C-type Animal Lectins Research Articles

Bottlebrush Glycopolymers from 2-Oxazolines and Acrylamides for Targeting Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin and Mannose-Binding Lectin.

Lectins are omnipresent carbohydrate binding proteins that are involved in a multitude of biological processes. Unearthing their binding properties is a powerful tool toward the understanding and modification of their functions in biological applications. Herein, we present the synthesis of glycopolymers with a brush architecture via a "grafting from" methodology. The use of a versatile 2-oxazoline inimer was demonstrated to open avenues for a wide range of 2-oxazoline/acrylamide bottle brush polymers utilizing aqueous Cu-mediated reversible deactivation radical polymerization (Cu-RDRP). The polymers in the obtained library were assessed for their thermal properties in aqueous solution and their binding toward the C-type animal lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and mannose-binding lectin (MBL) via surface plasmon resonance spectrometry. The encapsulation properties of a hydrophobic drug-mimicking compound demonstrated the potential use of glyco brush copolymers in biological applications.

A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2

Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1-3/4GlcNAcβ1-2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca(2+) in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.

Current concepts on anti-Phospholipase A2 receptor antibody in Idiopathic membranous nephropathy.

Implication for health policy/practice/research/medical education:Phospholipase A2 receptor, is normally expressed in podocyte membrane. In fact the phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family such as the mannose receptor. PLA2R regulates a number of biological responses produced by secretory phospholipase A2s (sPLA2s). Determining anti-PLA2r serum levels in patients with nephrotic syndrome should designate a probable diagnosis of idiopathic membranous nephropathy and in patients in which idiopathic membranous nephropathy had a pathology confirmation may be a determining factor to exclude secondary forms of the disease. However, it is too early to abandon a kidney biopsy in patients with nephritic syndrome, and PLA2R as the newly introduced serologic study needs confirmations to finds its proper place in the field of clinical medicine.

Crystal Structure of Human Lectin-like, Oxidized Low-Density Lipoprotein Receptor 1 Ligand Binding Domain and Its Ligand Recognition Mode to OxLDL

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

Lymphocyte activation receptors: new structural paradigms in group V of C-type animal lectins

The structure-function relationship in group V of C-type animal lectins remains incompletely understood despite the new structures of NK (natural killer) cell receptors that have been solved recently. Recombinant, soluble forms of rat and human NKR-P1 and CD69 that we obtained after in vitro refolding were analysed by Fourier transform-ion cyclotron resonance MS and heteronuclear NMR ((1)H-(15)N correlation). In NKR-P1, calcium may not be removed by chelating agents because of the very high affinity of binding. In CD69, incorporation of calcium causes a structural shift in several amino acids important for the interaction with carbohydrates. Structural studies have also allowed us to understand an interesting preference of these receptors for either linear (NKR-P1) or branched (CD69) carbohydrate sequences.

A new C-type animal lectin isolated from Bothrops pirajai is responsible for the snake venom major effects in the isolated kidney

We investigated the biochemical and biological effects of a new C-type galactoside specific lectin termed BPL that was isolated from the snake venom of Bothrops pirajai. This lectin was purified using size exclusion HPLC followed by an immobilized lactose affinity column. The purified BPL was homogeneous by reverse phase HPLC and SDS-PAGE. We evaluated the nephrotoxicity of the whole venom of B. pirajai and its lectin. The whole venom of B. pirajai (10 μg/mL) showed similar results as those observed for BPL (3, 10 and 30 μg/mL) evaluated by the perfused rat kidney method. They caused reductions in perfusion pressure (Control 120 = 110.28 ± 3.69; BP 120 = 70.70 ± 2.40 ∗; BPL3 120 = 113.20 ± 4.40; BPL10 120 = 67.80 ± 3.00 ∗; BPL30 120 = 64.90 ± 3.50 ∗ mmHg; ∗: P <0.05), renal vascular resistance, urinary flow, glomerular filtration rate (Control 90 = 0.695 ± 0.074; BP 90 = 0.142 ± 0.032 ∗; BPL3 90 = 0.314 ± 0.064; BPL10 90 = 0.250 ± 0.038 ∗; BPL30 90 = 0.088 ± 0.021 ∗ mL g −1 min −1; ∗: P < 0.05) and sodium (Control 120 = 81.28 ± 0.26; BP 120 = 55.71 ± 5.72 ∗; BPL3 120 = 80.94 ± 0.93; BPL10 120 = 65.23 ± 1.47 ∗; BPL30 120 = 76.03 ± 1.70 ∗%; ∗: P < 0.05), potassium and chloride tubular transport. Neither whole venom nor purified BPL induced direct vasoactive effects in perfused arteriolar mesenteric bed, and BPL did not potentiate bradykinin contraction in the ileum. We postulate that both B. pirajai and BPL promoted the same renal effects probably caused by the release of inflammatory mediators.

