N-terminal Lectin Domain Research Articles

Abstract A001: Mapping the interaction between C-type lectin domain group 14A and Multimerin 2

Abstract Introduction: Understanding the molecular basis of protein-protein interactions involved in the regulation of angiogenesis enhances our understanding of cancer pathogenesis and enables the development of novel anti-cancer therapies. In this study, we focused on the transmembrane spanning glycoprotein C-type lectin domain group 14A (CLEC14A), which is exclusively expressed on the tumor endothelium and is important in regulating tumor growth. CLEC14A promotes endothelial tube formation and migration in vitro, in addition tumor growth is retarded in CLEC14A knockout mice. CLEC14A, through its N-terminal C-type lectin domain (CTLD) interacts with the extracellular matrix protein Multimerin 2 (MMRN2) and heparan sulfate proteoglycans. Administration of antibodies which block the CLEC14A-MMRN2 interaction decreases tumor growth in vivo indicating the importance of this interaction, however the biological significance of CLEC14A’s binding to heparan sulfate proteoglycans is not known. Aims: The purpose of this study was to identify the area and key residues on CLEC14A which interact directly with MMRN2. Mapping the CLEC14A-MMRN2 interface would enable the design of small molecule inhibitors of this interaction, a potential anti-cancer strategy. Methods: We employed the Alpha fold predictive modelling algorithm to analyze the predicted structure of CLEC14A. Solvent-facing residues within a previously identified critical loop region were mutated to alanine and binding to MMRN2 was determined. Flow cytometry was used to assess the cell surface expression of these CLEC14A mutants, and far western blotting was employed to determine their capacity to bind MMRN2. Furthermore, we studied whether these mutants interacted with heparin-bound agarose beads in pulldown assays. Results: The alanine-scanning approach resulted in the identification of specific residues of CLEC14A which directly interacted with MMRN2. Furthermore, we identified the epitope on CLEC14A for a biologically active antibody previously reported to block the CLEC14A-MMRN2 interaction. Additionally, we identified critical residues that are essential for cell surface trafficking of CLEC14A. Importantly, the CLEC14A mutants that do not bind to MMRN2 retain their capacity to bind heparin. Conclusion: By leveraging predictive modelling software, we have characterized the molecular interaction between CLEC14A and MMRN2. Our findings may inform anti-angiogenesis drug design in the future. Citation Format: Aleen Baber, Victoria L Birmingham, Roy Bicknell, Philip R Morrison. Mapping the interaction between C-type lectin domain group 14A and Multimerin 2 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A001.

Poster session 1Roles of calreticulin and its mutants associated with MPN disorders in immune responses

MHC class I-β2m heterodimers (MHC-I) in the ER are typically loaded with peptides derived from cytosolic proteolysis, facilitated by the peptide-loading complex (PLC). Calreticulin (CRT) is an ER-localized lectin chaperone that is also an established member of the PLC. CRT is comprised of an N-terminal lectin domain, a proline-rich central P domain, and a negatively charged C-terminal domain (CTD) that also contains an ER-retention motif. Somatic frameshift CRT mutations (CRT-FS) have been shown to be associated with myeloproliferative neoplasms, a type of chronic, incurable blood tumor that can transform to acute myeloid leukemia. All mutations generate a common novel C-terminal domain that lacks the ER-retention signal and renders it positively charged. To study the effect of this mutation on antigen presentation by MHC-I, we first generated CRT-null HEK293T and found reduced surface MHC-I levels and association of MHC-I with low-affinity pep- tides. This phenotype was rescued by expression of CRT but not CRT-FS. CRT-FS was found to be secreted and no interaction with the PLC was detected, along with reduced recruitment of MHC-I. On retaining CRT-FS in the ER, it was found to associate with the PLC but did not restore MHC-I interaction with the PLC or surface MHC-I levels. Based on biochemical analysis and analysis of the PLC structure, we hypothesize that the altered C-terminal domain of CRT affects protein-protein interactions in the PLC that that render the complex ineffectual. Thus, the downregulation of MHC-I in CRT-FS-expressing tumor cells allows them to evade immune surveillance. We have used a patient-derived MPN cell line to generate cell lines lacking CRT expression, expressing CRT or CRT-FS, and have recapitulated the phenotypes reported above. These cell lines will be used to identify tumor-associated antigens and to investigate how extracellular CRT and CRT-FS influence innate immune responses, thus identifying potential immunotherapy targets.

Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism

The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.

Some Aspects in Mechano-Biology of Platelet and Leukocyte in Blood Flows

For hemostasis and thrombosis, some proteins, such as Von Willebrand Factor (VWF, a multimeric plasma glycoprotein synthesized in endothelial cells and megakaryocytes and secreted to circulation or attached to endothelial cells), the metalloprotease ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), P-selectin (one of three selectin family members with a N-terminal C-type lectin domain, an epidermal growth factor (EGF)-like module, a series of consensus repeat (CR) units, a transmembrane segment and a short cyto-plasmic domain) and β2 integrin. In adhesion and aggregation of circulating platelets towards to the sites of vascular injury, VWF on vascular wall captures and activates the circulating platelets through interaction with platelet receptor glycoprotein Ibα (GPIbα). The activated platelets will secrete P-selectin, mediating flowing leukocytes to be captured first to and then rolled on the platelets. Activation of β2 integrin on leukocytes makes the rolling cells slow down and adhere firmly to platelet. Pathological hemodynamic environment may cause platelet-induced inflammation overreaction of leukocytes, leading to mechanical instability of thrombotic plaque. Above mentioned events all are referred to their respective un-well known mechano-chemistry processes. For better understanding on the mechano-chemistry mechanism of interaction of leukocyte with platelet under flows, we have investigated the force-dependent Structure-function of VWF-A domain, force-regulated cleavage of A2 domain of von Willebrand factor (vWF) by ADAMTS13, P-selectin secretion from activated platelet, and P-selectin-mediated Activation of β2 integrin on leukocytes under shear stresses, and so on through AFM and flow chamber experiments and molecular dynamics simulation. Our data showed that, these events mentioned above were biphasic force-dependent. Increasing force stabilizes the globular VWF-A conformation first and then makes it become a spread one with higher affinity with platelet receptor glycoprotein Ibα (GPIbα), the force-regulated cleavage of VWF-A2 domain by ADAMTS13 maybe closely related to the induced-fit of ADMATS13 and VWF-A2, Force triggers and quickens P-selectin-induced Activation of β2 integrin on leukocytes.

Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: Engineering a minimalistic allosteric system

For many biological processes such as ligand binding, enzymatic catalysis, or protein folding, allosteric regulation of protein conformation and dynamics is fundamentally important. One example is the bacterial adhesin FimH, where the C-terminal pilin domain exerts negative allosteric control over binding of the N-terminal lectin domain to mannosylated ligands on host cells. When the lectin and pilin domains are separated under shear stress, the FimH-ligand interaction switches in a so-called catch-bond mechanism from the low- to high-affinity state. So far, it has been assumed that the pilin domain is essential for the allosteric propagation within the lectin domain that would otherwise be conformationally rigid. To test this hypothesis, we generated mutants of the isolated FimH lectin domain and characterized their thermodynamic, kinetic, and structural properties using isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, and X-ray techniques. Intriguingly, some of the mutants mimicked the conformational and kinetic behaviors of the full-length protein and, even in absence of the pilin domain, conducted the cross-talk between allosteric sites and the mannoside-binding pocket. Thus, these mutants represent a minimalistic allosteric system of FimH, useful for further mechanistic studies and antagonist design.

Novel mannose binding natterin-like protein in the skin mucus of Atlantic cod (Gadus morhua)

This study presents the first report of purification of natterin-like protein (Nlp) in a non-venomous fish. The peptide identities of purified cod Nlp were confirmed through LC-MSMS and matched to a cod expressed sequence tag (EST). A partial cod nlp nucleotide sequence was amplified and sequenced based on this EST. Multiple sequence alignment of cod Nlp showed considerable homology with other teleost Nlps and the presence of an N-terminal jacalin-like lectin domain coupled with a C-terminal toxin domain. nlp expression was higher in skin, head kidney, liver and spleen than in other tissues studied. Hemaggluttination of horse red blood cells by Nlp was calcium dependent and inhibited by mannose. A Vibrio anguillarum bath challenge however, did not alter the expression of cod nlp transcripts in the skin and gills. Further functional characterization is required to establish the significance of this unique protein in Atlantic cod and other teleosts.

