Primary structure of a protein isolated from reef shark (Carcharhinus springeri) cartilage that is similar to the mammalian C-type lectin homolog, tetranectin.

During the course of characterization of low molecular weight proteins in cartilage, we have isolated a protein from reef shark (Carcharhinus springeri) cartilage that bears a striking resemblance to the tetranectin monomer originally described by Clemmensen et al. (1986, Eur. J. Biochem. 156, 327-333). The protein was isolated by extraction of neural arch cartilage with 4 M guanidine hydrochloride, dialysis of the extract to bring the guanidine to 0.4 M (reassociating proteoglycan aggregates), followed by cesium chloride density gradient removal of the proteoglycans. The amino acid sequence had 166 amino acids and a calculated molecular weight of 18,430. The shark protein was 45% identical to human tetranectin, indicating that it was in the family of mammalian C-type lectins and that it was likely to be a shark analog of human tetranectin. The function of tetranectin is unknown; it was originally isolated by virtue of its affinity for the kringle-4 domain of plasminogen. Sequence comparison of human tetranectin and the shark-derived protein gives clues to potentially important regions of the molecule.     

The cartilage-derived, C-type lectin (CLECSF1): structure of the gene and chromosomal location.

Cartilage is a tissue that is primarily extracellular matrix, the bulk of which consists of proteoglycan aggregates constrained within a collagen framework. Candidate components that organize the extracellular assembly of the matrix consist of collagens, proteoglycans and multimeric glycoproteins. We describe the human gene structure of a potential organizing factor, a cartilage-derived member of the C-type lectin superfamily (CLECSF1; C-type lectin superfamily) related to the serum protein, tetranectin. We show by Northern analysis that this protein is restricted to cartilage and locate the gene on chromosome 16q23. We have characterized 10.9 kb of sequence upstream of the first exon. Similarly to human tetranectin, there are three exons. The residues that are conserved between CLECSF1 and tetranectin suggest that the cartilage-derived protein forms a trimeric structure similar to that of tetranectin, with three N-terminal alpha-helical domains aggregating through hydrophobic faces. The globular, C-terminal domain that has been shown to bind carbohydrate in some members of the family and plasminogen in tetranectin, is likely to have a similar overall structure to that of tetranectin.     

Mutational analysis of affinity and selectivity of kringle-tetranectin interaction. Grafting novel kringle affinity ontp the trtranectin lectin scaffold

C-type lectin-like domains are found in many proteins, where they mediate binding to a wide diversity of compounds, including carbohydrates, lipids, and proteins. The binding of a C-type lectin-like domain to a ligand is often influenced by calcium. Recently, we have identified a site in the C-type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4. This study provides further insight into molecular determinants of importance for binding selectivity and affinity of C-type lectin kringle interactions.     

Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor

The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes.Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 A resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not alpha-helical, while in tetranectin they form an alpha-helical coiled-coil.     

Tetranectin Is a Novel Marker for Myogenesis during Embryonic Development, Muscle Regeneration, and Muscle Cell Differentiationin Vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiationin vitro.We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophicmdxmice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiationin vitro.Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

Distinct functions of DC-SIGN and its homologues L-SIGN (DC-SIGNR) and mSIGNR1 in pathogen recognition and immune regulation

Antigen presenting cells express C-type lectins that are involved in pathogen capture, processing and antigen presentation to induce immune responses against these pathogens. However, it is becoming clear that pathogens have evolved to subvert the function of some C-type lectins to escape immune surveillance. An important C-type lectin family is represented by DC-SIGN and its homologues in human and mouse. Here we discuss the structure in relation to the pathogen binding specificity of the SIGN receptors and the function of these receptors in mouse and humans.     

C型凝集素

C型凝集素（C-type lectin）代表一个识别碳水化合物配体依赖于钙离子（Ca2＋）参与的糖原结合蛋白家族,含有一个或多个一级结构和二级结构同源的碳水化合物识别结构域。随着研究的深入,越来越多的C型凝集素能够识别体内的非糖类的配体,包括蛋白质和脂类等。这些C型凝集素在维持机体稳态、免疫防御以及免疫监视等重要生理病理过程中发挥着重要作用。就C型凝集素的结构、分类和在免疫系统中的功能作一介绍。     

Cloning and characterization of the Atlantic salmon serum lectin,a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.     

Cloning and characterization of the Atlantic salmon serum lectin,a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.     