Cloning, expression, and structural analysis of recombinant BJcuL, a c-type lectin from the Bothrops jararacussu snake venom

The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins. BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth. However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities. Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced. The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3). The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs). An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0 mM IPTG induction. The recombinant BJcuL was recovered and denatured in a buffer with 6 M urea and purified on a nickel-affinity column. Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer. The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.

Carbohydrate and Non-Carbohydrate Ligands for the C-Type Lectin-Like Receptors of Natural Killer Cells. A Review

The superfamily of C-type animal lectins is defined by a sequence motif of the carbohydrate- recognition domains (CRDs) and comprises seven groups of molecules. The soluble proteins are group I proteoglycans, group III collectins, and group VII containing the isolated CRDs. Type I membrane proteins include group IV selectins and group VI macrophage receptors and related molecules. Type II membrane proteins are group II hepatic lectins and group V natural killer cell receptors. The latter group has recently attracted considerable attention of the biomedical community. These receptors are arranged at the surface of lymphocytes as homo- or heterodimers composed of two polypeptides consisting of N-terminal peptide tails responsible for signaling, transmembrane domain, neck regions of varying length, and C-terminal lectin-like domains (CTLDs). Since this group is evolutionarily most distant from the rest of C-type animal lectins, the sequence of the C-terminal ligand-binding domain has diversified to accommodate other ligands than calcium or carbohydrates. These domains are referred to as natural killer domains (NKDs) forming a large percentage of CTLDs in vertebrates. Here are summarized the data indicating that calcium, carbohydrates, peptides, and large proteins such as major histocompatibility complex (MHC) class I can all be ligands for NKDs. The wide range of ligands that can be recognized by NKDs includes some new, unexpected compounds such as signal peptide-derived fragments, heat shock proteins, or oxidized lipids. The biological importance of this extended range of recognition abilities is also discussed. A review with 134 references.

Mammalian phospholipase A2: phospholipase A2 receptor.

The phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family such as the mannose receptor. PLA2R regulates a variety of biological responses elicited by secretory phospholipase A2s (sPLA2s). Group IB sPLA2 acts as an endogenous ligand to induce cell proliferation and lipid mediator production. Analysis of PLA2R-deficient mice suggested a potential role of the sPLA2-IB/PLA2R pathway in the production of proinflammatory cytokines during endotoxic shock. PLA2R is also involved in the clearance of sPLA2s, especially group X sPLA2, to protect their exaggerated reactions by potent enzymatic activities. In circulation, the soluble form of PLA2R is constitutively present as an endogenous inhibitor for mammalian sPLA2s.

Structure–Function Analysis of C-Type Animal Lectins

C-Type carbohydrate recognition domains (CRDs) are common modules in Ca 2+ -dependent animal lectins. Different C-type CRDs bind a wide variety of sugar ligands, reflecting the diverse biological processes that they mediate. The CRDs are often clustered in oligomers of lectin polypeptides, so they bind to complex, multivalent ligands with high affinity. Proteins containing C-type lectin-like domains (CTLDs) can be identified by sequence analysis, which can lead to predictions of possible sugar-binding activity. This chapter describes procedures that provide empirical methods for demonstrating sugar-binding activity in CTLDs and provides a means of preparing quantities sufficient for biochemical characterization and structural analysis. Approaches to determining the specificity of sugar binding and screening for high-affinity ligands are discussed. The chapter also discusses the expression system of C-type CRDs, production of native CRDs in Escherichia coli (E. coli), production as inclusion bodies in E. coli followed by renaturation, affinity chromatography of CRDs, solid-phase-binding assays of C-type CRDs, radiolabeled CRDs as probes for identification of high-affinity ligands, and identification of ligands by affinity chromatography on immobilized CRDs.

