP-C,with -55% identity at the amino acid level, have been previouslycharacterized from rodents. In humans, however, only one formof MBP has been characterized. In this paper we report studieselucidating the evolution of primate MBPs. ELISA and Westernblot analyses indicated that rhesus and cynomolgus monkeys havetwo forms of MBP in their sera, while chimpanzees have onlyone form, similar to humans. Two distinct MBP cDNA clones wereisolated and characterized from a rhesus monkey liver cDNA library.Rhesus MBP-A is closely related to the mouse and rat MBP-A,showing 77% and 75% identity at the amino acid level, respectively.Rhesus MBP-A also has three cysteines at the N-terminus, similarto mouse and rat MBP-A and human MBP. Rhesus MBP-C shares 90%identity with the human MBP at the amino acid level and hasthree cysteines at the N-terminus, in contrast to two cysteineresidues found in rodent MBP-C. A stretch of nine amino acidsclose to the N-terminus, absent in both mouse and rat MBP-A,but present in rodent MBP-C, chicken and human MBPs, is alsofound in the rhesus MBP-A. The phylogenetic analysis of rhesusand other mammalian MBPs, coupled with the serological datasuggest that at least two distinct MBP genes existed prior tomammalian radiation and the hominoid ancestor apparently lostone of these genes or failed to express it. collectin rhesus monkey mannose-binding protein MBP cDNA mannan-binding protein     

Differential recognition of core and terminal portions of oligosaccharide ligands by carbohydrate-recognition domains of two mannose-binding proteins

Two different mannose-binding proteins (MBP-A and MBP-C), which show 56% sequence identity, are present in rat serum and liver. It has previously been shown that MBP-A binds to a range of monosaccharide-bovine serum albumin conjugates, and that, among oligosaccharide ligands tested, preferential binding is to terminal nonreducing N-acetylglucosamine residues of complex type N-linked oligosaccharides. In order to compare the binding specificity of MBP-C, an expression system has been developed for production of a fragment of this protein which contains the COOH-terminal carbohydrate-recognition domain. After radioiodination, the domain has been used to probe natural glycoproteins, neoglycoproteins, and neoglycolipids. Like MBP-A, MBP-C binds several different monosaccharides conjugated to bovine serum albumin, including mannose, fucose, and N-acetylglucosamine, although binding to the last of these is relatively weaker than observed for MBP-A. The results of binding to natural glycoproteins and to neoglycolipids containing oligosaccharides derived from these proteins are most compatible with the interpretation that MBP-C interacts primarily with the trimannosyl core of complex N-linked oligosaccharides, with additional ligands being terminal fucose and perhaps also peripheral mannose residues of high mannose type oligosaccharides. This binding specificity is thus quite distinct from that of MBP-A. The presence of multiple MBPs with distinct binding specificities in preparations derived from serum and liver explains conflicting conclusions which have been reached about carbohydrate recognition by these proteins.     

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.     

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.     

Exon structure of a mannose-binding protein gene reflects its evolutionary relationship to the asialoglycoprotein receptor and nonfibrillar collagens

Cloned cDNAs encoding mannose-binding proteins isolated from rat liver have been used to isolate one of the genes encoding this group of proteins. This gene, which encodes the minor form of binding protein (designated MBP-A), has been characterized by sequence analysis. The protein-coding portion of the mRNA for the MBP-A is encoded by four exons separated by three introns. The NH2-terminal, collagen-like portion of the protein is encoded by the first two exons. These exons resemble the exons found in the genes for nonfibrillar collagens in that the intron which divides them is inserted between the first two bases of a glycine codon and the exons do not have the 54- or 108-base pair lengths characteristic of fibrillar collagen genes. The carbohydrate-binding portion of MBP-A is encoded by the remaining two exons. This portion of the protein is homologous to the carbohydrate-recognition domain of the hepatic asialoglycoprotein receptor, which is encoded by four exons. It appears that the three COOH-terminal exons of the asialoglycoprotein receptor gene have been fused into a single exon in the MBP-A gene. The organization of the MBP-A gene is very similar to the arrangement of the gene encoding the highly homologous pulmonary surfactant apoprotein, although one of the intron positions is shifted by a single amino acid. The 3' end of a mannose-binding protein pseudogene has also been characterized.     

Multivalent ligand binding by serum mannose-binding protein.

The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.     

Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A). Homology of binding site architecture with mammalian and chicken hepatic lectins

Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA. To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells. The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type. The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required. These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP. Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.     

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.     

Introduction of mannose binding protein-type phosphatidylinositol recognition into pulmonary surfactant protein A.