Mass spectrometric characterisation of post-translational modification and genetic variation in human tetranectin

Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein primarily involved in tissue remodeling and development, was scanned for covalent modifications and sequence heterogeneity, using a combination of mass spectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of different mass and abundance in plasma tetranectin, all of higher mass than that calculated from the cDNA sequence. To identify and locate residues accounting for the heterogeneity, samples of tetranectin were subjected to proteolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry and, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due to sequence heterogeneity at position 85 and the presence of a partially sialylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is encoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixture of Ser85 and Gly-85 sequence variants. Mass spectrometric analysis of enzymatic and mild acid hydrolysates of an N-terminal glycopeptide showed that the composition and partial covalent structure of the O-linked oligosaccharide prosthetic group is < or =N-acetylhexosamine < or =[hexose, (sialic acid)0-3].     

C-type lectin-like domains.

Carbohydrate-recognition domains of C-type (Ca2+-dependent) animal lectins serve as prototypes for an important family of protein modules. Only some domains in this family bind Ca2+ or sugars. A comparison of recent structures of C-type lectin-like domains reveals diversity in the modular fold, particularly in the region associated with Ca2+ and sugar binding. Some of this diversity reflects the changes that occur during normal physiological functioning of the domains. C-type lectin-like domains associate with each other through several different surfaces to form dimers and trimers, from which ligand-binding sites project in a variety of different orientations.     

Isolation and cloning of a C-type lectin from the hexactinellid sponge Aphrocallistes vastus: a putative aggregation factor

Among the sponges (Porifera), the oldest group of metazoans in phylogenetic terms, the Hexactinellida is considered to have diverged earliest from the two other sponge classes, the Demospongiae and Calcarea. The Hexactinellida are unusual among all Metazoa in possessing mostly syncytial rather than cellular tissues. Here we describe the purification of a cell adhesion molecule with a size of 34 kDa (in its native form; 24 kDa after deglycosylation) from the hexactinellid sponge Aphrocallistes vastus. This adhesion molecule was previously found to agglutinate preserved cells and membranes in a non-species-specific manner (Müller, W. E. G., Zahn, R. K, Conrad, J., Kurelec, B., and Uhlenbruck, G. [1984] Cell adhesion molecules in the haxactinellid Aphrocallistes vastus: species-unspecific aggregationfactor. Differentiation, 26, 30--35). The fact that the aggregation process required Ca(2+) and was inhibited by bird's nest glycoprotein and D-galactose but not by D-mannose or N-acetyl-D-galactosamine suggests that this cell adhesion molecule is a C-type lectin. To test this assumption, two highly similar C-type lectins were cloned from A.vastus. The deduced polypeptides of the two cDNA species isolated classified these molecules as C-type lectins. The calculated M(r) of the 191 aa long sequences were 22,022 and 22,064, respectively. The C-type lectins showed highest similarity to C-type lectins (type-II membrane proteins) from higher metazoan phyla; these molecules are absent in non-Metazoa. The two sponge C-type lectins contain the conserved domains known from other C-type lectins (e.g., disulfide bonds, the amino acids known to be involved in Ca(2+)-binding, as well as the amino acids involved in the specificity of binding to D-galactose) and a hydrophobic N-terminal region. The N-terminal part of the purified C-type lectin was identical with the corresponding region of the deduced polypeptide from the cDNA. It is proposed that the A.vastus lectins might bind to the cell membrane by their hydrophobic segment and might interact with carbohydrate units on the surface of the other cells/syncytia.     

Primary structure characterization of Bothrops jararacussu snake venom lectin

The complete amino acid sequence of the lectin from Bothrops jararacussu snake venom (BJcuL) is reported. The sequence was determined by Edman degradation and amino acid analysis of the S-carboxymethylated BJcuL derivative (RC-BJcuL) and from its peptides originated from enzymatic digestion. The sequence of amino acid residues showed that this lectin displays the invariant amino acid residues characterized in C-type lectins. Amino acids analysis revealed a high content of acidic amino acids and leucine. These findings suggest that BJcuL, like other snake venom lectins, possesses structural similarities to the carbohydrate recognition domain (CRD) of calcium-dependent animal lectins belonging to the C-type -galactoside binding lectin family.     

Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of ¹⁵N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.     

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.     

Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli

The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIgamma and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of beta-strands which is typical of C-type lectins. Finally, both RegIIIgamma and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.     

Genomic analysis of C-type lectins

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell-cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.