Phospholipase A 2 receptor: a regulator of biological functions of secretory phospholipase A 2

The phospholipase A 2 receptor (PLA 2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family that includes the mannose receptor. PLA 2R regulates a variety of biological responses elicited by specific types of secretory PLA 2s (sPLA 2s). Group IB sPLA 2 (sPLA 2-IB) acts as an endogenous PLA 2R ligand to induce cell proliferation, cell migration, and lipid mediator production. Analysis of PLA 2R-deficient mice has suggested a potential role of the sPLA 2-IB/PLA 2R pathway in the production of pro-inflammatory cytokines in endotoxic shock. PLA 2R is also involved in the clearance of sPLA 2s, including group X sPLA 2 (sPLA 2-X) and a particular type of snake venom sPLA 2, and clearance suppresses their potent enzymatic activities. In the circulation, the soluble form of PLA 2R is constitutively present as an endogenous inhibitor of sPLA 2s. This review will focus on recent findings on the roles of PLA 2R in regulating sPLA 2 functions and summarize what is known about the other binding proteins for mammalian and snake venom sPLA 2s.

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.

Erratum: C-type lectin receptors on dendritic cells and langerhans cells

Nature Reviews Immunology 2, 77–84 (2002) The human CD69 gene maps to the natural-killer gene complex on chromosome 12p, not chromosome 19 as stated incorrectly in this Review. For further details, see López-Cabrera et al. Molecular cloning, expression and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors.

Identification of a Soluble Form Phospholipase A2Receptor as a Circulating Endogenous Inhibitor for Secretory Phospholipase A2

Venomous snakes have various types of phospholipase A(2) inhibitory proteins (PLIs) in their circulatory system to protect them from attack by their own phospholipase A(2)s (PLA(2)s). Here we show the first evidence for the existence of circulating PLI against secretory PLA(2)s (sPLA(2)s) in mammals. In mouse serum, we detected specific binding activities of group IB and X sPLA(2)s, which was in contrast with the absence of binding activities in serum prepared from mice deficient in PLA(2) receptor (PLA(2)R), a type I transmembrane glycoprotein related to the C-type animal lectin family. Western blot analysis after partial purification with group IB sPLA(2) affinity column confirmed the identity of serum sPLA(2)-binding protein as a soluble form of PLA(2)R (sPLA(2)R) that retained all of the extracellular domains of the membrane-bound receptor. Both purified sPLA(2)R and the recombinant soluble receptor having all of the extracellular portions blocked the biological functions of group X sPLA(2), including its potent enzymatic activity and its binding to the membrane-bound receptor. Protease inhibitor tests with PLA(2)R-overexpressing Chinese hamster ovary cells suggested that sPLA(2)R is produced by cleavage of the membrane-bound receptor by metalloproteinases. Thus, sPLA(2)R is the first example of circulating PLI that acts as an endogenous inhibitor for enzymatic activities and receptor-mediated functions of sPLA(2)s in mice.

Crystal Structure of the C-type Lectin-like Domain from the Human Hematopoietic Cell Receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.

