Detailed N-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognises both mannosylated and sulphated ligands through its C-type lectin domains (CTLDs) and cysteine-rich (CR) domain, respectively. It is widely expressed among different tissues and by certain cell types in vivo. Our previous study suggested that the glycosylation, especially terminal sialylation, regulated the functional specificities of MR. In the current investigation, the distribution of MR among various mouse tissues was studied and the N-linked glycosylation of spleen MR was analysed. Our results showed that spleen expressed the most abundant MR, consistent with its wide distribution in different cell types in this organ. Spleen MR was heterogeneously N-glycosylated. The majority of the glycans were sialylated in the α2 → 6-linkage and both Neu5Ac and Neu5Gc sialic acids were detected. Most glycans were bi-antennary (74%) with ∼22% tri-antennary and most were core fucosylated (68%). About 13% contained α-galactose. In the lung, MR exhibited more terminal sialic acids in the α2 → 3- rather than in the α2 → 6-configuration. Our study provides a profile of MR N-linked glycosylation that will facilitate our understanding of their physiological role on MR biology in vivo.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Fine discrimination in the recognition of individual species of phosphatidyl-myo-inositol mannosides from Mycobacterium tuberculosis by C-type lectin pattern recognition receptors

The Mycobacterium tuberculosis (M.tb) envelope is highly mannosylated with phosphatidyl-myo-inositol mannosides (PIMs), lipomannan, and mannose-capped lipoarabinomannan (ManLAM). Little is known regarding the interaction between specific PIM types and host cell C-type lectin pattern recognition receptors. The macrophage mannose receptor (MR) and dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells engage ManLAM mannose caps and regulate several host responses. In this study, we analyzed the association of purified PIM families (f, separated by carbohydrate number) and individual PIM species (further separated by fatty acid number) from M.tb H(37)R(v) with human monocyte-derived macrophages (MDMs) and lectin-expressing cell lines using an established bead model. Higher-order PIMs preferentially associated with the MR as demonstrated by their reduced association with MDMs upon MR blockade and increased binding to COS-1-MR. In contrast, the lower-order PIM(2)f associated poorly with MDMs and did not bind to COS-1-MR. Triacylated PIM species were recognized by MDM lectins better than tetra-acylated species and the degree of acylation influenced higher-order PIM association with the MR. Moreover, only higher-order PIMs that bind the MR showed a significant increase in phagosome-lysosome fusion upon MR blockade. In contrast with the MR, the PIM(2)f and lipomannan were recognized by DC-SIGN comparable to higher-order PIMs and ManLAM, and the association was independent of their degree of acylation. Thus, recognition of M.tb PIMs by host cell C-type lectins is dependent on both the nature of the terminal carbohydrates and degree of acylation. Subtle structural differences among the PIMs impact host cell recognition and response and are predicted to influence the intracellular fate of M.tb.     

The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor

The mannose receptor (MR) binds foreign and host ligands through interactions with their carbohydrates. Two portions of MR have distinct carbohydrate recognition properties. One is conferred by the amino-terminal cysteine-rich domain (Cys-MR), which plays a critical role in binding sulfated glycoproteins including pituitary hormones. The other is achieved by tandemly arranged C-type lectin domains that facilitate carbohydrate-dependent uptake of infectious microorganisms. This dual carbohydrate binding specificity enables MR to bind ligands by interacting with both sulfated and non-sulfated polysaccharide chains. We previously determined crystal structures of Cys-MR complexed with 4-SO(4)-N-acetylglucosamine and with an unidentified ligand resembling Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]). In continued efforts to elucidate the mechanism of sulfated carbohydrate recognition by Cys-MR, we characterized the binding affinities between Cys-MR and potential carbohydrate ligands using a fluorescence-based assay. We find that Cys-MR binds sulfated carbohydrates with relatively high affinities (K(D)=0.1 mM to 1.0 mM) compared to the affinities of other lectins. Cys-MR also binds Hepes with a K(D) value of 3.9 mM, consistent with the suggestion that the ligand in the original Cys-MR crystal structure is Hepes. We also determined crystal structures of Cys-MR complexed with 3-SO(4)-Lewis(x), 3-SO(4)-Lewis(a), and 6-SO(4)-N-acetylglucosamine at 1.9 A, 2.2 A, and 2.5 A resolution, respectively, and the 2.0 A structure of Cys-MR that had been treated to remove Hepes. The conformation of the Cys-MR binding site is virtually identical in all Cys-MR crystal structures, suggesting that Cys-MR does not undergo conformational changes upon ligand binding. The structures are used to rationalize the binding affinities derived from the biochemical studies and to elucidate the molecular mechanism of sulfated carbohydrate recognition by Cys-MR.     