Structural and Functional Insights into Three Undercharacterized Fimbrial Adhesins

Adhesion to biotic and abiotic surfaces is an essential step in the infectious cycle of many human pathogens. To this end, both Gram-positive and Gram-negative bacteria have developed a variety of strategies to overcome innate clearance mechanisms and persist within the host. The expression and biogenesis of Chaperone-Usher Pathway (CUP) pili represent one such strategy. In addition to mediating the formation of bacterial biofilms, these proteinaceous extracellular appendages contain, at their tip, a two-domain adhesin that is composed of an N-terminal lectin domain and a C-terminal pilin domain.

Abstract B064: The effects of tumor-associated calreticulin mutants on antigen presentation

Abstract The phenomenon of antigen processing and presentation aids the immune system in distinguishing between normal and malignant cells. In cancer cells, Major Histocompatibility Complex (MHC) proteins present tumor-associated antigens (TAAs) that may serve as targets of immunotherapy. Antigen presentation by MHC class I-β2m heterodimers (MHC-I) requires peptide association to occur in in the endoplasmic reticulum (ER). Binding is facilitated by the Peptide Loading Complex (PLC), which consists of the TAP transporter, tapasin, the thiol-oxidoreductase ERp57, and a lectin chaperone, calreticulin (CRT). CRT associates with MHC-I via the single, conserved, glycan present on MHC-I, drawing it into the PLC to facilitate peptide loading. CRT is comprised of an N-terminal lectin domain, a proline-rich central P domain, and a negatively charged C-terminal domain (CTD) that contains an ER retention motif. Two recent studies report a mutant CRT associated with myeloproliferative neoplasms (MPNs). These MPNs carry a somatic frame-shift CRT mutation (CRTFS) that generates a novel CTD that lacks the ER-retention signal and renders it positively charged. Previous studies have shown that ablation of CRT expression in mouse embryonic fibroblast cells result in decreased surface expression of MHC-I, which is reversed on reconstitution of full-length CRT. Interestingly, reconstitution of CRT lacking the CTD did not restore MHC-I expression, indicating a role for the CTD in MHC-I expression. We therefore suspect that antigen presentation in MPNs carrying the CRTFS is altered. To address the role of CRTFS in antigen presentation in a human cell line, we have generated a CRT knockout of HeLaM cells using CRISPR/Cas9 technology. The expression of MHC-I in HeLaM cells dramatically increases on exposure to interferon-γ and we are currently assessing the alterations in MHC-I expression in these CRT knockout cells using various biochemical techniques. Future experiments include analysis of the peptide repertoire displayed on the surface of these cells as well as changes in the molecular interactions of the mutant CRT with other members of the PLC. After a preliminary study in HeLaM cells, we will study the effect of CRTFS in antigen presentation in MPN cell lines to identify unique TAAs to facilitate the specific targeting of these cells by immunotherapy. Citation Format: Najla Arshad, Peter Cresswell. The effects of tumor-associated calreticulin mutants on antigen presentation. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B064.

Molecular Mechanism of Flocculation Self-Recognition in Yeast and Its Role in Mating and Survival

ABSTRACTWe studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-bridge” hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating.

Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains “mammalian-specific insets” that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.

Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin

The cholesterol-dependent cytolysins (CDCs) attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modeling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.

Sugar-Binding Properties of the Two Lectin Domains of LEC-1 with Respect to the Gal.BETA.1-4Fuc Disaccharide Unit Present in Protostomia Glycoconjugates

Galβ1-4Fuc disaccharide unit was recently reported to be the endogenous structure recognized by the galectin LEC-6 isolated from the nematode Caenorhabditis elegans. LEC-1, which is another major galectin from this organism, is a tandem repeat-type galectin that contains two carbohydrate recognition domains, the N-terminal lectin domain (LEC-1Nh) and the C-terminal lectin domain (LEC-1Ch), and was also found to have an affinity for the Galβ1-4Fuc disaccharide unit. In the present study, we compared the binding strengths of LEC-1, LEC-1Nh, and LEC-1Ch to Galβ1-4Fuc, Galβ1-3Fuc, and Galβ1-4GlcNAc units as well as to LEC-6-ligand N-glycans by using frontal affinity chromatography (FAC) analysis. The two lectin domains of LEC-1 exhibited the highest affinity for Galβ1-4Fuc, though sugar-binding properties differed somewhat between LEC-1Nh and LEC-1Ch. Furthermore, these two domains had significantly lower affinities for the LEC-6-binding glycans. These results suggest that the endogenous recognition unit of LEC-1 is likely to be Galβ1-4Fuc, and that the endogenous ligands for LEC-1 are different from those for LEC-6.

Cross-Link Formation between Mutant Galectins of Caenorhabditis elegans with a Substituted Cysteine Residue and Asialofetuin via a Photoactivatable Bifunctional Reagent

LEC-1 is the first tandem repeat-type galectin isolated from an animal system; this galectin has two carbohydrate recognition domains in a single polypeptide chain. Because its two lectin domains have different sugar-binding profiles, these domains are thought to interact with different carbohydrate ligands. In our previous study, we showed that a mutant of LEC-1 in which a cysteine residue was introduced at a unique position in the N-terminal lectin domain (Nh) can be cross-linked with a model glycoprotein ligand, bovine asialofetuin, by using a bifunctional photoactivatable cross-linking reagent, benzophenone-4-maleimide. In the present work, we applied the same procedure to the C-terminal lectin domain (Ch) of LEC-1. Cross-linked products were formed in the cases of two mutants in which a cysteine residue was introduced at Lys¹⁷⁷ and Ser²⁶⁸, respectively. This method is very useful for capturing and assigning endogenous ligand glycoconjugates with relatively low affinities to each carbohydrate recognition domain of the whole tandem repeat-type galectin molecule.

Molecular characterization of a novel pathogen-responsive receptor kinase-like in Citrus limon

Infection by pathogenic microorganisms is a source of biological stress in plants. Understanding the interaction between plants and microbial infection at molecular level might contribute to our understanding for the effective control of the disease. Here, we isolated a novel putative receptor kinase-like protein (Citrus limon P5) that is the first Lec-receptor kinase-like protein isolated in lemon during a pathogen infection. C. limon P5 cDNA fragment was detected by differential display assay in C. limon during Capnodium citri (sooty mold) infection. The deduced amino acid sequence of P5 full-length cDNA revealed a 83% sequence homology with a receptor kinase-like protein from Arabidopsis thaliana characterized by a N-terminal lectin domain and C-terminal serine/threonine conserved domain. The inhibition of the pathogen-responsive P5 by the protein kinase inhibitor staurosporine and by a mitogen-activated protein kinase inhibitor (PD 98059) indicated a defense mechanism in C. limon against pathogens mediated via signal transduction pathway. To our knowledge, this is the first evidence that C. limon uses this putative defense mechanism against pathogens. The role of this protein is discussed as a starting point to understanding the molecular mechanisms in the C. limon in response to C. citri infection.

Specific residues in the N‐terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone-usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.

Identification of a Second, Non-conserved Amino Acid That Contributes to the Unique Sugar Binding Properties of the Nematode Galectin LEC-1

The basic disaccharide structure recognized by galectin family members is the lactosamine-like structure Galbeta1-4(3)Glc(NAc). The 32-kDa galectin LEC-1 of the nematode Caenorhabditis elegans is composed of two domains, each of which is homologous to vertebrate 14-kDa-type galectins. The N-terminal lectin domain of LEC-1 recognizes blood group A saccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAc), whereas this saccharide is poorly recognized by the C-terminal domain. Using a combination of site-directed mutagenesis and analysis of the sugar-binding profile by frontal affinity chromatography, we previously found that Thr41 in the N-terminal lectin domain of LEC-1 is important for its affinity for A-hexasaccharide. Thr41 is located on beta-strand S3, next to the three beta-strands S4-S6, where the conserved amino acids form the binding site for the basic Galbeta1-4(3)Glc(NAc) structure. Here, we report that a second amino acid, Asp133, in the N-terminal lectin domain of LEC-1, located on the beta-strand S2 adjacent to that containing Thr41, is important for LEC-1-specific recognition of A-hexasaccharide. These results suggest that amino acid residues other than those located on the three beta-strands S4-S6, contribute to the unique sugar binding specificity of individual galectins.