Functional analysis of phospholipase A2 receptor by gene knockout studies

Phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family and mediates a variety of biological responses elicited by group IB secretory phospholipase A2 (sPLA2-IB). In the present study, we have shown the evidence that a novel type of sPLA2, sPLA2-X, also acts as one of the high-affinity ligands for mouse PLA2R. We then generated PLA2R-deficient mice and found that the knockout mice exhibited the resistance to an endotoxic shock with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha and IL-1 beta. In situ hybridization analysis revealed that the expression of PLA2R transcript was markedly enhanced in type II alveolar epithelial cells and a subset of splenic lymphocytes in accordance with the elevated expression of sPLA2-IB and TNF-alpha mRNAs during endotoxic shock. In addition, the elevated expression level of TNF-alpha transcript was significantly reduced by the deficiency of PLA2R, suggesting that PLA2R plays a role in the regulation of TNF-alpha expression in these cell types. We then synthesized a specific sPLA2 inhibitor, indoxam, which blocked the binding of sPLA2-IB and X to PLA2R. Indoxam was found to suppress the elevation of the plasma level of TNF-alpha and prolonged the survival of endotoxin-challenged mice. The inhibitory effects of indoxam were abolished by the deficiency of PLA2R, demonstrating the involvement of PLA2R in the progression of endotoxic shock. We also detected and characterized a soluble form of PLA2R protein in the plasma of mouse with anti-PLA2R antibody, and showed its potential role as an endogenous sPLA2 inhibitor. Taken together, a series of studies with PLA2R-knockout mice have elucidated a critical role of PLA2R in the regulation of the development of endotoxic shock.

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

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

Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout ( Oncorhynchus mykiss)

Utilizing a splenic cDNA library and rapid amplification of cDNA 5′ ends (5′-RACE), a C-type lectin gene was cloned from a homozygous cloned rainbow trout. The 1176 bp cDNA contains a 714 bp open reading frame from which a 238-amino-acid (aa) (27 kDa) protein was deduced. It was confirmed that this protein belongs to the C-type animal lectins, and is a type II membrane receptor. The predicted protein from this sequence contains a 48 aa cytoplasmic domain, a 20 aa transmembrane domain (TM), a 46 aa stalk region and a 124 aa carbohydrate-recognition domain (CRD). The stalk region contains a leucine-zipper, and an N-glycosylation site was also found in the CRD. Sequence alignment and phylogenetic analysis of the CRD indicate that the protein has similarity with human dendritic cell immunoreceptor (DCIR), gp120 binding C-type lectin (gp120BCL) and mammalian hepatic lectins. The N-terminus (aa 4–183) has similarity with NKG2, a group of C-type lectin receptors important in human natural killer cell function. The genomic DNA (gDNA) containing this gene was amplified and sequenced. The 4569 bp gDNA contains five exons and four introns. The first three exons encode the cytoplasmic domain, the TM and stalk region, respectively. Unlike the other type II C-type lectin receptors in which the CRD was encoded by three exons, the CRD of this lectin was encoded by two exons. A transposon Tc1-like fragment was found in intron III. Intron IV is composed of a simple repeat. Tissue-specific expression of the gene was studied by RT–PCR, and it was mainly expressed in spleen and peripheral blood leukocyte (PBL). Using AluI to digest the fragment containing exon I, intron I and exon II, an RFLP was produced between the sequences of this gene in two cloned fish, OSU 142 and Arlee (AR). Seventy-one doubled haploids (DH) of OSU X AR were screened, and the gene was mapped to linkage group XIV on the published map (Young et al., Genetics 148 (1998) 839).

Expression of Isolated C-Type Carbohydrate Recognition Domains

A galactose-binding lectin from the venom of the snake Trimeresurus stejnegeri consists of isolated carbohydrate recognition domains, belonging to group VII of the C-type animal lectins. As a first step toward determining the tertiary structure of the galactose-specific lectin, we produced the lectin in Escherichia coli. By in vitro refolding and affinity chromatography, modest amounts (8 mg/liter) of active recombinant proteins were obtained. The recombinant protein was homogeneous, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. Its amino acid sequence without the initiated methionine at the N-terminus was also characterized by mass spectrometry. The data of hemagglutination and enzyme-linked lectin binding assays demonstrated that the recombinant lectin showed similar sugar-binding activity as the native protein. In addition, fluorescence spectroscopy and circular dichroism also showed obviously their structural similarity.

Primary structures of two hemagglutinins from the marine red alga, Hypnea japonica

As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys 5-Cys 62 and Cys 12-Cys 89. Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro 19, Arg 31 and Phe 52 of hypnin A-1 as compared with Leu 19, Ser 31, and Tyr 52 of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).