Structural Basis for Multiple Sugar Recognition of Jacalin-related Human ZG16p Lectin

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1–3Man and Glcβ1–3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using ¹⁵N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.     

Glycosylation of acetylxylan esterase from Trichoderma reesei

The nature of the N- and O- linked glycosylation of acetylxylan esterase (AXE) of the Trichoderma reesei strain Rut-C30 has been characterized using different enzymatic, chromatographic, and mass spectrometric techniques. The combined data showed that the AXE N-glycan is phosphorylated and highly mannosylated. The predominant N-glycans on the single glycosylation site on AXE can be represented as GlcNAc(2)Man((1-6))P. The linker-substrate binding domain peptide separated from the core by papain digestion is heavily O-glycosylated and consists of mannose, galactose, and possibly glucose as monosaccharide and disaccharide substituents. In addition to glycosylation, sulfation was observed in the linker region. Both N- and O- linked glycans show remarkable heterogeneity. Three isoforms of AXE, separated by 2D SDS-PAGE, are described with pI values of 5.0, 5.3, and 5.9. The three isoforms can be explained by posttranslational modification of the enzyme by glycans, phosphate, and sulfate. Advancing the knowledge on the nature of the glycans produced by T. reesei is elementary for its use as a host for the expression of heterologous glycoproteins of industrial and pharmaceutical importance.     

Characterization of a binary tandem domain F-type lectin from striped bass (Morone saxatilis)

Among other functions, lectins play an important role in the innate immune response of vertebrates and invertebrates by recognizing exposed glycans on the surface of potential pathogens. Despite the typically weak interaction of lectin domains with their carbohydrate ligands, they usually achieve high avidity through oligomeric structures or by the presence of tandem carbohydrate-binding domains along the polypeptide. The recently described structure of the fucose-binding European eel agglutinin revealed a novel lectin fold (the "F-type" fold), which is shared with other carbohydrate-binding proteins and apparently unrelated proteins from prokaryotes to vertebrates, and a unique fucose-binding sequence motif. Here we described the biochemical and molecular characterization of a unique fucose-binding lectin (MsaFBP32) isolated from serum of the striped bass (Morone saxatilis), composed of two tandem domains that exhibit the eel carbohydrate recognition sequence motif, which we designate F-type. We also described a novel lectin family ("F-type") constituted by a large number of proteins exhibiting greater multiples of the F-type motif, either tandemly arrayed or in mosaic combinations with other domains, including a putative transmembrane receptor, that suggests an extensive functional diversification of this lectin family. Among the tandem lectins, MsaFBP32 and other tandem binary homologues appear unique in that although their N-terminal domain shows close similarity to the fucose recognition domain of the eel agglutinin, their C-terminal domain exhibits changes that potentially could confer a distinct specificity for fucosylated ligands. In contrast with the amniotes, in which the F-type lectins appear conspicuously absent, the widespread gene duplication in the teleost fish suggests these F-type lectins acquired increasing evolutionary value within this taxon.     

The mannose receptor ligands and the macrophage glycome

A unique glycan-binding protein expressed in macrophages and some types of other immune cells is the mannose receptor (MR, CD206). It is an endocytic, transmembrane protein with multiple glycan-binding domains and different specificities in binding glycans. The mannose receptor is important as it has major roles in diverse biological processes, including regulation of circulating levels of reproductive hormones, homeostasis, innate immunity, and infections. These different functions involve the recognition of a wide range of glycans, and their nature is currently under intense study. But the mannose receptor is just one of many glycan-binding proteins expressed in macrophages, leading to an interest in the potential relationship between the macrophage glycome and how it may regulate cognate glycan-binding protein activities. This review focuses primarily on the mannose receptor and its carbohydrate ligands, as well as macrophages and their glycomes.     

Macrophage lectins in host defence

Macrophage lectins contribute to host defence by a variety of mechanisms. The best characterised, mannose receptor (MR) and complement receptor three (CR3), are both able to mediate phagocytosis of pathogenic microbes and induce intracellular killing mechanisms. The regulation of the effector functions induced via MR is complex, and may involve both host and microbial factors. Therefore, MR is likely to play a dynamic role in the response to infection; it may act as a classical pattern recognition receptor in phagocytosis, whereas other poorly characterised factors may make a more decisive contribution to its function in physiologic settings. In contrast, the lectin site of CR3 appears to lack host-derived ligands and may be a true pattern recognition receptor. Further studies are required to evaluate the roles of other macrophage lectins in recognition of and responses to microbes.     

Crystal structure of a β-prism II lectin from Remusatia vivipara

The crystal structure of a β-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4??. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.     

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.     

Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4(+) T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.     