P-selectin knockout: a mouse model for various human diseases.

P-selectin is a transmembrane adhesion receptor specific to platelets and endothelial cells. It has an N-terminal lectin domain that recognizes specific carbohydrate moieties on monocytes, neutrophils and some other subsets of leukocytes. P-selectin is stored in granules and is expressed on the plasma membrane only after the cells are stimulated by vascular injury or during inflammation. Physiologically P-selectin is likely to be involved in the recruitment of leukocytes that promote wound healing and fight infection. There are many disorders in which the excessive recruitment of leukocytes is characteristic, including chronic inflammation, atherosclerosis, arthritis, diabetes, asthma and reperfusion injury. Because certain cancer cells also express the ligand for P-selectin it is possible that this receptor is involved in metastasis. To study the specific role of P-selectin in these pathological processes, we have prepared a mouse lacking P-selectin through gene targeting. Leukocyte interaction with the vessel wall is defective in these animals as leukocytes do not roll in the mesenteric venules and their extravasation at sites of inflammation and vessel injury is limited. We are testing these animals in models of the various diseases mentioned above in order to evaluate when the absence of P-selectin is beneficial.

Recognition of the N‐terminal lectin domain of FimH adhesin by the usher FimD is required for type 1 pilus biogenesis

In this work we discover that a specific recognition of the N-terminal lectin domain of FimH adhesin by the usher FimD is essential for the biogenesis of type 1 pili in Escherichia coli. These filamentous organelles are assembled by the chaperone-usher pathway, in which binary complexes between fimbrial subunits and the periplasmic chaperone FimC are recognized by the outer membrane protein FimD (the usher). FimH adhesin initiates fimbriae polymerization and is the first subunit incorporated in the filament. Accordingly, FimD shows higher affinity for the FimC/FimH complex although the structural basis of this specificity is unknown. We have analysed the assembly into fimbria, and the interaction with FimD in vivo, of FimH variants in which the N-terminal lectin domain of FimH was deleted or substituted by different immunoglobulin (Ig) domains, or in which these Ig domains were fused to the N-terminus of full-length FimH. From these data, along with the analysis of a FimH mutant with a single amino acid change (G16D) in the N-terminal lectin domain, we conclude that the lectin domain of FimH is recognized by FimD usher as an essential step for type 1 pilus biogenesis.

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 (NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow (BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein (GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.

Identification of the Amino Acid Residue in the Nematode Galectin LEC-1 Responsible for Its Unique Sugar Binding Property: Analysis by Combination of Site-Directed Mutagenesis and Frontal Affinity Chromatography

The basic disaccharide structure recognized by galectin family members is the lactosamine-like structure Galbeta1-4(3)Glc(NAc). In galectins, eight highly conserved amino acid residues participate in the recognition of this basic structure. Each galectin seems to mediate diverse biological functions due to recognition of different modifications of the basic disaccharide Galbeta1-4(3)Glc(NAc), but there is very little information about which amino acid residue in galectin is responsible for recognizing these modifications. The 32-kDa galectin LEC-1 of the nematode Caenorhabditis elegans is composed of two domains, each of which is homologous to vertebrate 14-kDa-type galectins. Although both lectin domains have an affinity for N-acetyllactosamine (Galbeta1-4GlcNAc)-containing, N-linked, complex-type sugar chains, the N-terminal lectin domain of LEC-1 recognizes blood group A saccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAc), whereas this saccharide is only poorly recognized by the C-terminal domain. Here, we used a combination of site-directed mutagenesis of the N-terminal lectin domain of galectin LEC-1 and an analysis of the sugar-binding profile by frontal affinity chromatography to identify the amino acid residues important for this recognition. Our results indicate that Thr(41) in the N-terminal lectin domain of LEC-1 is important for its affinity for A-hexasaccharide.