Pulmonary surfactant protein A (SP-A) and mannose-binding protein A (MBP-A) are collectins in the C-type lectin superfamily. These collectins exhibit unique lipid binding properties. SP-A binds to dipalmitoyl phosphatidylcholine (DPPC) and galactosylceramide (GalCer) and MBP-A binds to phosphatidylinositol (PI). SP-A also interacts with alveolar type II cells. Monoclonal antibodies (mAbs PE10 and PC6) that recognize human SP-A inhibit the interactions of SP-A with lipids and alveolar type II cells. We mapped the epitopes for anti-human SP-A mAbs by a phage display peptide library. Phage selected by mAbs displayed the consensus peptide sequences that are nearly identical to 184TPVNYTNWYRG194 of human SP-A. The synthetic peptide GTPVNYTNWYRG completely blocked the binding of mAbs to human SP-A. Chimeric proteins were generated in which the rat SP-A region Thr174-Gly194 or the human SP-A region Ser174-Gly194 was replaced with the MBP-A region Thr164-Asp184 (rat ama4 or hu ama4, respectively). The mAbs failed to bind hu ama4. Rat ama4 bound to an affinity matrix on mannose-sepharose but lost all of the SP-A functions except carbohydrate binding and Ca2+-independent GalCer binding. Strikingly, the rat ama4 chimera acquired the PI binding property that MBP-A exhibits. This study demonstrates that the amino acid residues 174-194 of SP-A and the corresponding region of MBP-A are critical for SP-A-type II cell interaction and Ca2+-dependent lipid binding of collectins.     

Comparative modeling of the three-dimensional structure of type II antifreeze protein.

Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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).     

cDNAs and deduced amino acid sequences of subunits in the binding component of mouse bactericidal factor, Ra-reactive factor: similarity to mannose-binding proteins.

The complement-dependent bactericidal factor, Ra-reactive factor, binds specifically to Ra polysaccharide, which is common to some strains of Gram-negative enterobacteria, and its is a complex of proteins composed of a polysaccharide-binding component and a component that is presumably responsible for the complement activation. The former component consists of two different 28-kDa polypeptides, P28a and P28b. We determined the partial amino acid sequences of P28a and P28b, and the results indicated that these polypeptides were similar to two species of mannose-binding protein, MBP-C and MBP-A (alternative names, liver and serum mannan-binding proteins, respectively), which have been isolated from rat liver and/or serum [Drickamer, K., Dordal, M. S., & Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., & Yamashina, I. (1987) J. Biochem. 101, 135-144]. Thus, we cloned the respective cDNAs, using as probes synthetic oligonucleotides for which the sequences had been deduced from the amino acid sequences of P28a and P28b and of rat MBP cDNAs. The primary structures of P28a and P28b deduced from the cloned cDNAs are homologous to one another. They have three domains, a short NH2-terminal domain, a collagen-like domain, and a domain homologous to regions of some carbohydrate-binding proteins, as has been reported for rat MBPs. Southern and Northern blotting analyses using these cDNAs indicated that the P28a and P28b polypeptides are the products of two unique mouse genes which are expressed in hepatic cells.     

Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain.

Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor. DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein. The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD. The tail peptide was released from the fusion protein using endoproteinase Arg-C. This method provides an alternative to chemical synthesis for the production of midlength peptides.     

Conformation of a trimannoside bound to mannose-binding protein by nuclear magnetic resonance and molecular dynamics simulations

A model of the carbohydrate recognition domain of the serum form of mannose-binding protein (MBP) from rat complexed with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside is presented. Allowed conformations for the bound sugar were derived from simulated annealing protocols incorporating distance restraints computed from transferred NOESY spectra. The resulting sugar conformations were then modeled into the MBP binding site, and these models of the complex were refined using molecular dynamics (MD) simulations in the presence of solvent water. These studies indicate that only one of the two major conformations of the alpha(1-->6) linkage found in solution is significantly populated in the bound state (omega = 60 degrees ), whereas the alpha(1-->3) linkage samples at least two states, similar to its behavior in free solution. The bound conformation allows direct hydrogen bonds to form between the sugar and K182 of MBP, in addition to other water-mediated hydrogen bonds. Estimates of binding constants of candidate complexes based on changes in solvent-accessible surface areas upon binding support the NMR and MD results. These estimates further suggest that the enthalpic gains of the additional sugar-MBP interactions in a trisaccharide as opposed to a monosaccharide are offset by entropic penalties, offering an explanation for previous binding data.     

Serum lectin with known structure activates complement through the classical pathway

Serum mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, was revealed to activate the complement system as measured by passive hemolysis using sheep erythrocytes coated with yeast mannan. In contrast, rat liver MBP, which shares many properties in common with serum MBP, could not activate complement at all. The activation by serum MBP was inhibited effectively by the presence of haptenic sugars and dependent absolutely upon the presence of C4, indicating that the activation is initiated by the sugar binding activity of MBP and proceeds through the classical pathway. The 25 NH2-terminal amino acid sequence of rat serum MBP determined in this study was completely matched with that of MBP-A deduced from cDNA sequence by Drickamer et al. (Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887), revealing that MBP-A is in fact identical with serum MBP. On the basis of the knowledge of primary structures and physicochemical properties of rat serum and liver MBPs, a possible mechanism of the complement activation by serum MBP is discussed with reference to close similarity in the gross structures of serum MBP and C1q.     

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.