Distinct glycosylation in membrane proteins within neonatal versus adult myocardial tissue

Mammalian hearts have regenerative potential restricted to early neonatal stage and lost within seven days after birth. Carbohydrates exclusive to cardiac neonatal tissue may be key regulators of regenerative potential. Although cell surface and extracellular matrix glycosylation are known modulators of tissue and cellular function and development, variation in cardiac glycosylation from neonatal tissue to maturation has not been fully examined.In this study, glycosylation of the adult rat cardiac ventricle showed no variability between the two strains analysed, nor were there any differences between the glycosylation of the right or left ventricle using lectin histochemistry and microarray profiling. However, in the Sprague-Dawley strain, neonatal cardiac glycosylation in the left ventricle differed from adult tissues using mass spectrometric analysis, showing a higher expression of high mannose structures and lower expression of complex N-linked glycans in the three-day-old neonatal tissue. Man₆GlcNAc₂ was identified as the main high mannose N-linked structure that was decreased in adult while higher expression of sialylated N-linked glycans and lower core fucosylation for complex structures were associated with ageing. The occurrence of mucin core type 2 O-linked glycans was reduced in adult and one sulfated core type 2 O-linked structure was identified in neonatal tissue. Interestingly, O-linked glycans from mature tissue contained both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), while all sialylated N-linked glycans detected contained only Neu5Ac.As glycans are associated with intracellular communication, the specific neonatal structures found may indicate a role for glycosylation in the neonatal associated regenerative capacity of the mammalian heart. New strategies targeting tissue glycosylation could be a key contributor to achieve an effective regeneration of the mammalian heart in pathological scenarios such as myocardial infarction.     

Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the β subunit (GIIβ) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIβ MRH domain and that the N-terminal GIIβ G2B domain is involved in the GIIα-GIIβ interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIβ MRH domains with a higher specificity for glycans with high mannose content.     

Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.     

Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of beta-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG. These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2). Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2). These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages. The results also indicated extension of the Man(5)GN(2) structure by a single alpha-1,6-linked mannose. The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no alpha-1,3 outer extensions.     

In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein

In vivo recognition of mannosylated proteins by hepatic mannose receptors and serum mannan-binding protein (MBP) was investigated in mice. After intravenous administration, all three different (111)In-mannosylated proteins were taken up mainly by liver, and uptake was saturated with increasing doses. (111)In-Man-superoxide dismutases and (111)In-Man(12)- and (111)In-Man(16)-BSA had simple dose-dependent pharmacokinetic profiles, whereas other derivatives ((111)In-Man(25)-, -Man(35)-, and -Man(46)-BSA and (111)In-Man-IgGs) showed slow hepatic uptake at <1 mg/kg. Purified MBP experiments in vitro indicated that these derivatives bind to MBP in serum after injection, which interferes with their hepatic uptake. To quantitatively evaluate these recognition properties in vivo, a pharmacokinetic model-based analysis was performed for (111)In-Man-BSAs, estimating some parameters, including the Michaelis-Menten constant of the hepatic uptake and the dissociation constant of MBP, which correlate to the affinity of Man-BSAs for mannose receptors and MBP, respectively. The dissociation constant of Man-BSA and MBP decreased dramatically with increasing density of mannose, but the Michaelis-Menten constant of hepatic uptake of Man-BSA was not so sensitive to the change in density. This suggests that the in vivo recognition of MBP has a stronger cluster effect than that of mannose receptors. Differences obtained here are due to the unique arrangement of carbohydrate recognition domains on each mannose-specific lectin available for mannosylated ligand recognition.     

Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain

C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewis^b and Man₃. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.     

On the stringent requirement of mannosyl substitution in mannooligosaccharides for the recognition by garlic (Allium sativum) lectin. A Surface Plasmon Resonance Study

The kinetics of the binding of mannooligosaccharides to the heterodimeric lectin from garlic bulbs was studied using surface plasmon resonance. The interaction of the bound lectin immobilized on the sensor chip with a selected group of high mannose oligosaccharides was monitored in real time with the change in response units. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal alpha-1,2-linked mannose residues. An increase in binding propensity can be directly correlated to the addition of alpha-1,2-linked mannose to the mannooligosaccharide at its nonreducing end. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied, exhibited the greatest binding affinity (Ka = 1.2 x 10(6) m(-1) at 25 degrees C). An analysis of these data reveals that the alpha-1,2-linked terminal mannose on the alpha-1,6 arm is the critical determinant in the recognition of mannooligosaccharides by the lectin. The association (k1) and dissociation rate constants (k(-1)) for the binding of Man9GlcNAc2Asn to Allium sativum agglutinin I are 6.1 x 10(4) m(-1) s(-1) and 4.9 x 10(-2) s(-1), respectively, at 25 degrees C. Whereas k1 increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k(-1) decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with Allium sativum agglutinin I with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition.